A346 ISK (MINK), KVLQT1 POTASSIUM CHANNEL Th-PM-H7 Th-PM-H8 COEXPRESSION OF minK MODIFIES hKV1.5 ACTIVATION IN IKs channels: minK/KvLQT1 heteromultimers have a larger unitary MAMMALIAN CELLS. ((Tao Yang, Sabina Kupershmidt, Dan M. Roden, Dirk conductance than homomeric KvLQT1 channels. J. Snyders)) Vanderbilt University, Nashville, TN 37232. ((Federico Sesti, and Steve A. N. Goldstein)) Section of Developmental Biology and Biophysics, Departments of Pediatrics and Cellular & Molecular Physiology, Coexpression of minK modifies K+ currents obtained with expression of Yale University School of Medicine, New Haven, CT. KvLQTI (resulting in slowed activation and an increase in current) and ofHERG IKs is a voltage-dependent K+ channel in human heart that opens during (resulting in increased current with no change in gating kinetics). In this study, we contraction to repolarize the myocardium. The channel is a heteromeric complex have examined the interaction ofminK with hKvl .5 stably transfected in L-cells, of the single P loop subunit KvLQTI and minK, a non-P loop protein with a single transmembrane segment (Barhanin et al., 1996. Nature. 384:78-80; Sanguinetti et and transiently transfected in HEK-293 cells. Current obtained with transfection al., 1996. ibid. 384:80-83). Mutations in both subunits have now been identified to by green fluorescence protein (GFP) alone was compared to that with minK + GFP. cause inherited Long QT syndrome (LQTS), a cardiac dysrrhythmia that In both systems, minK shifted the voltage dependence of activation - 10-15 mV predisposes to sudden death (Splawski et al. Nature Genetics. 1997. in press). positive, and attenuated slow inactivation. MinK strikingly slowed activation, Recently, we showed that the IKs pore is formed by contributions of both without altering deactivation: for example, at +50 mV, control activation T was subunits (cf., Tai and Goldstein, this volume) using cysteine mutation and Cd2+ or 2.3±0.1 ms vs 5.4±0.6 ms with minK Usual hKvl .5 currents were observed with Zn2+ to probe side-chain exposure. Here we use heterologous expression of expression of an hKvl .5 mutation lacking the C-terminal 57 amino acids, but no wildtype and engineered subunits in Xenopus oocytes to explore the single channel effect of coexpression with minK was found. We conclude that minK modifies characteristics of IKs and homomeric KvLQTl channels. Application of transition function of hKvl.5 IL cell metals to channels carrying substituted cysteines is used to confirm the identity of Kvl.5+GFP IL cell Kv1.5+mimK+G the channels under study. We find the unitary current through KvLQTI homomers channels expressed in is too small to be resolved by direct measurement and can only be evaluated by mammalian cells, and that variance analysis; in contrast, currents through single IKs channels are observed the interaction between y i-I mV l Vs10 V directly. Our findings contradict those of Romey et al. (JBC. 1997. 272:16713-6). the two proteins likely IKs channels formed with minK mutants that lead to LQTS in humans are now involves the C-terminal under study. domain of hKvl .5. bOrM

SMOOTH MUSCLE REGULATORY PROTEINS II Th-Posl Th-Pos2 TRANSGENIC MOUSE HEARTS WITH A POINT MUTATION IN a- VISUALIZATION OF TROPOMYOSIN TURNOVER AND INCORPORATION IN ADULT TROPOMYOSIN SHOW ALTERED MYOFILAMENT SENSITIVITY TO CARDIAC MYOCYTES ((Daniel Lt Michele and Joseph M. Metzger)) Department of CALCIUM, INDEPENDENT OF PHOSPHORYLATION STATE. ((C.C. Evans,+ Physiology, University ofMichigan, Ann Arbor, Mt 48109. B.M. Wolska+, M. Muthuchamy*, D.F. Wieczorek*, R.J. Solaro+)) 'University of Illinois at Chicago, College of Medicine, Chicago, IL 60612, *University of Adult contractile cells turn over their contractile proteins with average half lives of several Cincinnati, College of Medicine, Cincinnati, OH 45267. days while maintaining the ability to produce force. Utilizing adenoviral gene transfer into adult cardiac myocytes in Wvno, we have remodeled the tropomyosin (Tm) within the We studied from adult mouse contractile apparatus with ectopically expressed tropomyosin isoforms. An adenoviral vector myofilaments nontransgenic (NTG) hearts that express (AdaxTnFLAG) was constructed with a CMV promoter driving expression ofhuman ea-Tm wild type a-tropomyosin (a-Tm) and from hearts ofadult transgenic mice (TG) over with a C-terminal FLAG. Western blot analysis oftotal protein from adult rat cardiac expressing a mutant a-Tm (Aspl75Asn) linked to familial hypertrophic myocytes infected with AdaxTnmFLAG showed no detectable aTmFLAG protein at day 1, but cardiomyopathy (FHC). Transgene expression was driven by the murine a-myosin aTmFLAG/total Tm increased to 40%/ by 6 days post gene transfer. Dual monoclonal heavy chain promoter and was restricted to the heart. TG mouse hearts demonstrated immunfluorescence and confocal microscopy using a FLAG epitope Ab and a troponin I or an a significant increase in Ca2" sensitivity in a Ca2"-activated myofibrillar MgATPase a-actinin Ab was used to visualize the incorporation ofthe cxTmFLAG into the contractile assay (A pCa50 = 0.1 1) compared to NTG mice. Force measurements from Triton X- apparatus. At day 2 post infection, aTmFLAG was first detected evenly expressed throughout 100 skinned fiber bundles prepared from the TG mouse hearts also had increased Ca2" the myocyte in a narrow band near the free end (pointed end) ofthe thin filament. This was sensitivity compared to fibers from NTG hearts (A pCa5o= 0.08). Phosphorylation in contrast to the troponin I Ab which labelled the entire thin filament from barbed to pointed oftroponin I with cyclic-AMP dependent protein kinase induced an equal decrease end. In addition, we have constructed an adenovirus vector containing a CMV promoter in Ca2" sensitivity of force in fibers from NTG (ApCa55 = 0.06) and TG (ApCa50 = driving expression of rat P-Tm (AdpTm). Western blot analysis indicated that 30-35% ofthe 0.07) hearts. Our preliminary studies also show no difference in maximum Ca2 - total Tm was the expressed P-Tm 5-7 days post infection with no P-Tm protein detected in activated force in fiber bundles from NTG and TG mouse hearts. These results control myocytes. Immunfluorescence using an Ab that recognizes both Tm isoforms showed indicate that although there is no difference in the maximum force generated by periodic staining was maintained in infected cells 6 days post infection suggesting that the myofilaments from TG hearts as compared to NTG hearts, TG heart myofilaments expressed Tm is incorporated into sarcomeres. We conclude that 1) adenoviral mediated are more sensitive to Ca2" whether they are phosphorylated or not. Increased Ca2+ gene transfer can be used to remodel Tm in the context ofthe adult cardiac myocyte, 2) Tm turnover and incorporation in adult cardiac myocytes appears to occur most readily at the free sensitivity, particularly under phosphorylating conditions as occurs during J3- end ofthe adrenergic stimulation, may predispose the myocardium to impaired relaxation and thin filament. contribute to altered cardiac function in patients with FHC during exercise or stress.

Th-Pos3 Th-Pos4 CHARACTERIZATION OF TN EXCHANGE IN MYOFIBRILS AND SOLUTION. HIGH RESOLUTION OF THE pCa/FLUORESCENCE RELATION OF DANZ- ((M. She', S. Frisbie', D. C. Trimble', L. C. Yu', and J. M. Chalovich')) 'NIAMS/NIH, TNC SUBSTITUTED RABBIT PSOAS SINGLE FIBERS. ((M Huang, D Bethesda MD 20892; 'E. Carolina Med. School, Greenville, NC 27834. Burkhoff, F Schachat, & P Brandt)) Depts. of Anatomy & Cell Biology, It has been shown earlier that fluorescently labeled troponin (Tn) can be exchanged into Biomedical Engineering and Medicine, Columbia U., and Cell Biology, Duke U. rabbit psoas fibers in order to simultaneously measure mechanical properties and the state of the regulatory proteins (Brenner & Chalovich, 1995 Biophys. J. A142). We We constructed an apparatus to continued to characterize this exchange process. X-ray diffraction patterns of single 1.0- measure fluorescence ratio fibers in rigor showed intensification of 385 A after 12 h of incubation with avidin- o/° change (AF) in single muscle attached Tn, an indication of Tn exchange. Fluorescence microscopy of myofibrils 0.8- fibers with further showed that the exchange of Tn under rigor conditions occurred mostly in the small changes in overlap region, as in muscle fibers. However, in the presence of exogenous S1, the I t 1 pCa. AF (0) of DANZ-TnC exchange occurred along the entire thin filament, requiring more than 1 h of incubation. 0 0.6i substituted fibers increases In relaxing condition, little exchange was observed. These results suggest that the from the first decrease in pCa exchange process is slow but can be facilitated by myosin binding to actin. Several 80o.4- Cy j(8 to until saturation at methods were attempted to estimate the rate of Tn exchange in solution. When the thin 7.7) pCa filaments were prepared using a radioactively labeled TnI, in - 3 h at 20 OC, the ° 0.2- / e 6. Fitting the AF points with one radioactivity could be totally displaced by a large excess of unlabeled Tn. The time c binding constant fails to fit the course of labeled-Tn exchange in the presence of excess Tn was also monitored via the 0.0- upper half well while fitting with decrease of fluorescence of lANBD-Tn and by the change in fluorescence energy two binding constants (3 transfer between IAANS-Tn (donor) and DABMI-actin (acceptor). Both systems 8.0 7.5 7.0 6.56 r.05.5 0.0 variable parameters) includes reported a similar characteristic time of exchange, - 40 min at 20 IC. The exchange pca all the points. The fitted pKs for process appeared to be faster in the presence of SI but this could not be accurately AF in the figure (fiber F0918A) are 7.21 and 6.41 and the nH's are 1.0 and 2.33 measured due to a decrease in the fluorescence change. The binding of LAANS-Tn to respectively. On average the DABMI-actin-tropomyosin was observed to occur in at least two steps; the sum of 2nd two binding events contribute equally to the step rates k2+IL2 was approximately 2.5s-'. The binding affinity was too high to allow maximum fluorescence change. Tension (0) pK is 5.97 and nH is 3.64. AF ready estimation of the first step through stopped-flow measurements. Taken together, precedes tension as reported previously (Guth & Potter, J. Biol. Chem. 262: the results indicate that the exchange ofTn is a slow process in the absence of SI and is 13267,1987). The high affinity event shows no cooperativity so its nH is fixed at facilitated by the binding of S I to actin. The extent ofthis acceleration is not yet known, 1.0 but the low affinity event is cooperative. The two binding events may be one likely to be a factor of 100 or more. of continuously increasing cooperativity. A347 SMOOTHSMOOTH~~~MUSCLEMUCL REUATRREGULATORY PRTENPROTEINS HII A347- Th-Pos5 Th-Pos6 THE USE OF YEAST ACTIN MUTANTS TO PROBE THE MEASUREMENT OF ACTIN FILAMENT REGULATION USING A INTERACTIONS AMONG THE CARDIAC THIN FILAMENT MYOSIN Si TITRATION AT NANOMOLAR ACTIN PROTEINS DURING THE CONTRACTION CYCLE ((V. Korman & CONCENTRATIONS ((R. NMaytum. MI.A. Geeves.)) Miax-Planck Institute for I. Tobacman.)) Depts. Internal Medicine & Biochemistrv, Univ. Iowa College of Molecular Physiology. 44137 Dortmund. Germany MIedicine, Iowa City, IA 522 12 It has recently been show'n (Kurzawa & Geeves. J.Mus.Res.Cell NMotil. 17 1) that F'ull understandiing of cardiac regulation depends uipon knowledge of how the regu- phalloidin stabilised pyrene labelled actin can be used to measure myosin St affinity latory proteins interact wsith the actini filament the contractile cycle. We have for actin at sufficiently low actin concentrations to allow accurate determination of during the St affinty in a stop-flow assav. Using these principles a titration system has been chosen to use yeast actini as a inodel system, which is amenable to mutagenisis, to explore how the regulators proteins interact with actin. We find that tropomyosili devloped allowing St binding to be monitored in a continous titration at similar low anid troponin bind to veast actin with affinities similar to those for muscle actin. (50 nMI) pyrene-actin concentrations and under conditions where kinetic and mixing are to measure and make the ATPase rate effects negligable. Using this technique it has been possible accurately Additionally, troponin tropomyosin actomyosin highly cooperative St binding curves to actin in the presence of Tropomvosin and senisitive to Ca+2. The yeast actin mutant, 4AC actin et al. 1992 J. Biol. (Tm) (Cook the complete regulatorv complex (Tm.Tn). The binding curves can be fitted to a Chem. 267:9430), in which the N-terminus has two additional negative charges- similar to muscle model based on that of Geeves & Halsall (Biophys. J. 67 273) and has allowed actin-has unaltered interactions with troponin, tropomyosin and determination of both the 'on' / 'off' equilibrium (KT) of the filamant and the Ca+2. However iii the case of another mutant actin, K315A R316A (Wertman apparent cooperative unit size (n). The results curve fitting shows that the values et al. 1992 Genetics 132:337). myosin St ATPase experiments show that there is determined for KT and n for Tm alone (7-8 and 0.16 respectively') and for the Tm.Tn an 3 fold increase in the for the approximatels Ca+2 affinity regulated thin fila- complex (11-12 and 0.14) in the presence of calcium are in agreement with previous inents coniprised of the wild type actin as compared to ones made of this mutant measurements with other methods. However in the absence of calcium although the actin. Ilowever, there is no signiificant difference between the binding affinities of determined value of KT (0.01) is in general agreement, that for n is considerably the tropomyosin-troponin complex for actin in either the presence or absence of lower (7-8) and similar in size to the structural repeat unit. The difference in the Ca+2. Additionally, the binding of tropomyosin both with and without myosin St value of n in the presence and absence of calcium can be explained by the mechanism also show' no significant change in bindiiig affinities between the two types of actin. of Tn control. In the presence of calcium the Tn complex is only weakly associated These results stuggest that actin stubdomain 3 residues. 315 and 316, are important to the actin filament and hence the cooperative unit size is largelv controlled by the for thin filaiiient activation despite little effect of these residues on protein binding persistance length of the Tm.Tn complex. In the absence of calcium Tnl is strongly in the presenice of Ca+2. bound to the filament every 7 actins both pushing the A7Tm.Tn into the 'off' state and restricting the Tm.Tn mobility due to this pining effect.

Th-Pos7 Th-Pos8 STERIC BLOCKING AND COOPERATIVE ACTIVATION MECHA- THE CALCIUM SENSITIVITY OF TNC STRUCTURAL CHANGES IN NISMS IN RABBIT PSOAS MUSCLE FIBERS. ((Y. Zhao, P.M.G. Swamy, SKINNED SKELETAL FIBERS: EFFECTS OF SARCOMERE LENGTH and M. of and Cell AND CROSSBRIDGE INHIBITION. ((Donald A. Martyn, C.J. Freitag and A.I. K.A. Humphries Kawai)) Department Anatomy Biology, Gordon.)) Departments of Bioengineering and Physiology and Biophvsics. University of College of Medicine, The University of Iowa, Iowa City, IA 52242 Washington, Seattle, WA 98195. The elementary steps of the cross-bridge cycle in which Troponin C was partially Dichroism was measured to monitor alterations in troponin C (TnC) structure during Ca extracted were sinusoidal in rabbit psoas muscle fibers. activation, with and without force inhibition, in single rabbit psoas fibers which were recon- investigated by analysis stituted with TnC labeled (Cys 98) with tetramethylrhodamine. Solutions The effects of on isometric tension and the rate constants of fluorescently 5' phosphate (P) expo- contained (in mrM) 133 Na + K, 15.0 EGTA, 5.0 MgATP, 1.0 Mg, 15 creatine phosphate, nential processes were studied at 200 mM ionic strength, 5 mM MgATP, pCa 15-50 MOPS. at pH 7.0, IS 170 mM and 10 C. Force was inhibited with 30 mM butanedione 4.20, pH 7.0, and at 200C. monoxime (BDM). During force generation sarcomere length (SL) was monitored by laser Step 4 Step 5 The results were analyzed diffraction, maintained by periodic length transients (0.2/sec) and held isometric at either 2.56±0.01 or 2.15±0.01 mm by adjustment of overall fiber length. Dichroism was maximal k4 with the cross-bridge scheme relaxation 9.2) and was minimal at pCa 4.0. Ca dependent changes in force and AMDP AM*DP + P during (pCa * AM*D at left, where A=actin, dichroism were fit with the Hill relation to determine the pCass and slope (n). pCasu and n k4 K5 M=myosin, and D=MgADP. of force at SL 2.56 mm were 5.8±0.02 and 1.92±0.1 (5 fibers); corresponding values obtained When TnC was extracted so that the average remaining tension was 11%, k4(rate at 2.15 mm were 5.61±0.02 and 1.99±0.15 (5 fibers). Thus, increasing SL increased the Ca constant of force generation) decreased to 0.4x, k/c (reversal of force generation) sensitivity of force. Surprisingly, altered SL had no effect on the Ca sensitivity of dichroism increased to to and the values of n for dichrosim were significantly lower: at 2.56 and 2.15 mm SL pCaso 2x, K4 (=k4lk/4) decreased 0.1 7x, and K5 (P association constant) and n were 5.40 ± 0.08, 0.74 ± 0.06 and 5.44 ± 0.03. 0.89 ± 0.04. respectively. At both did not change. The activation factor cc, which represents the fraction of cross- SL's inhibition of force with 30 mM BDM decreased the pCaso and n of force. but had no bridges participating in the cycling, decreased from I to 0.14 with TnC extraction. effect on the changes in dichroism. The data indicate that in skeletal fibers (1) inhibition The decrease of cc is consistent with the steric and indicates of crossbridges with BDM has little effect on TnC structure at any level of activation. as blocking mechanism, measured by changes in dichroism, and (2) shifts in the Ca sensitivitv of force with SL are that some of the cross-bridges disappear from the active cycling pool. The fact not caused by corresponding changes in Ca binding to TnC.Supported by NIH 51277 and that the equilibrium constant decreased is consistent with the cooperative activa- NIH 52558. tion mechanism (graded activation hypothesis) among thin filament regulatory units that consist of troponin complex, tropomyosin and seven actin molecules.

Th-Pos9 Th-PoslO MODULATION OF THIN FILAMENT ACTIVATION USING AN CALCIUM DEPENDENCE OF ISOMETRIC FORCE MEASURED IN SINGLE THIN INACTIVATED CARDIAC TROPONIN C IN SKINNED SKELETAL FILAMENTS USING GLASS MICRONEEDLES. ((D.M. Lee, L.S. Tobacman*, and MUSCLE FIBERS. ((C.A. Morris, L.S. Tobacman*, and E. Homsher.)) Dept. E. Homsher)) Department of Physiology, Medical School, UCLA, Los Angeles, CA, 90095. Physi'ology, UIC,A, I,os Angeles, CA 90095 and *Dept. Int. MIed.. U. Iowa, Iowa City, IA *Department of Internal Medicine,University of Iowa,Iowa City, IA, 52242. 52242. We have used glass microneedles to measure pN forces exerted by heavy meromyosin (lvIv) heads pulling on single, regulated thin filaments in an in vitro motility assay. Thin filaments Calcium is kiioivn to rnoduilate tie level of fiber actisation but the precise mechanisni wepe reconstituted usingskeletal F-actin with cardiac troponin and tropom osin and exhibited of Ca +sensitive force generation, in vitro (bottom left). At saturating Cal (pCa5), the maximum regulation reniainis tindeterriminei. Ca2+ may control the nurnber of crossbridges that force exerted on regulated thin filaments was 12.36 ± 0.4 pN (mean ± sem) per En of thin bind to the thin filament and proceed through the crossbridge cycle, or it may regulate the filament in contact witlzthe motility surface. Thin filament sliding speed was anso measured over actomyosin kinetics ishile the number of cycling crossbridges remains relatively constant at the same range of ICa 1, thus allowing a direct comparison to be made between the calcium varyiiig levels of actiuation, or both. In the present study we have used an inactive mutant regulation of sliding speedand isometnc force. The shift of the force-pCa curve to the right of cardiac the speed-pCa curve (bottom right) by approximately one pCa unit is comparable to results troporiiti C (C'BMIITnC), that is unable to bind Ca'+, to determine the role of thin obtained using glycerinated muscle fibers. We have extended these studies to measure the tilament activation in muscle contraction. Extraction of endogenous skeletal TnC (sTnC) maximal force produced by thin filaments reconstituted with cardiac proteins containing a and reconstittution with cardiac TnC (cTnC).or mixtures of cTnC andiCBMIITnC, were tropomn T (TnT) mutant associated with familial hypertrophic cardiomyopathy (FHC), i.e., performed in skiniiied fibers. have, altered the level of activation Ile79Asn missense mutation. Previous studies have demonstrated that the presence of the rnuscle WVe therefore, Ile79Asn TnT mutant in the troponin complex increases thin filament sliding speed by around bv varying only the number of crossbridges that can interact with the thin filament at 50% in the in vitro motility assay (Lin et al, JCI. 97:1-8, 1996). Using the microneedle saturating [Ca2+]. Reconstitutiori swith cTnC alters the Ca2+ sensitivity from that seen technique the maximumforce exerted on thin filaments containing the lle79Asn TnT (at pCa 5) swith endogenoims sTnC. In the presence of cTnC the pK is redtuced from 6.8 to 6.6 and was 9.38± 0.35 pN/pm (mean ± sem). It is possible that the reduced isometric force (p<0.01vs. the to Reconstitution with ratios of reduces the control) and increased filament sliding speed are associated with altered actomyosin kinetics, 1Hill coeff. frorn 2.4 1.8. CBMIIiTnC:cTnC i.e., an increased rate of cross isometric a linear fashion bridge dissociation. The presence of this TnT mutation, in vivo, tensiomi in (25% CBMIITnC- 67%; 50% CBMIITnC- 42%; 75% may compromise pressure development and the efficiency of cardiac output, whh could be,tpe CBMIIITnC- 24Il). The rate of tetision redevelopment (kt,) has been shoswn to increase stimulus for the hypertrophy associated with this disease. (NI grantsAR30988 HL38834L). as is in skinned skeletal muscle In the of nonlinearly [Ca2+] increaseud fibers. presence 12 1.010 c[nC, our observed k,. is coiistamit at 3s-' above pCa 6.6(<0.6 P0), while below pCa 6.6 Speedi increases to a of at Addition of E t n=< c R ° 75 kt, iiiaxinirinui 7.5s-1 pCa 4.5. 50% CB3MIITnC reduces z 8 pK 7i1 the level of activation bs 58% but kt, falls by 14%c. Replacement with 75% CBMIITnC drops the force by 76% while kt, is redtuced by 35%. We are cturrently investigating the n effect of thin filarnent activation iiri unloaded shortening selocity. (NIH grants AR30988 o-1.066 (EH)/HL38834 (I,T)). IZ at6 pK-6.1995 9 8 7 6 5 9 8 7 6 5 pCapC A348 SMOOTHSOT MUSCLEUCERGLTRREGULATORY PROTEINSRTISIH Th-Posll Th-Posl2 THE EFFECTS OF MUTANT TROPONIN T GENE TRANSFER ON EFFECTS OF ISOFLURANE ON SUBMAXIMUM Ca2*-ACTIVATED FORCE ADULT CARDIAC MYOCYTE STRUCTURE AND FUNCTION OF THE CONTRACTILE PROTEINS IN SAPONIN-SKINNED ARTERIAL ((E.MN. Rust. F.P. Albayya. J.Ml. Mietzger.)) Department of Physiology. University STRIPS FROM DEVELOPING RABBITS. ((J.Y. Su and H. Liu)) Dept of of Mlichigan. Ann Arbor. MII. 48109-0622 Anesthesiology, Box 356540, Univ. of Washington, Seattle, WA 98195 Adenovirus vectors are a highly effective means to introduce foreign genes into Isoflurane has been shown to enhance the submaximum Ca2+-activated force in adult cardiac myoc tes in primary culture. Previous experiments have shown that both the stabilitv of the cardiac myocyte contractile assembly and cardiac contractile saponin-skinned femoral arterial strips from young adult rabbits, possibly by function are unaltered by adenovirus infection per se. This approach is now being activating PKC and CaM kinase 11 (Anesthesiology 85:A569, 1996). The used to study cardiac contractile protein structure-function relationships after gene purpose of this study was to examine (1) whether isoflurane had a similar effect transfer of the myofilament protein troponin T (TnT). Recombinant adenovirus vec- on other conduit arteries such as the aorta, and (2) the responsiveness of the tors have been constructed wvhich contain the embryonic isoform of rat cardiac TnT aorta from developing rabbits to isoflurane. Aortic strips (1-1.5 mm width), (eTnT) under control of the CNIV promoter (AdCNMIVeTnT). In addition. tsvo mu- isolated from rabbits of <5 days, 3-4 weeks, and young adults, were mounted tations associated with the disease familial hypertrophic cardiomyopathy. Ile79Asn on photodiode force transducers. The strips were then immersed in relaxing (179.N) and Arg92Gln (R92Q). are being studied. Both TnT mutations are located solution containing 300 pg/ml or 500 pg/mI saponin to permealize the in the region of calcium-insensitive binding to 0-tropomyosin and have similar clin- sarcolemma ('skinned"). The skinned strips were activated by buffered ical phenotypes. Western blot analvsis of samples collected from control cardiac solutions containing various free [Ca2+] concentrations, and effects of isoflurane myocytes and myocytes infected with either AdCMIVeTnT or AdCNIX'eTnTJazQ on submaximum (ED30) Ca2+-activated force were examined. We found that demonstrated expression of the eTnT or eTnTR92Q protein only in the infected my- 0.1 mM Ca2+-activated maximum force was about 7-fold smaller in skinned ocytes. Immunofluorescence labeling of myocytes infected wvith AdCNIN'eTnT using a monoclonal TnT antibodv showed staining that was indistinguishable from con- strips from rabbits <5 days old than from older developing rabbits. Isoflurane at trol myocytes. indicating the expressed protein is incorporated into the myofilament. 3% decreased the pCa 5.6 (ED30)-activated force in skinned strips. In skinned Studies are underwvay to examine the expression and incorporation of the *vild-type strips from rabbits of different developmental stages, there was no significant and mutant eTnT proteins into the contractile assembly. and to assess the effects difference in the pCa-tension relationship (expressed as % of the maximum on contractile structure and function. force) and the isoflurane effect. (Supported by NIH-GM48243)

Th-Posl3 Th-Posl4 INTRACELLULAR RADIAL AND LONGITUDINAL DIFFUSION STRUCTURE OF DOMAIN 4B OF CALDESMON DETERMINED COEFFICIENTS FOR PARVALBUMIN IN FROG SKELETAL MUSCLE. BY 3D NMR ((Y.Gao, O.Copeland, P.A.J.Huber. M.EL-Mezgueldi, S.B.Marston ((D. Maughan & R. Godt)) Department of Molecular Physiology & and B.A.Levine.)) University of Birmingham, Birmingham , B15 2TT. UK and Biophysics, U. Vermont, Burlington VT 05405, and Department of Imperial College Sch. Med. at NHLI, Dovehouse St. London SW3 6LY, UK Physiology & Endocrinology, Med. College Georgia, Augusta GA 30912. The C-terminal 150-170 residues of caldesmon (domain 4) interact with actin- tropomyosin and Ca2+.calmodulin (CaCM). This part of the molecule lacks ex- Parvalbumin content of single fibers from semitendinosus muscles of tended secondary structural features and seems to be rather flexible, but with several Rana temporaria was measured using SDS-PAGE. Total concentration (of well defined folds in the peptide chain leading to a compact structure (Mornet et al., isoforms IVa & b) was 7.4±2.4 g/l fiber, or 0.9±0.3 mmol/l myoplasmic Biochemistry 34:1893-1901; 1995). The domain 4b peptide 658-756 (658C) and a water. Diffusion coefficients were estimated from the time course of synthetic peptide Leu-693-Trp722 (LW30) were studied by NMR spectroscopy. Full parvalbumin diffusion 1) out of skinned fibers into droplets of relaxing spectral assignment was achieved for both peptides. Ordered structure was appar- ent in the regions flanking Trp692 and Trp722. Altering the solution pH from 5.8 to solution, and 2) between two juxtaposed skinned fibers under oil. Both 7.2 we have observed that the induced increase in amide proton exchange is greater isoforms diffused at the same rate. Method 1 produced artifactually for the residues adjacent to Trp-692. The linker peptide between these hydropho- elevated values due to dilution of myoplasm by relaxing solution. Method bic residues contains a proline rich sequence Ser702-Pro-Ala-Pro-Lys-Pro7O7.which 2 provided better estimates of intracellular diffusion coefficients because by interresidue Nuclear Overhauser Effects (NOEs) is shown to adopt an extended no artificial solutions were used. The value for radial diffusion, 1.9 (±0.3) x (trans X-pro) conformation. This region may thus be able to act as both spacer 10-7 cm2 s-1, was not significantly different than that for longitudinal and swivel joint allowing for the interaction of the two Trp regions with actin and diffusion, 1.7 (±0.4) x 10-7 cm2 S-1 (4°C). The -3-fold reduction in mobility of CaCM. Further NOE study of LW30 indicates that the region 713-716 (GDVS) 1 forms a turn which may be important in forming the interface between caldesmon parvalbumin in undisturbed myoplasm compared to water (6.5 x 0- cm2 and actin domain 1. The two sequences around the Trp residues 692 and 722 have s-5 at 40C, determined using pre-loaded 3% agarose cylinders) is due to been shown to be distinctly involved in contacts with actin and CaCMf. Mutations the high intracellular concentration of both non-diffusible and diffusible in the linker (S702D) weaken interaction at both Trps equally. A nonsense mutation constituents (affecting both tortuosity and viscosity). The mobilities of (691EWLTKT696 to PGHYNN) weakens the contacts of this site with actin and smaller particles are less affected by these constituents. NIH support. totally abolishes contact of this site with CaCM. We conclude that the C-terminus of caldesmon forms multi-point contacts with either actin or CaCM.

Th-Posl5 Th-Pos16 A NOVEL Ca2+-BINDING PROTEIN ASSOCIATED WITH CALDESMON A PREDICTOR OF ACTIVITY IN CALMODULIN STRUCTURAL IN SMOOTH MUSCLE THIN FILAMENTS CONGENERS: THE PROPENSITY FOR HELIX-COIL STRUCTURE ((S.Marston,G.Notarianni,J.Collins,J.Leszyk.)) Imperial College Sch. Med. at IN THE LINKER RESIDUES 77-81 ((F. Pitici. C.F. Wong and H. NHLI,Dovehouse St,London,SW3 6LY,UK Department of Biological Chemistry,University Weinstein.)) Dept. Physiology and Biophysics. Mount Sinai School of Medicine. of Maryland, Baltimore, MD, 21201, USA, and Worcester Foundation, Shrewsbury, MA 01545, USA. New York. NY 10029 We have extracted and purified the Ca2+-binding protein (CaBP) from smooth muscle thin Biochemical and structural evidence confirms the critical role of the linker region filaments by DEAE-cellulose chromatography in 6M urea and phenyl-Sepharose chromatog- (residues 77-81) that connects the two globular domains of calmodulin (CaMl). in raphy. At pCa5, 250C, 50mM KCI approximately stoichiometric CaBP reverses caldesmon conferring functional adaptability. However, the underlying structure-function re- inhibition and potentiates ATPase to up to 160% of the uninhibited level whereas calmod- lationship determinant of the CaM-like phenotype has not yet been identified. A ulin produces very little reversal of inhibition. In these conditions CaBP forms a complex recentlv developed helix-coil transition theory [Misra and WVong. Proteins 28:344, in Ca2+ with caldesmon-actin-tropomyosin, whilst Ca2+-calmodulin binding to caldesmon was used to characterize the of the linker of of re- dissociates it from actin, suggesting a different mode of action of the two proteins. Native gel 1997] propensity region CaMI and electrophoresis (in Tris/borate pH 8.5/20% glycerol) shows that CaBP binds to caldesmon lated mutant constructs to adopt helical or coil structures. Four sets of mutants that in Ca2+ with a greater affinity than calmodulin. Unlike calmodulin, CaBP also binds to have alterations in the linker region or in the adjacent helices have been considered caldesmon in the presence of EGTA. CaBP has a molecular weight of 18kD and comigrates [Putkev et al.. J. Biol. Chem. 263: 11242. 1988: Persechini et al., ibid. 264:8052. with calmodulin on SDS gels. Partial sequencing and Mass spectroscopy (MALDI-MS) 1989; VanBerkum et al., ibid. 265:3750, 1990; Gulati et al., ibid. 268:11685, 1993: shows the CaBP is indistingushable from calmodulin, raising the possibility that CaBP is Meador et. al.. Nat. Str. Biol. 2:943, 1995]. The calculated local flexibility of the a transcriptionally modified form of calmodulin. We have investigated phosphorylation of linker is in qualitative agreement with the experimentally observed ability of the calmodulin by casein kinasell since calmodulin is phosphorylated by casein kinase II at thr CaM-related constructs to activate various CaM-dependent enzymes. Several point 79 and ser 81 in vivo in the liver (Quadroni et al, J.Biol.Chem. 1994:269:16116-16122). The maximum level of calmodulin phosphorylation using our smooth muscle casein kinasell mutations can be proposed on this basis to achieve a modulation of the CaM-like preparation was 10-15% In autoradiograms of native gels bovine brain calmodulin partly phenotype. phosphorylated with [32P]ATP bound to caldesmon in the presence of EGTA, a property characteristic of CaBP. Further characterisation of the phosphorylated form of calmodulin and expressed thr/ser to asp mutations at 79 and 81 will be reported. SMOOTH MUSCLE REGULATORY PROTEINS IIH A349 Th-Posl7 Th-Posl8 INTERACTIONS AND CONFORMATIONAL STUDIES OF PEVK IDENTIFICATION OF MOLECULAR CONTACTS BETWEEN SEGMENT OF HUMAN FETAL SKELETAL MUSCLE TITIN ((G. NEBULIN AND CALMODULIN ((C. L. Shih. K. D. Linse. and K. WN'ang.)) Gutierrez, A. van Heerden. and K. Wang.)) Dept. of Chemistry and Department of Chemistry and Biochemistry. University of Austin at Texas. Biochemistry, University of Texas, Austin, TX 78712. Austin. Tx 78712. The extension of elastic titin filaments. especially in the PEV'K region of the Nebulin appears to serve both as a length-regulating protein ruler and as a I-band segment in the sarcomere, is thought to be responsible for the generation of calcium/calmodulin-mediated regulatory protein on the thin filaments of the skeletal resting tension of skeletal and cardiac muscle. We have previously obtained a 2.3 muscle sarcomere. The nebulin polypeptide consists of nearly 200 tandem repeats kb cDNA clone by screening a human fetal muscle library with a mAb (RT11) that of a 35 residue module. These modules are believed to serve as the basic actin recognizes an I-band epitope. This sequence is enriched in proline. glutamine, binding domains along nebulin's length. The structural basis of nebulin's interac- valine and lysine and shows high degree of similarity with published PEVK tions with calmodulin remains uncertain. To identify molecular contacts between sequences of rabbit skeletal muscle. We have expressed and purified a 53 kD nebulin and calmodulin, we have studied the interaction of calmodulin with cloned fragment (TP1) of this proline-rich region in E. Coli as a soluble protein. Studies nebulin fragments, ND8 (two modules) and ND66 (four modules). from the single with solid phase and surface plasmon resonance assays indicate that TPl interacts repeat region near the C-terminus of nebulin. Fluorescence titration of conjugated with actin and cloned nebulin fragments. Interestingly, the nebulin/PEVK calmodulin with both clones revealed calmodulin binding sites in the micromolar interaction is abolished bv Ca/CaM or Ca/S100. These data suggest that PEVK range. Chemical crosslinking of ND8/ND66 and calmodulin by EDC produced pre- region of titin mav also be involved in interfilament association with thin filaments dominately complexes of 1:1 molar ratio. Sites of crosslinking are being determined which in turn mav modulate titin extensibility in a calcium sensitive manner. by protein sequencing. As a complementary approach, a set of peptides correspond- Conformational studies of TP1 by NMR and CD indicate that PEVK in aqueous ing to different regions of one nebulin module were synthesized and tested for their solution has open. flexible conformations. Urea and thermal titrations cause grad- interaction with calmodulin using capillarv electrophoresis techniques. WVe are able ual and reversible changes of its CD without any evidence of melting commonly to identify a nine amino acid region as an essential element of the nebulin modules observed for compact protein domains. We suggest that in aqueous solution PEVK that bind to calmodulin in a calcium dependent manner. We speculate that this assumes a limited number of open, yet relatively stable conformations in equilib- sequence region plays an important role in untethering actin from nebulin in the rium. The 'random coil" depiction of PEVK in the recent literature is inadequate presence of calcium/calmodulin. and misleading. Further evaluation of these specific conformations and factors which influence this equilibrium is likely to facilitate the understanding of the generation and modulation of titin elasticity.

Th-Posl9 Th-Pos20 SMALL-ANGLE SCATTERING STUDIES OF THE ROLE OF THE REGULATORY DOMAIN OF PKCo ON ITS Ca2+-DEPENDENCE OF CALMODULIN-MYOSIN LIGHT CHAIN ENZYMATIC PROPERTIES AND LOCALIZATION. ((N. Takizawa, KINASE INTERACTIONS ((J. K. Krueger, Nicholas A. Bishop. Donald K. D.J. Schmidt. T. Kambara. R. Ikebe. and MI.lkebe.)) Department of Physiology. Blumenthal*. Gang Zhi**. James T. Stull"5. Jill Trewhella.)) Los Alamos University of MIassachusetts Mledical Center. WN'orcester MIA 01655 National Laboratory. *University of Utah, and **UT Southwestern Medical Center Protein kinase C alpha (PKCCo) is a Ca2+ and diacylglycerol dependent isoform of We have completed a set of small-angle scattering experiments aimed at testing the PKC. Its N terminal domain consists of the autoinhibitory domain. diacylglycerol hbpothesis that myosin light chain kinase (MLCK) activation bv calcium-calmodulin binding site (2 zinc finger domains), and a Ca2+ binding site; and is thus responsible (CaNI) involves a 2Ca2+6CaM6MLCK intermediate structure [Bavley et al. (1996) for regulation of enzvmatic function. WVe produced various mutant PKCas lacking Protein Science 5. 1215; Peerson et al. (1997) Protein Science 6. 794]. We measured regions of the regulatory domain to investigate the function of each region on both scattering data from mixtures of CaM and MLCK enzyme as a function of Ca2+ enzymatic activity and subcellular localization. The following mutants were studied: concentration. The MLCK enzyme was an active truncation mutant of skeletal Vi-, lack of the VI region: AI-. lack of the autoinhibitorv region: ZF 1- lack of the muscle MLCK [Krueger et al., (1997) Biochemistry 36, 6017]. We used the forward first of two Zn2+ finger domains; ZF-, lack of both Zn2+ finger domains (entire Cl). scattering to evaluate binding of CaNI to MILCK and the scattering intensity as a The mutant kinases were expressed in sf9 cells and isolated. WNhile PMIA/Ca2+ function of angle (I(Q) versus Q) to determine to overall shapes of any complexes sensitivity of V- wvas similar to the wild tvpe. it was abolished for Al- and the that formed. We also completed a parallel set of experiments substituting for MLCK activity in Ca2+ was rather decreased for ZF 1- and ZF-. PMIA dependence was the two different peptide sequences: the peptide corresponding to the CaMN-binding diminished with deletion of the autoinhibitory sequence. Interestingly. phospholipid domain of MLCK (MLCK-I), and a truncated version of MLCK-I that is missing sensitivity was biphasic. i.e.. the sensitivity diminished in Al- and resumed with the conserved Trp residue that binds to the C-terminal domain of CaM. These data deletion of the Zn2+ finger domain(s). Translocation of the kinase mutants was reveal differences between CaM-peptide versus CaM-enzyme interactions providing studied by immunofluorescence (anti-catalytic domain) and 3D digital imaging. as insights into the protein determinants of MILCK binding. The data from the trun- wcell as cell fractionation. Wrild tvpe and Vl- translocated from cytosol to membrane cated MILCK-I peptide also provides insight into the roles of its N- and C-terminal upon PMIA stimulation but ZF 1- and ZF- showed impaired translocation; thus domains in binding and inducing the conformational collapse of CaM. indicating that deletion of just one Zn2+ finger domain is sufficient to interfere with PMIA dependent translocation. (Supported by NIH Grants HL47530, HL56218 and HL14523)

CARDIAC MUSCLE: STRUCTURE AND MECHANICS Th-Pos2l Th-Pos22 THE TIME COURSE OF SHORTENING UNDER LOAD IS REGULATED BY THE AGE-INDUCED DEPRESSION OF MYOCARDLAL CROSS-BRIDGE LEVEL OF THIN FILAMENT ACTIVATION IN SKINNED CARDIAC MYOCYTES. KINETICS ((D.P. Fitzsimons, J.R. Patel, M.S. Hollander and RL. Moss)) ((K.S. McDonald and R.L. Moss)) Dept of Physiology, Univ of Wisconsin, Madison, WI Department ofPhysiology, University ofWisconsin Medical SchooL Madison, 53706. WI 53706 Length traces are curvilinear during isotonic shortening of skinned cardiac myocytes during submaximal Ca2W-activations, i.e., velocity slows as shortening proceeds. In We tested the hypothesis that the kinetics ofcross-bridge interaction are this study, we tested the hypothesis that curvilinear shortening arises from an internal progressively depressed as a consequence ofthe age-induced alteration in as a load which varies function of the level of thin filament 0 ventricular myosin heavy chain (MHC) expression. We examined the rate of activation. Single skinned rat myocytes were attached E 5 pc 58 tension development (kc8) foilowing the photorelease ofcaged Ca2e and maximal between a force transducer and position motor by placing 10 unloaded shortening velocity (Vo) in sldnned left ventricular (LV) myocardium the ends ofthe myocyte in stainless steel troughs. m from 3-, 9- 21- and 33-month (mo) old male Fischer 344 x Brown Norway Fl Myocytes (n=7) were Ca2 -activated to yield -0.30 of 0 15 pCa5.NEN 1 hybrid rats. Foilowing flash photolysis, kc.force increased with force and was tension and were over 20 peak shortening velocities measured on the level ofactivation in four to a range of loads using a servo system to clamp force. 8 dependent all age groups. Relative that observed in the 3-mo at - at - Isotonic was curvilinear as shortened group, kc. 500/% Po and 900/. Po was significantly shortening myocytes Z 6 lower in the 21- and 33-mo rats during force clamps at low loads (pCa 5.8, see Fig.). 4 9-, (P<0.05). Furthermore, compared to the 3- Myocyte activations at pCa 5.8 in the presence ofNEM- - 2 mo old rats, Vo, which is an index of cross-bridge detachment, was significantly SI (a strongly-bound crossbridge analog) increased both L_ 0i _ reduced in the 33-mo rats, but not in the 9- and 21-mo old rats. MHC protein the level of thin filament activation and reduced the 100 150 200 250 analysis revealed an age-dependent upregulation of P-MHC and concomitant curvilinearity ofthe length trace during force clamps Time (msec) downregulation ofa-MHC expressions. In addition, we observed no age- (Fig.). These results suggest that the progressive slowing dependent alteration in the expression ofmyosin light chain isoforms and thin of loaded shortening during submaximal Ca2+-activations filament proteins. These data suggest that the progressive age-dependent arises in part from an internal load, most likely due to slowly detaching crossbridges, that varies with the level of thin filament activation. depression in cross-bridge kinetics is associated with the increased phenotypic expression of 3-MHC. Supported by NI HL54581 and AHA 97GB90 A350 CARDUC MUSCLE:ADSTRUCTURES T ANDD MMECHANICS Th-Pos23 Th-Pos24 CROSS-BRIDGE INTERACTION KINETICS ARE MODULATED BY SPERMINE REDUCES Ca2+SENSITIVITY IN RAT CARDIAC MYOCYTES. MYOSIN HEAVY CHAIN EXPRESSION IN SKINNED RAT ((S.P. Harris, J.R. Patel, and R.L. Moss)) University of Wisconsin, Dept. of MYOCARDIUM ((J.R Patel, D.P. Fitzsimons and R.L. Moss)) Dept. of Physiology, Madison, Wl 53706. Physiology, University ofWisconsin Medical School, Madison, WI 53706 Due to their interactions with inward rectifier K+ channels (Lopatin et al., Nature, 372, 1994), it has been proposed that the naturally-occurring We investigated the imnpact ofaltered rat ventricular myosin heavy chain (MHC) polyamines (spermine, spermidine, and putrescine) are modulators of cardiac expression on (1) both the submaximal and maximal rates oftension development membrane excitability. In addition, Ventura et al. (Am. J. Physiol., 267,1994) (ke.) and the half-time oftension relaxation from a given level ofsteady-state force showed that polyamines reduce contractility in isolated cardiac myocytes, an following flash photolysis ofphotolabile caged Ca+ compounds in skinned effect attributed to reductions in intracellular Ca2+. To investigate whether myocytes; (2) the maxima shrtening velocity (i.e., V.) in skinned myocytes; and (3) polyamines affect myofilament Ca2+ sensitivity, we simultaneously measured the time course of myocyte shortening and Ca2+ transients in living myocytes both in cell shortening and intracellular Ca2+ transients in rat ventricular myocytes that had been loaded with the Ca2+ indicator, fura-2. Myocytes were obtained via the absence and presence ofthe p-agonist isoproterenol. Complete remodeling of enzymatic digestion of rat ventricles, loaded with the acetoxymethyl ester of ventricular MHC expression by thyroid deficiency from - 80% sa-MHC/20%/o ,B- fura-2, and electncally stimulated (0.2 Hz) to contract. In the presence of 1 MHC to 100% P-MHC reduced the rates of submaximal and maximal tension mM extracellular Ca2 , spermine (150-500 pjM) reversibly reduced cell development by three-fold, prolonged the apparent half-time for relaxation from shortening in a dose-dependent manner. Intracellular Ca20 transients, as submaximal force by a factor of two, and reduced Vo by five-fold. Altering the assessed by fura-2 fluorescence ratios (340:380), were also reduced by spermine. However, analysis of the time course for spermine's effects on cell phenotypic expression ofMHC had no significant effect on either the extent of shortening and intracellular Ca2+ showed that decreases in cell shortening myocyte shortening or the diastolic and systolic fura-2 fluorescence ratios. However, preceeded reductions in fura-2 fluorescence. Furthermore, in the presence of the half-times of myocyte shortening and relengthening were increased and the 2 mM extracellular Ca2+, 250 piM spermine reduced cell shortening by 24 ± decline in fura-2 fluorescence ratio was significantly slower both in the absence and 4.0% (n=6), but steady state fura-2 fluorescence ratios were reduced by 6.2 in the presence of isoproterenol. Based on these results, we conclude that there is a ± 2%. These data suggest that the negative inotropic effects of spermine may MHC-specific modulation of the kinetics of interaction of actin and myosin. be mediated in part by reductions in myofilament Ca2+ sensitivity and that, in Supported by NIH HL54581 and AHA 97GB90 addition to effects on cardiac excitability, polyamines may be modulators of cardiac contractility. Supported by NIH HL47053.

Th-Pos25 Th-Pos26 CALCIUM DEPENDENCY OF FORCE REDEVELOPMENT IN RAT RIGOR ACTIVATION OF CONTRACTION IN CARDIAC CARDIAC MYOCYTES ((C. Tasche, E. Meyhofer, B. Brenner)) Medical MYOFIBRILS AT LOW PH ((K.W. Yancey, D. Zhang and D.R. Swartz.)) School Hannover, Dept. of Clinical Physiology, 30625 Hannover, Germany Anatomy Department IL Medical School Indianapolis. IN 46202 It is a goal of our study to gain further insight into the molecular mechanisms of Striated muscle contracts in the absence of Ca2+ at low [MgATP]. This process force generation and regulation in cardiac muscle and to compare these findings has been termed rigor activation and is well characterized in skeletal muscle. This to our results from skeletal muscle. To measure the mechanical properties of mechanism of activation has not been well studied in cardiac muscle, where it likely single isolated heart muscle cells we designed a setup with force transducer and has a role in the pathophysiology of ischemic heart disease. In the ischemic heart, piezoelectric translator sufficiently sensitive to record mechanical parameters there is a cessation of the heart beat followved by a slow rise in diastolic pressure. from single isolated heart muscle cells. This slow rise has been termed ischemic contracture and has been ascribed to ac- Since forces of cardiac myocytes are in the pN-range we had to develop a tivation either by a slow Ca2+-activated or rigor activated contraction. We tested suitable force transducer with a thin stecl foil (2 mmx2 mm; 25 im thick) the hypothesis that rigor activation results in an increase in contractile activity at serving as cantilever beam. We monitored the bending of the beam by reflecting a loy pH values associated with the ischemic heart. The ATPase activity of rat car- laser beam off the steel foil and recording its deflection with a photodiode diac myofibrils as a function of [MgATP] at pCa 9.0 was measured at several pH detector. Our force transducers have a resolution of about 50 nN, stiffness of levels (pH 5.4 - 7.4). At pH 7.0, the ATPase activity as a function of [MgATP] about 2x 1O2 m/N and resonance frequency of 3 kHz. With this system we was bell-shaped with a maximum at 8 - 10 uaM MgATP with further increases in measured the force redevelopment after rapid release-restretch maneuvers from MgATP resulting in lower activity. The same response was observed at lower pH's skinned single heart muscle cells from rats. The cells were mounted, using a one with greatest activity at about 10 jaM. Interestingly. lower pH did not result in a component polyurethane glue, between our force transducer and a piezo substantial decrease in myofibrillar ATPase activity even at pH 3.4. To determine translator. Our initial measurements show force-pCa-relations with pCa50 around if the increase in ATPase activity was associated with contractile activity, sarcom- 6 and a Hill coefficient of 1.6. In agreement with Wolff et al. (Circ. Res. 76 ere lengths were measured in myofibrils under the same conditions as the ATPase (1995): 154-160), but diffcrent from Hancock et al. (Biophys. J. 70 (1996): measurements. The extent of shortening was greatest at 8 ja.sI MgATP and less at 2819-2829), which both used trabeculae and not myocytes like in this study, we both lower and higher MgATP and this response was observed at pH levels of 5.4, also find a calcium dependency of the rate-constant of force redevelopment. For 6.0, 6.4 and 7.0. These studies suggest that the peak ATPase activity associated our cells we find a 4-fold increase in k,,d,0 between pCa 6.8 and 4.6. with low MgATP was coupled to sarcomere shortening. These results support the hypothesis that rigor activation of contraction and the subsequent shortening occur at low pH associated with the ischemic heart. Supported by AHA # 9630028N.

Th-Pos27 Th-Pos28 MATHEMATICAL MODELING OF ACTIVE EMBRYONIC Ca2+--deic)endesice of diastolic proljertics of cardiac sarcomeies ((13rilrio MYOCARDIUM MECHANICS. ((O. Solovyova. L. Katsnelson, T. Romanchenko, P. D.Stiyvers & llenk E.L.J. ter Keurs.)) Cardiovascular Rtesearch Group - Faculty of Tsyvian, S. Routkevich, V. Mvarkhasin, B.B. Keller'.)) Institute of Physiology, Lral Medicine - University of Calgary - 3330 Hospital Drive NW - Calgary, Alberta, T2N\lN - Branch, RAS, Ekaterinburg 620016, Russia and 'Universitv of Rochester, Rochester, NY Canada 14642 Objective: recent findings from rat cardiac trabeculae have revealed that mechanlicl Originallv wve published a mathematical model of the mechano-chemical coupling in properties of the sarcomeres vary during the diastolic period separating two stinislilte(i cardiomyocite (Izakov et al., Circ Res, 1991: Katsnelson & Mlarkhasin. JMICC, 1996). twitches: a significant increase in leiigth and in stifrioess of the sarcorrieres teas deteiitd Later on Ca kinetics in SR was additionally incorporated into that model. The model simultaneotisly with a continuous decline of cytosolic Ca2+-concenitratioti ([Cii2+ IX) li,lis adequately simulated basic phenomena in active mature myocardium mechanics. 250 nM. Variations of stiffness and of [Ca'+ ]i occurred with similar tirqetcourse suggest- Now we analvze. with the help of this model. the influence of some specific embryonic ing that mechanical properties of the sarcomeres were dependent on [Ca2* ]i in a range morphological features on the mechanical performance of active mvocardium. We consider where probability of attachment of cross-bridges (Xb) between actin and myosin was loss the following features: 1) embryonic myocardium has a more amorphous cytoskeleton vs. (J.Physiol. 1997, 502, 3, 661-677). The present sttidy was conducted in order to tst adult one (i.e. it has lower series stiffness); 2) there is a higher surface-to-volume ratio in this hypothesis. Method: we controlled the free Ca2+.concentration ([Ca2+ ]) ssrroondiiig both embryonic cardiomvocite and its intracellular organelles than in mature ones, 3) the sarcomeres in skinned cardiac trabeculae of rat. We measuLred sarcoinere stiffness svlieit immature SR occupies a smaller part in cellular volume vs. SR in adult. By changing the [Ca2+] wsas changed between 0 and 430 nM. Sarconoere Length (SL) and Force'(F) irce values of corresponding parameters (in comparison with the simulation of the adult measured by laser diffraction techniqtues (resolution: 4 nm) and a silicon straiii gaiigi (res myocardium) we successfully simulate important mechanical properties distinctive of olution: 0.63 MN) respectively. Sarcomere stiffness was calculated from 500 lIz siniioisldl embryonic mvocardium. Specificallv, the changes cause the following events in the model: perturbations of mnuscle length and spectral analysis of corresponding F and SI, sintisosdis 1) isometric tension becomes smallervwhereas the time of contraction and relaxation Resuilts: The stiffness was independent of [Ca2+] below 70 iiM. Ilowever, betwveen 711 intl increases: 2) load dependence of relaxation disappears; 3) mvocardium inactivation in 200 nM, stiffness declined with increase in [Ca2+J anid increased steeply with [Ca2+] ilsosl response to fast small deformations (3-5% of the length) practicaliv disappears as sell. All 200 nM. The data fitted to the sumr of two sigmoidal functions: one showing a sigrsoiildil the above distinctions betveen embrvonic and adult myocardia were observed in decrease of stiffisess witli a Kd = 160 ± 13 nM (slope (ni) = -2.6 ± 0.7) atid one eqlivisilvtll physiological experiments. Our numerical experiments wvith the above model predict that to the fiinctioii describing the [Ca2+] dependent forrniation of Xb (Kd = 3.4 ± 0.3 pSI. ill more significant deformations of the embryonic mvocardium fiber (20%'c and higher) would = 2.2 ± 0.2). Using this model, we could reprodtice accurately the results obtained pit(t i- result in sizable inactivation. We have confirmed this model prediction in our recent ously from intact muscle. Conclusion: below 250 nM, sarcomere stiffness is goveriied b) phvsiological experiments. a visco-elastic system which is independent of Xb formation and which becomes stiffer as The work is supported by the CRDF Award RNI-420 and RFBR Award 96-04-50042. [Ca2+] surrounding the myofilaments decreases. CARDIAC MUSCLE:STRUCTtURE MECHANICS ('APnIAe MU-('UANTCq A351A3L w1F* Th-Pos29 Th-PWsO

Involvement of titin in stiffness-[ Ca'+] relationship of cardiac A MEHTOD TO ESTIMATE MYOFIBRILLAR RESPONSIVENESS TO sarcomeres ((Bruno D.Stuyvers; Jian Ping Jin; Henk E.D.J. ter Keurs.)) Ca2+ IN ISOLATED RAT VENTRICULAR MYOCYTES. Cardiovascular Research Group Faculty of Medicine University of Calgary - ((K. Hongo'), Y. Kusakaril)2), M. Konishi2), S. Kurihara2) and S. 3330 Hospital Drive NW - Calgary, Alberta, T2N- 4N1 Canada Mochizukil))) Dept. Med. IV') and Dept. Physiol.2), The Jikei University, School of Medicine,Int. Tokyo, Japan. It has been shown that titin generates 90% of passive stiffness in cardiac muscle at sarcomere length (SL) < 2.1 gm. Studies of rat cardiac trabeculae have shown that We used a tetanic contraction of isolated ventricular myocytes to assess variations of Ca2+ concentration ([Ca2+]) below 250 nM affected sarcomere stiffness myofibrillar responsiveness to Ca2+. Enzymatically isolated rat (Sarc Stiff) following a sigmoidal relation. We have tested the hypothesis that ventricular myocytes were loaded with fura-2 AM (4 yt for 10 min); fura-2 fluorescence was monitored during excitation at 340nm and stiffness-[Ca2+] relationship originated in alteration of the architecture of titin in the of intracellular sarcomere. that of titin for actin was modulated by [Ca2+] in situ, 380nm and the 340/380 fluorescence ratio (a function Assuming affinity were simultaneously (23-250C). a of titin to and from thin filaments in response [Ca2+]) and cell length monitored possible attachment/detachment Following treatment with thapsigargin (0.2 pM), myocytes were to a in below 250 nM was tested in intact sarcomere. Skinned change [Ca2+] stimulated at 10 Hz for 10 sec to produce tetanic contraction and an muscles were exposed to a cloned fragment of rat cardiac titin (Ti I-II) showing instantaneous plot of fluorescence ratio versus cell length (F/L strong affinity for F-actin in vitro. Identical approach was conducted with Ti I-I, trajectory) was constructed. The F/L trajectory followed the same path another fragment of titin with much lower affinity for actin than Ti I-II. It was during cell shortening and re-lengthening, suggesting that dynamic anticipated that Ti I-11 would compete with native titin for same binding site(s) equilibrium between intracellular[Ca2+1 and cell length was achieved on actin and would, therefore, alter the response to a change in [Ca2+] of native during tetanus. Increasing extracellular[Ca2+1 from 1 to 8 mM did not titin-based system leading to a detectable modification of stiffness-[Ca2+] relation. change the F/L trajectory. To test whether myofibrillar responsiveness SStiff was determined from spectral analysis of force and SL sinusoids induced by to Ca2+ could be detected using the F/L trajectory, we used EMD57033 500 Hz oscillations of muscle length. Stiffness-[Ca2+] relation was studied in saponin (a Ca2+ sensitizer) and 3-isobutyl-1-methylxanthine (IBMX) (a PDE skinned trabeculae at [Ca2+] varying below 450 nM in presence of Albumin, Ti I-I inhibitor). EMD57033 (1 shiftedF/Lthe trajectory to the left and Ti Ti 111 (2 MM) induced a significant decrease of both Kd and slope of (sensitization), whereas IBMX (200 iM) shifted the F/L trajectory to the the sigmoidal1I11. relation; Ti and Albumin (2 MM) did not affect the right (desensitization), in agreement with previous results obtained stiffness-[Ca2+]relation. Ti I-II induced aI-I and decrease of Ca2+ using skinned preparations. We conclude thatF/Lthe trajectory could ] specific significant to in isolated stiffness-[Ca2+of SStiff. Such alteration resulted from a competition process be used to estimate myofibrillar responsivenessCa2+ dependence probably he inotropic agents. between cloned fragment and equivalent segment of natsve molecule of titin. myocytes and might useful for screening of

Th-PTs3l Th-Pos32 MEASUREMENTOFFORCEFROMCARDIACMYOCYTES USINGACMOS COOPERATIVE ACTIVATION IN CARDIAC MUSCLE: INFLUENCE OF MICRO-ELECTROMECHANICAL FORCE TRANSDUCER. Lin,((G. R.E. SARCOMERE LENGTH. (David P. Dobesh and Pieter P. de Tombe)). The University Palmer, Pister, and K.P. Roos))Depts. ofElectricalEngineering, Physiology of Illinois at Chicago, 60307. & TheKS..Cardiovascular Research Univ. of Calif. at Los Angeles, CA., 90095. Lab, It has been well established that an alteration in calcium responsiviness of the contractile apparatss underlies the Frank-Starling relation of the heart. However, there is The measurement of active force from isolated cardiac myocytes is difficult due the uncertainty whether the level of cooperativity is affected by changessarcomerein cell's size relative to conventional tension transducers. Bandwidths no greater was measured in skinned smnll length (SL). Accordingly, active force development (F) rat than a few 100 Hz can be achieved due to the large mass of attachment arms which cardiac trabeculae as a function of free [Ca2+] at five SL's(1.85-2.25 mM free restrict studies to slow orsteady-stateconditions. We havedevelopedasubmersible, [Mg2+]; 13 OC). Only muscle preparations with minimal force ron-down during the miniaturized force transducer capable of measuring active force from skinned entire protocol were included in the analysis. SL was measured and controlled by on- isolated rat cardiac myocytes at fidelities 10 KHz. Micro-electromechanical line computer video micrometry within 0.01 pm. Care was taken to ensure a sufficient 2 number of data points in the steep portion of the F-[Ca2+] relationship at every SL systems ("MEMS') technology permits the production of a piezoresistive force (figure). Average results obtained from four trabeculse are shown in the table; transducer in standard CMOS. A signal to noiseratio of over10:1 was achieved maximum F (Fmax) extrapolated to zero at SL=0.82 pm. Analysis by multiple linear with a Wheatstone bridge and an on-chip CMOS aniplifier. The sensingmechanism regression revealed that both maximum F and EC50 were strongly dependent on SL involves the deflection of a cantilever beam with straina gauge at the base. This (p-cOOl). However, the slope of this relationship (HUIl coefficient) was not dependent beamis partofathree-dime nsionaloxidem icrostructure with aluminuminterconnect on SL (p=0.6). We conclude that alterations in SL result in an increase in the calcium hinges that are fabricatedsimultaneously with the electronics. The entire system responsiviness of the cardiac sarcomere without includes off chipam plification and filtering. The systemresponse is about 1.5 V/pm affecting the level of cooperativity. in both air and aqueous environnents. Thermal micro-actuators have also been SL Fmax (%) Hill EC50 (pM) fabricated and integrated into these devices. Preliminary studies with myocytes 2.25 140.9±6.4 7.2+1.2 3.25±0.10 (D attached to a of s with silicone sealant have measured forces at full 2.15 131.3±5.3 6.6±0.6 3.52±0.10 L 0 pair micro-beam U- and intermediate levels withCaecontaining activating solutions. These tests confirm 2.05 123.8±6.0 7.1±0.4 3.76±0.11 that this novel miniaturized transducer system is fully capable of characterizing 1.95 114.2±5.6 6.8±0.7 4.09±0.11 °* cardiac myocyte mechanics at high fidelity. Supported by AHA-GLAA. 1.85 100.0±3.9 6.8±0.7 4.52±0.12 free [Ca2+

Th-Pos33 Th-Pos34 CONTRACTILE PARAMETERS OF TWITCHING CARDIAC MUSCLE AGE-RELATED DIFFERENCES IN VOLATILE ANESTHETIC EFFECTS ON PREPARATIONS IN A MUSCLE CULTURE SYSTEM ((P.M.L. Janssen,J. FORCE IN CARDIAC MUSCLE ((YS. Prakash, A. 0. Prestle, Lehnart, J.C. Lynker, G. Hasenfuss.)) Department of Cardiology and REGULATION vanTrappen, Angiology,S.E.University of Freiburg, Freiburg, Germany Isbir, C.B. Mantilla, MJ. Cody, P.R. Housmans and G.C. Sieck)) Depts. of Anesthesiology and Physiology, Mayo Clinic, Rochester, MN 55905. (Spon. E.M. In the intact heart, a wide array of triggers (e.g. pharmacological interventions. mechan- Gallant). ical stress, gene manipulation) induces alterations in gene expression that can impact on contractile function. In order to study these changes, contractile function needs tobe as- In the present study, we determined whether greater myocardial sensitivity of neonates to sessed from the moment the trigger occurs until a subsequent altered protein expression is halothane is attributable to greater anesthetic effect on force regulation. In triton-X achieved (typ. > 24 hours) Thus, long term culture of an intact stimulated muscle prepara- skinned rat cardiac muscle maximally activated at pCa 4.0, wemeasured1) the rate of tion would provide a powerful tool to assess the resulting contractile response. Accordingly, force and restretching, and 2) isometric we a in which a studied for periods redevelopment (k,,) followingrapid shortening developed system twitching muscle preparation can be stiffness at two-state we > 48 hours (including > 100 hours). Right ventricular trabeculae from rabbit hearts were maximal activation and in rigor. Based on Huxley's model, mounted in a sterile muscle chamber that allowed continuous on-line monitoring of contrac- derived the fraction of attached cross-bridges (ae) and apparent rate constants for cross- tile parameters. At a temperature of 37 C, calcium concentration of 1.75 mM, stimulated bridge attachment at 0.5 Hz, and sarcomere length approx. 2.1 pm, average initial developed force (Fdev) was Neonate A"Wt (fwp) and detachment 13.7 mN/mm2, comparable to other studies using a similar preparation under the specified Control Halotiane Control HElothane (gw) Halothane conditions. Over time, Fdev remained remarkably constant; after 48 hours the muscles 5.4± 0.3 4.0± 0.4* 8.2±0.2 7.1 ± 0.5* decreased ats, 1 and developed 98.1% of the initial Fdev on average (n=4), while both time to 50% relaxation 4.2 ±0.7 2.1 ± 0.6* 6.8 ± 0.3 4.7 ± 0.4* f,,p to a greater extent and time to peak tension showed no significant change. After at least 48 hours in culture, responses to an increase in calcium to 2.5 mM (i.e. increase in Fdev) and l-adrenergic N,,, 1.2 ±0.7 1.9 ±0.6 1.4± 0.3 2.4± 0.4* in neonates, (Table); stimulation with isoproterenol (i.e. increase in both Fdev and rate of relaxation) remained ah% 0.8±0.1 0.5±0.1* 0.9± 0.1 0.7±0.1* however, gw was similar to those observed in freshly dissected preparations. Thus, the described system pro- pCasp 5.7 ± 0.1 5.2± 0.1* 5.5 ±: 0.1 5.3±0.1* nminimally affected. In vides a novel tool with a wide array of possible applications (study of e.g. long term effects Values are means± SE.*indicates significant difference. other studies, we of pharmacological interventions, changes in contractile function due to alterations in gene- found that halothane the producing 50% and protein expression, pathophysiological processes). decreases pCaso (Ca+concentration maximum force) to a greater extent in neonates (Table). We conclude that greater myocardial sensitivity of neonates to volatile anesthetics reflects, at least in part, altered cross-bridge cycling and decreased myofibrillarCa2+sensitivity. Supported by the Mayo Foundation. A352 A352CADAMUCESTUTRANMEHNSCARDIAC MUSCLE: STRUCTURE AND MECHANICS Th-Pos35 Th-Pos36 THREE-DIMENSIONAL STRUCTURE OF THE A-BAND AND ELECTROPHORETIC SEPARATION AND QUANTITATION OF CARDIAC MYOSIN LOCALISATION OF MyBP-C IN CARDIAC MUSCLE ((P. K. Luther.)) HEAVY CHAIN ISOFORMS IN EIGHT MAMMALIAN SPECIES. ((Peter J. Reiser and Blackett Laboratorv. Irntperial C'ollege. london. U.K. William 0. Kline)) Oral Biology, Ohio State University, Columbus, OH 43210. Contractile activitv in sertebrate striated rnuiscle occurs in the A-band due to Myosin heavy chain (MHC) isoforms have a major role in determining contractile interactions between the mvosini and actin filamnents. which are spatially organised properties of muscle. Two MHC isoforms (MHC-a and MHC-,f) are expressed in in a hexagonal lattire. It is irnportant to stui(dy the high resolution 31) structure mammalian cardiac muscle. The molecular masses of these two isoforms are both of the for several reasons. In particular, we do not know the 3D arrange- approximately 223 kDa and differ from each other by as little as 0.2% (McNally et A-bandl al., J. Mol. Biol. 210:665-671, 1989). MHC-a and MHC-,6 have, therefore, been ment of myosin bin(ling protein C (NIyBP-C, C-protein), which in cardiac muscle difficult to separate electrophoretically, particularly arduous in some species. A gel is phosphocrlated. and occurs along 7 to 11 transverse bands of periodicity 43 nm electrophoretic protocol that results in the separation of MHC-a and MHC-8 has (approx). Knowledge of the 3D struictture of NyvBP-C may also help to shed light on been developed and successfully applied to eight mammalian species. The protocol its function. In the present studvy 3D reconstruiction of the A-band in rat heart pap- is based on a simple, non-gradient SDS polyacrylamide gel system. One gel format illary muscle in the qtuiescenit state has beeis carried out from electron inicrographs consistently separates MHC-a and MHC-,6 in mouse, rat, guinea pig, canine, pig, of longitudinal cryosections. The micrographs show excellent order, and more than baboon and human myocardium. Another format separates rabbit MHC-a and MHC- 10 orders of the 43 nni repeat are seen along the meridian. The reconstruction was ,f. The glycerol concentration of the separating gel was determined to be crucial for achieved tusing selected lattice views, of the type 10, 11 and 21, obtained by tilt- separating these two MHC isoforms. The amount of separation between MHC-a ing the sections alonig the myofibril axis. For the analysis, corresponding A-band and MHC-,@ in all of these species is sufficient for the reliable determination, by regions spanniing 160 nm from the edge of the bare-region were selected from the scanning densitometry, of the relative amounts of these two isoforms in samples micrographs. The iusiages were aseraged over the A-band lattice, but no averag- as small as 1pg, when coupled with silver staining. The linearity of silver staining ing was applied along the filament length. Sinice vertebrate myosin filaments have of MHC-a and MHC-,8 over a six-fold range of gel sample loads was tested for three 3-fold rotational svnsmetry. this symmetry was uised to extend Fourier space. The species. The mean correlation coefficients for the linear regression between sample 3D image reveals several interesting features. The myosin crossbridlges appear to load and densitometric area were 2 0.97 for both isoforms. The mean coefficient be mainly close to the thick filament backbone. A regular feature along the myosin of variation for the determination of the relative amounts of the two isoforms was filarnent, with a well-defined 3D structure and approximately 43 nm periodicity, is 9.9% over the same loads. A complete description of sample preparation and of It is likely gel composition, preparation and running conditions will be presented. Supported also seen. that this is NMyBP-C. Fturther details of its local structure and a Grant-in-Aid from the American Heart the results of immtjnolabelling bv MyBP-C antibodies will be presented. Supported by Association. bh the British Ileart Irsundation.

Th-Pos37 Th-Pos38 TRIADIN-1 IS PRIMARILY CONFINED TO THE JUNCTIONAL SARCOPLASMIC (SR) DIACYLGLYCEROL ENHANCES EXCITATION CONTRACTION COUPLING OF CANINE CARDIAC MYOFIBERS. (('C. Thompson, 'A.C-Y. Shen, 2Y. Kobayashi, AND MYOFILAMENT CALCIUM SENSITIVITY IN CARDIAC MYOCYTES. 2L.R. Jones and 'A.O. Jorgensen)) 'Dept. of Anatomy and Cell Biology, U. of Toronto, ((YeQing Pi, R. Sreekumar, XuPei Huang and Jeffery W. Walker)) Department of Toronto, M5S 1A8, Canada. 2Krannert Inst. of Cardiology, Dept. of Biochemistry and Physiology, University of Wisconsin, Madison, WI 53706 Molecular Biology, Indiana U. School of Medicine, Indianapolis, IN 46202. Our previous work demonstrated that controlled photolytic release of the Triadin is an SR protein which interacts with calsequestrin, the ryanodine receptor (RyR) and diacylglycerol analogue, diC8, dramatically increased twitch amplitude in junctin and is believed to play a role in excitation-contraction coupling (Zhang et al, J. Biol. electrically-paced adult cardiac myocytes, whereas perfusion of diC8 or phorbol Chem. 272:23389). Previous studies have suggested that three isoforms of triadin, having M,s esters decreased twitch amplitude (Pi et al., Circ. Res. 81: 92-100, 1997). We have of 35, 40 and 92 kDa, are expressed in rabbit cardiac myofibers (Guo et al. J. Biol. Chem. now investigated the effects of photoreleased diC8 on intracellular Ca>+ transients 271:458). Immunolocalization studies using an antibody that recognized all three isoforms of measured with fluo-3 or fura-2. Levels of diC8 that produced about a 4-fold increase triadin suggested that generic triadin colocalizes with the RyR in the junctional SR (jSR) and in twitch amplitude produced only a 2-fold increase in the Ca2> transient amplitude. corbular SR (cSR) of rabbit cardiac myofibers (Carl et al. J. Cell Biol. 129:673). To Both determine if the subcellular distribution of triadin-I differs form that of generic triadin, the changes developed with a similar time course (tl/2=2 min at 22°C), and both distribution of triadin-I and generic triadin was compared to either that of phospholemman were inhibited by >85% following treatment with the protein kinase C inhibitor (PLM, a marker of the sarcolemma (SL) and T-tubules), the RyR (a marker of jSR and cSR), chelerythrine (4 I,M). Amplitudes of caffeine-induced Ca,2 transients were unaltered or junctin (a marker of jSR), in canine cardiac myofibers by indirect double by diC8, indicating no change in SR Ca2> content. Intracellular pH measured with the immunofluorescence labeling. Our results suggest that like junctin, labeling for both triadin-I fluorescent indicator, BCECF, was also not altered by diCg. Moreover, inhibition of and generic triadin are primarily localized as intense foci along T-tubules and the SL of atrial myocyte Na/H exchange activity with EIPA was without effect on the myocyte and ventricular myofibers. However, in contrast to previous studies in rabbit, only occasional response to diC8. We conclude that activation of protein kinase C by diacylglycerol foci of weak intensity were detected for triadin-1 and generic triadin in the interior regions of can initiate a substantial positive inotropic response in cardiac myocytes by a canine atrial and ventricular myofibers where cSR is densely distributed. These results mechanism that involves enhancement of both excitation-contraction coupling and suggest that triadin-I and generic triadin, like junctin, are very densely distributed in jSR but myofilament Ca>2 sensitivity. The latter effect does not appear to be due to the well- are either absent or at best sparsely distributed in cSR of canine myofibers. Studies are in characterized effect of alkalinization on myofilament Ca>2 regulatory proteins. progress to determine whether the difference in the distribution of triadin in canine and rabbit (Supported by NIH and AHA-Wisconsin). cardiac myofibers is due to species differences. Supported by HSFO, (AOJ), and NIH (LRJ)

Th-Pos39 LOCALIZATION OF Gs AND ADENYLYL CYCLASE IN THE TRANSVERSE-TUBULE OF VENTRICULAR CARDIOMYOCYTES: IMPLICATIONS FOR cAMP-DEPENDENT SIGNALING ((Nlihavl A. Laflamme and Peter L. Becker.)) Dept. of Physiology. Emnory UnIiversity School of MIedicirne. Atlanta. CA

he transverse tuibtules are a structural adaptation of the cardiac sarcolemmal mem- brane that facilitates excitation-contraction (EC) coupling. A number of proteins directly involved in FC cotuplirig have been shown to reside in the transverse-tubular miiemmibramie or adjaretit struictuires. Using immtuiuofltiorescence microscopy, we have fouind that G,5 arid adeirnylyl cyclase, key elements in the 3-adrenergic signal trans- dtiction cascade. are abtiridantly and liomoge'ieously distributed throughout the transverse tubtilar netssork and the intercalated disk regiots of carrdiac myocytes. Cs, in particular. appears to be sastly itiore abtindant wsithin the tranisverse tubu- lar itiernbrane thain in the peripheral sarcoletiima. In dual-labeled nivocytes, the dlistribution of Cs5 was contrasted witis that of wheat germ agglutinin (W'GA) bind- ing sites, an indricator of cell suirface. XNhile the fltuorescent \\,GA probe exhibited intense stainiiig of the peripheral sarcolemtisa. the simultaneously acquiired anti- (.s pattern isicluderl quantitatively less periphemal labeling and relatively intense transverse ttubular labeling. Ihe location of these elemnents of the cAP11 -signaling cascade within a fes iriicrons of every irnotropic target suggests that control and action of this second nmessenger could be qtcite local. Ithus. in addition to their role in excitatioii-contraction coupling. transverse ttubtules appear to be the primary site for signaling by inotropic agents. %I'VARnTAVA D.ISK ArfiPATIWnPMVQeWanOAICDV5l%I%jn (1(!VnV- AAlil2(1z Th-Pos40 Th-Pos4l MECHANISMS OF MYOCYTE DYSFUNCTION IN HEART PROTECTIVE EFFECTS OF L-ARGININE IN SIMULATED FAILURE FOLLOWING MYOCARDIAL INFARCTION IN RATS ISCHEMIA AND REPERFUSION IN GUINEA PIG VENTRICULAR ((Even Holt', Theis TonnessenN'. Per K. Lunde', J. Andrew Wasserstroin2. Ole MYOCYTES ((A.W.H. Au, G.R. f'errier and S.E. Howlett.)) Dalhousie M. Sejersted', and Geir Christensen'.)) 'Institute for Experimental Medical University, Halifax, Nova Scotia, ('anada Research, University of Oslo, N-0407 Oslo, Norway and 2Department of Medicine (Cardiology), Northwesterii University Medical School, Chicago, IL, USA. NO is released in myocardial ischemiiia and reperfusioii and may modulate cel- lular respontses to ischemia and reperfusion in the lieart. WVe determined effects Duriiig congestive heart failure (CHF) following snyocardial infarction the car- of L-arginine (L-Arg), the precursor for nitric oxide (NO) production by NO syn- diornyocytes have reduced contractility. We hypothesize that this may be caused thase (NOS), on currents and contractions in ventricular myocytes during simnulated by reduced contribution of calcium from the sarcoplasmic reticulum (SR) to elicit ischemia and reperfusion. Currents were recorded with switch clamrlp and cell short- contraction. Myocvtes were isolated from the left ventricle six weeks after of ening was measured with a video edge detector. Recordings were made for 10 min in ligation normal Tyrode's solution. Then cells were exposed to a simulated ischemic the left coronary artery in W%7istar rats. End diastolic pressure and heart were Tyrode's weight solution for 20 acidosis. lactate ac- raised in to sham rats. Peak Ca2+ currents min (e.g. hypoxia. hyperkalemia, hypercapnia. signiificantly CHF-rats compared L-type cumulationi, no glucose) and reperfused with normal solution for 30 min. were not whereas with field stimulation calcium tran- Tyrode's significantly changed, peak Three experimental conditions were examined: no drug; 200 sients measured were reduced The 1) 2) uM1 L-Arg in by fura-2 by 19a% (P<0.01). reduced transient ischeniia; or 3) 200 uM L-Arg plus the NOS inhibitor, L-NAME in ischemia. was reflected in reduced fractional (3 uM) shortening by 31a% in CHF (P<0.01). Sitisulta- In the absence of drug, contractions were abolished in ischemia and the magnitude of neous nieasurements of fura-2 transients and mechaniical shortening did not reveal Ca current was reduced. Upon reperfusion, contractions recovered and temporarily atiy alteration in the Ca2+ mrryofilament sensitivity in CHF. Time to peak contrac- exceeded control values. In addition, ischemia-reperfusion iilduced the arrhyth- tion (TTP) aind time to 90a% relaxation were significantly prolonged ini CHF-cells mogenic transient inward current (Iti). I,-Arg attenuated Ca current reduction in (P<0.01). We also examinied the contribution of the SR-Ca2+ -ATPase to removal ischemia and potentiated the overshoot in contractions in early reperfusion. These of cytosolic Ca2+ during relaxation by superfusing cells with 1 yM thapsigargin effects were not antagonized by L-NAME. L-Arg also decreased the incidence of Iti which inhibits the Ca2+ -ATPase. Relaxation time was prolonged significantlv less from 90.5 to 53.6 percent (n=21-28 cells). However, when cells were exposed to I- in CHF-cells by this drug (P<0.01). This suggests that other mechanisms, prob- Arg plus L-NAME, the effect of L-Arg was abolished (incidence of Iti=100 percent; ably the Na+/Ca2+ exchanger contributes significantly to the relaxationi rate in n=7). 'These results suggest that effects of L-Arg on Ca current and contractions CHF. In conclusion, we found normal L-type Ca2+ current density, but significantly in ischemia may be mediated by a mechanism other than NO synthesis. However, depressed SR Ca2+ release and reuptake in CHF. NO may protect the myocardium in iscliemia and reperfusion by reduction in the incidence of Iti.

Th-Pos42 Th-Pos43 Na-Ca EXCHANGE MEDIATED CONTRACTIONS EXHIBIT POSITIVE STAIRCASE REDUCED PROTEIN KINASE A-DEPENDENT TROPONIN-I PHOSPHORYLA- IN LV MYOCYTES FROM FAILING HUMAN HEART. TION IN FAILING HUMAN HEARTS. ((Daniel R. Zakhary", Christine S. ((J.A. Mattielo, K.B. Margulies, V. Jeevanandam and S.R. Houser)) Department of Moravec" and Meredith Bond")) Department of Physiology and Biophysics, Case Physiology, Temple University School of Medicine, Philadelphia, PA 19140 Westem Reserve University; Department of Molecular Cardiology', Lemer Research Contractions caused by the direct contribution of Ca influx via reverse-mode Na-Ca Institute, and Center for Anesthesiology Research', Cleveland Clinic Foundation. exchange were examined in isolated LV myocytes from failing human hearts. During whole-cell voltage clamp experiments, cells were dialyzed with low resistance patch To examine the role of altered protein phosphorylation in human heart failure, we have pipettes (Ripet,e =2 MQ) filled with a high Na solution ([Na]ppe,, - 20 mM). determined the stoichiometry of cardiac troponin-I (cTn-I) phosphorylation in hearts Thapsigargin (100 nM) and nifedipine (1 uM) were used to eliminate SR function and from 7 patients with dilated cardiomyopathy (F) and from 6 nonfailing donor hearts Ca current, respectively. Contractions were elicited with 1 second voltage damp steps (N). Phosphorylation of cTn-I by protein kinase A (PKA) influences the affinity of the to +100 mV. Following several minutes without stimulation (holding potential - -50 troponin complex for Ca' and consequently acts to alter the sensitivity of force tonic contractions mediated direct contribution reverse mV) by of mode Na-Ca production to Ca'. In this study we provide the first measurements of stoichiometry of exchange exhibited positive staircase (example illustrated in left panel). The peak contraction magnitude increased with succeeding voltage steps (right panel). Large, cTn-I phosphorylation in the human heart. 'Back-phosphorylation' using PKA beat-dependent effects of Na-Ca exchanger mediated contractions suggest that catalytic subunit or cyclic-AMP showed a significant reduction in the number of sites intracellular Ca buffers may compete with contractile proteins for Ca influx mediated on cTn-I phosphorylated in situ in F, compared to N. The calculated stoichiometry of by the exchanger, and/or that the rate of the reverse-mode Na-Ca exchange reaction bound P. was 0.8 +/- 0.4 and 1.1 +/- 0.2 mol P/mol cTn-I in F and N, respectively may be highly sensitive to diastolic [Cali. (p<0.05). Maximal stoichiometry, obtained after treatment with alkaline phosphatase, 10 m was not different between the two populations; 1.8 +/- 0.4 in F and 1.7 +/- 0.2 mol P/ >b ~~~~-5OmV50m mol cTn-I in N. Westem blot analysis and ELISA demonstrated unchanged cTn-I protein levels, and in addition, total PKA activity in the membrane-enriched fraction 0 was unaltered in the failing hearts. However, type II PKA regulatory subunit (RII) a~~~~~~~~~ protein levels were decreased by 30% (p<0.05) in thisfraction in F compared to N, but not in total heart homogenates. These results indicate that PKA-dependent cTn-I _E_;t // phosphorylation is decreased the failing human heart under basal conditions, which 0 5 2 246 810121416 Time (seconds) Episode may be explained by an altered distribution of RII.

Th-Pos45 FKBP12-DEFICIENT EXHIBIT CONGENITAL DILATED CARDIOMYOPATHY ROLE OF G PROTEIN SIGNALING IN CARDIOMYOPATHY; PHYSIOLOGIC RECEPTOR FUNCTION. ((W. Shou, B. Aghdasi, D. MEASUREMENTS FROM TRANSGENIC MOUSE HEARTS ((A.J. Baker, C. Armstrong, Q. Guo, Bao, Charng, Mathews*, M. Schneider, S. Hamilton and M. Redfem, V.M. Figueredo, E.C. Keung, S.A. Camacho & B.R. Conklin)). Depts. Matzuk)) Departments Pathology, Molecular Physiology and Biophysics, and Medicine, Radiology, Cardiology, & Gladstone Institute, Univ. Calif San Francisco. CA 94143. Baylor College Medicine, Houston, (Spon. by Mandel) Human dilated cardiomyopathy (DCM) is associated with increased signaling by Gi- FKBP12 (FK506-Binding Protein, kDa) was originally identified as an immunophilin which coupled receptors. However, a causal role for Gi signaling in DCM has not been drug (FK506 rapamycin) induced immunosuppression. Later, it was found that interacts with intracellular calcium release shown. Our goal was to test the that chronically increased G; FKBP12 channels (CRC) including the skeletal hypothesis signaling and the inositol causes a form of DCM ryanodine receptor (RyRI) 1, 4, 5-triphosphate receptor (IP3R). Because with impaired myocardial contractile function. rapamycin depleted RyRI shows altered single channel behavior and The approach was to measure myocardial contractile function in a transgenic drugs strong on excitation-contraction (E-C) coupling in skeletal muscle, mouse that allows G, signaling to be controlled. The mouse expresses a Gi-coupled FKBP12 postulated to have an important role in skeletal muscle excitation-contraction receptor (GR) designed to be activated solely by the drug spiradoline. A tetracycline- (E-C) coupling. define the biological function of FKBP12, we have recently generated controlled expression system regulates GR expression. Spiradoline treatment results in FKBPI12-deficient mice using embryonic stem cell technology. The majority of FKBP12- acute slowing of heart rate and complete atrio-ventricular block, which are known birth to heart Mutants deficient mice duc failure. exhibit severe cardiac defects but effects of increased Gi signaling. Prolonged G, signaling via this receptor causes a normal phenotypically skelelal imiuscle. The FKBP12-deficient hearts have 4-chamber dilation, lethal form of congestive heart failure with severe Contraction of cardiac mssiesip excessively prominent trabeculations, deep iniertrabecular recesses, thinned left ventricular walls edema and histopathological features of DCM. To from control& mice ventricular transgenic septal defects, which histologically resembles the fetal-type of dilated determine the effects of Gi signaling on myocardial 1 cardiomnyopathy noncompaclion of leftventricular myocardium (NLVM), a human inherited function, isolated cardiac muscle strips from the right control transgenic Intriguingly, both skeletal muscle RyRI and cardiac muscle RyR2 from FKBP12- ventricle were studied during isometric contraction. alterations channel function. FKBP12 depleted RyRl and RyR2 Increased G, signaling was associated with a slowed \ FKBP12-deficient have increased mean open time, and the channels show frequent time course of contraction and relaxation (see figure). opening subconductance states. These studies indicate an important biological role of FKBP12 In 2 experiments, the twitch time to peak was E D function cardiac development, and suggest a model in which FKBP12 modulates increased 34 ± and channel function. The altered by 3%; the half relaxation time 'c (RyR2) RyR2 function may contribute was increased 40 ± Conclusion: to dysfunction FKBP12-deficient mice. Future studies of FKBP12-deficient by 1%. G, ° skeletal will causes cardiomyocytes myotubes provide further insight on the role of FKBP12 in signaling a form of DCM with impaired 05 02 04 excitation-contraction (E-C) coupling. contractionand relaxation. tanei 0. A354 CARDIAC PATHOPHYSIOLOGYPATHOPHYSIOLOGY Th-Pos46 Th-Pos47 FUNCTIONAL EFFECTS OF HYPERTROPHIC CARDIOMYOPATHY TROPONIN I DEGRADATION AND COVALENT COMPLEX MUTATIONS IN ALPHA-TROPOMYOSIN ON Ca2+ REGULATION OF FORMATION WITH MYOCARDIAL ISCHEMIA/REPERFUSION. THIN FILAMENTS ((C S Redwood. I F Purcell. G Esposito, H WVatkins. S B NMarston ((J.L. McDonough, D.K. Arrell, Y. Gawad*, J.E. Van Eyk.)) Department of and W Bing.)) University of Oxford, Oxford 0X3 9DS, UK and Imperial College Sch. Physiology, Queen's University, Kingston and 'Skye Pharmatech, Toronto, Canada Med. at NHLI. London. 5W3 6LY, UK Single amino acid substitutions Aspl75Asn and GluI80Glv in alpha-tropomvosin (TM), Degradation and/or loss of troponin (Tn) from the myofilament may be responsi- have been shown to cause familial hypertrophic cardiomyopathv. To investigate the effects ble for the reduction in maximum force observed in triton-skinned trabeculae fiber of these mutations on protein function, wild type (XVT) and FHC-mutant TMs were ex- bundles obtained from ischemia/reperfused rat heart. Tissue and effluent samples pressed in E.coli with an N-terminal ala-ser leader swhich mimics N-terminal acetylation and from Langendorff-perfused hearts were obtained following 15 or 60 min ischemia (I) gives normal head-to-tail interactions. The mutant tropomyosins migrated slightly faster with 45 min reperfusion (R). The major degradation product isolated by TnC affin- than WT on SDS gels. Binding of expressed %VT and the mutants to actin was similar to ity chromatography of 60I/45R tissue was identified by western blotting, amino acid natural In vitro motility assays were with recombinant incor- tropomyosin. performed TM sequencing, and electrospray mass spectrometry (ESMS) to be TnI residues 1-193. porated into skeletal muscle actin-phalloidin filaments moving over skeletal muscle heavy meromyosin. The motility parameters of actin-T.M filaments in the absence of troponin This 22 kDa product is present in myocardial tissue following both 15I/45R and (TN) were identical for WVT and the two mutants. In relaxing conditions (pCa9), troponin 60I/45R, and is the primary TnI product found in the reperfusion effluent following added to actin filaments reconstituted with either WVT or mutant TM resulted in normal 601/45R. Additional Tnl degradation products of 14 and 15 kDa were not observed switch-off, with a decrease in the fraction filaments moving from >80% to <20%. Hosvever in 15I/45R tissue, but were isolated from 60I/45R tissue by anti-TnI antibody affin- in activating conditions (pCa5), TN had only a minor effect upon actin-TM(WT) filament ity chromatography (81-7, monoclonal anti-TnI, epitope 139-148). Western blot velocity (increased bv 5±15±1% n=10), svhereas the velocities increased by 18±3%c(n=7) wvith analysis suggested that these products have the same C-terminal cleavage as the 22 actin-Aspl75Asn TMI filaments and bv 21±2%o(n=8) svith actin-Glul8OGly T'M filaments. kDa fragment, while having different degrees of N-terminal truncation. As well, TnT XVe then tested the effect of NENM-S-1 svhich switches TMI to the fullv on state. 8nM NENI- degradation products and a western were iso- S-1 had no activating effect on the velocity of actin filaments reconstituted wsith either NVT Tnl-TnT complex (identified by blot) or mutant TM in the presence of TN thus indicating that the thin filaments svere already lated. The Tnl-TnT complex migrated at approximately 52 kDa on 6M urea tricine fully switched on. This suggests that the difference observed in the activation between SDS-PAGE but ESMS determined a mass of 33 kDa. The complex did not dis- XVT and mutant TM reflects a difference in the on state conferred by the FHC mutations. sociate with 6M urea and 1mM DTT at 100°C, suggesting a covalent interaction. presumably via altered interaction with troponinT. The severity of ischemia dictates the extent of degradation and covalent complex formation of Tnl and TnT, which could contribute to the myofilament dysfunction associated with ischemia/reperfusion.

Th-POS48 Th-Pos49 SENSITIVITY OF THE NA+/K+ PUMP CURRENT TO CARDIAC EFFECTS OF CARDIAC MYOFIBRILLAR ATPase ACTIVITY ON GLYCOSIDES IN VENTRICULAR MYOCYTES FROM HUMAN STRUCTURAL INCORPORATION OF CARDIAC TROPONIN C INTO FAILING HEART. ((F. Verdonckt, J. Vanhaecke2, K. Mubagwa3, UT. CARDIAC MYOFIBRILS IN PIGS AFFLICTED WITH MALIGNANT Flameng3, T. Stankovicova4, K. Sipido4.)) iIRC, Univ. Leuven, Campus Kortrijk, HYPERTHERMIA. ((Y.M. LIOUt'2, C.M. WU2, AND M.J. WU3.)) Department of Zoology', National Chung Hsing University, Institute of Biochemistry2, National Chung Kortrijk, B-8500, Belgium; 2Dept. Cardiology, Univ. Leuven, Leuven, B-3000; Hsing University, Taichung, Taiwan, and Taiwan Livestock Research Institute3, Tainan, 3CEHA, Univ. Leuven, Leuven, B-3000; 4Dept. Experimental Cardiology, Univ. Taiwan. Leuven, Leuven, B-3000 It has been known that during cardiac contraction the actomyosin interaction may gen- Digitalis is part of the current treatment of chronic congestive heart failure. We erate a positive feedback on the thin filament. It will be of interest to study the effects of have investigated the relative contribution of Na+/K+ pump isoforms with high the actomyosin ATPase activity on the structural interactions among regulatory proteins (HA) and low (LA) affinity for cardiac glycosides (dihydro-ouabain, DHO; digoxin) in the thin filament. Recently, we have shown that cardiac myofibrils isolated from the to the of in heart MH-sensitive porcine hearts contain the greater myofibrillar ATPase activity as compared generation Na+/K+ pump current (I,) end-stage failure. Single with the normal controls (Y.M. Liou, et al. Biophys. J. 70:A171, 1996). Using this model myocytes were isolated from the explanted hearts of transplant recipients. Ip was of the MH-sensitive swine, we started to examine the effects of cardiac myofibrillar ATPase measured at -20 mV as the K+, (5.4 mM)-activated whole-cell current after inhi- activity on the structural incorporation of cTnC into the thin filament. CDTA, a strong bition of all passive K+,-sensitive currents. The pump was maximally activated by divalent cation-chelating reagent, was used to remove cTnC from cardiac myofibrils in nor- inserting 100 mM Na+i in the pipette solution. The capacity of the cells was 307 mal and MH-sensitive porcine hearts. Satisfactory extraction of cTnC (>95%) from the +/- 19 pF (mean +/- SENI; n = 40 from 4 hearts) and maximal lp density was 0.87 normal porcine cardiac myofibrils could be obtained with CDTA at pH 8.4. However, the pA/cm2 (n = 27). The DHO concentration-Ip inhibition curve showed a biphasic same CDTA treatment only removed 80% cTnC from the MH-sensitive cardiac myofibrils. which is indicative for the existence of at least two distinct and In addition, a complete re-incorporation of cTnC could be obtained in the normal control, shape (HA LA) while a to partial re-insertion ( 70%) in the MH-sensitive cardiac myofibrils. Thus, the cTnC of pump Ullits. Ip generated by HA pumps amounted 14% of total Ip. Apparent the MH-sensitive porcine cardiac myofibrils is more resistant to extraction with the CDTA KD (K'D) values of DHO for HA and LA pumps were 6 x 10-9 M and 2 x 10-6 M solutions. The resistance of the cTnC extraction might reflect the structural stability of respectively. K'D values of LA pumps in human and guinea-pig cells differed by a cTnC incorporated with other regulatory proteins in the thin filament. The stronger incor- factor of 10 which was caused by a slower dissociation rate in the human cells. K'D poration of cTnC into cardiac myofibrils could be due to the effects of the higher myofibrillar values of digoxin for HA and LA pumps were 10-9 M and 3 x 10-7 M, respectively. ATPase activity in the MH-sensitive porcine hearts. Conclusions: in our experimental conditions (high [Na+]i) digoxin, at therapeuti- cally active concentrations, was estimated to block 7-10% of total Ip, and inhibition was almost exclusively cauised by suppression of HA pumps.

Th-Pos5O Th-Pos5l T-TVPE CALCIUM CURRENT INDUCES CONTRACTION IN FAILED HUMAN VENTRICULAR CALCIUM CURRENT IN LEFT AND RIGHT VENTRICLES DURING NORMAL AND MYOCYTES. DEFECTIVE CHICK HEART DEVELOPMENT. Nichols and T.L. Medical ((John P. Gaughan, Valluvan Jeevanandam, Steven R. Houser)) ((C.A. Creazzo)) Temple University School of Medicine, 3420 Noeth Broad St., Philadelphia, PA 19140 College ofGeorgia, Augusta, GA 30912. (Sponsored by T.L. Creazzo) (Spon. by C. Philips) Low-threshold, voltage-dependent calcium current has not been reported in human ventricular myocytes. We isolated left Ablation of the cardiac neural crest during early embryonic chick development results in ventricular myocytes from failed human explants. A piece of left ventricular free wall (10 x 8 cm) was cannulated and failure of the outflow tract to undergo septation producing a lethal heart defect known as retroperfused through a surface vein with calcium-free Krebs solution for 30 min. followed by coliagenase-containing Krebs solution (I 80 u/ml) for approx. 40 min. Isolated cells were filtered, centrifuged and resuspended in fresh Krebs persistent truncus arteriosus (PTA). Myocytes from pooled left and right ventricles (LV & solution containing 200 pM Cai until use. The resulting rod-shaped myocytes had clear striations and did notdemonsteane RV) of embryonic day (ED) 11 hearts with PTA have reduced Ca2itransients and L-type Can2 spontaneous contractions. current. (Creazzo et.al. 1997; Aiba and Creazzo, 1992). Kirby et.al (1990) have observed that Voltage clamp experiments were carried out from holding potentials of -90 and -50 mV the outflow tract in PTA hearts generally overrides the RV possibly increasing the workload of in Na-and K'-free solutions in the presence that ventricle. This may lead to differences in Cas2 current between LV & RV. To test this of compound 7943 (Kanebo), a specific hypothesis, L-type Ca"2 current was measured in LV & RV of ED 11 and ED15 ventricular blocker of the Na/Ca exchanger with Bai from normal hearts and hearts with PTA. The were cultured for (see fig.) or Cai as charge carrier. From a myocytes myocytes ovemight holding potential of -90 mV, steps to +ve use in perforated patch voltage clamp experiments. No significant difference in the magnitude potentials elicited large slowly-inactivating ofthe current was seen between the LV & RV of normal hearts at EDI I at 0 mV (1.63±10.17; inward calcium currents. From -50 mV, 1.39+0.19 At the average current in the normal RV similar but smaller currents were evident. pA/pF respectively). ED15, 10pM nitrendipine abolished currents and (1.05±0.09pA/pF) was significantly smaller than in the normal LV (1.45±0.13pA/pF) at OmV contractions from -50 mV while from -90 (p=0.01). There was no significant reduction in the average current of either the normal LV or mV a rapidly activating and inactivating T- normal RV from EDI1 to ED15. There was a significant reduction in the average current of type current remained (mean 247.2 + 91.7 pA, n=5). 100 pM NiCI2 abolished the T- RV myocytes from hearts with PTA from ED 11 (1.24±0.12pA/pF) to ED15 (0.75.0.06) at type current. T-type current in the presence OmV (p<0.01). Current in LV myocytes from hearts with PTA was not significantly reduced of nitrendipine elicited smaller and slower from EDI1 to ED15 at 0 mV. The average current in both LV and RV myocytes from hearts contractions compared to those triggered by with PTA was mV than the current in LV L-type current. significantly smaller at 0 average the respective and These results show that T-type calcium current is present in failed human ventricular RV myocytes from normal hearts at EDI I and ED15. The voltage dependence of activation myocytes and may contribute to the initiation ofcontraction. and voltage dependence of inactivation were not significantly different among any of the groups. In agreement with our hypothesis, we saw a greater decrease in L-type Can" current in the RV of PTA hearts between EDII and ED15 compared to LV at EDII and ED15. Supported by NIH HL36059. VA0nTAV raiPATUnPUV.QlInInurn ir aiujuuunV-i "DDA Agg Th-Pos52 Th-Pos53 MULTI-PHOTON CONFOCAL MICROSCOPY OF THE TRANSVERSE TUBULAR SYSTEM OF THE POST-FED HYPERTROPHIED PYTHON HEART; A CHRONICALLY PACED DOG MYOCYTES. (M. Conklin, V. Centonze', M. Wolff', and R. POTENTIAL MODEL OF HUMAN HEART FAILURE. Coronado). Departments of Physiology, 2Cardiology, and 'Integrated Microscopy ((*KE Quinn, #H Birmingham, #N Zecevic, $S Secor, $JM Diamond, *BE Resource (IMR); University of Wisconsin, Madison, WI 53706. Ehrlich)) *Yale University, New Haven, CT, #University ofCT, Farmington, Dilated cardiomyopathy was produced in dogs by chronic rapid pacing at 220-250 CT, $UCLA, Los Angeles, CA. beats/min for -32 days. Enzymatically-dissociated myocytes from control, sham operated and fast-paced hearts were stained with the non-penetrating membrane dye Cardiac hypertrophy occurs in Burmese pythons consuming meals 25% of FM4-64 to monitor the transverse-tubular system. Z-series consisting of optical their body weight. At 3 days post-feeding the heart increases 30-50% in mass. sections spaced 0.5 gm apart were taken with a 40X 1.3 NA objective in a multi- The increase in cardiac mass returns close to the original size as the snake photon confocal microscope equipped with a solid state Nd:YLF laser (Microlase) delivering a femtosecond pulse at 1047nm. The inset shows the 3-D reconstruction returns to a fasted state. The hypertrophied hearts ofpost-fed snakes undergo of a z-series from a control (left) and a failing (right) myocyte. Both cells twitched changes which, upon morphometric analysis, appear to be similar to changes with external stimulation. Arrays of highly-stained dots in 2-D images and well- that are seen in the hypertrophied, failing hearts ofhuman patients. In both ordered arrays of pillars in 3-D human hearts in failure and in the post-fed snake there is a significant increase reconstructed images were observed in both the surface area and volume ofmitochondria per unit volume of in control and sham operated cells. myocytes. To determine whether these morphological changes correlated These pillars are -0.3 gim across, i- with stand -1.6 apart and their length functional changes, biochemical measurements were made in hearts from gm fasted controls and snakes 2, 4 and 6 after fed a meal. The results is -5 gm. The density and I days being dimensions suggest pillars represent showed a significant increase in cytochrome C activity, a marker enzyme for single tubules extending into the cell. mitochondrial oxidation rates, 6 days post-feeding. The similarity ofthe In failing cells, the t-system was far less regular and was completely absent in many morphological response to stress in human and snake heart suggest that cells. The latter could be due to tubules pinching-off from the plasmalemma into the python heart can be a useful model for the failing human heart. Additional cytosol thus rendered inaccessible to the dye. The ryanodine receptor itself does not components of myocyte morphology and function will be compared as the appear to be modified in failing cells since Ca2' sparks were unchanged. The absence snake heart after of a fully functional transverse tubular system may contribute to the reduced Ca2' hypertrophies feeding and in the regression of hypertrophy. transient seen in failing dog myocytes. Supported by AHA and NIH.

Th-PosS4 Th-Pos55 USE OF NONLINEAR DYNAMICS IN THE STUDY OF SICK SINUS SYNDROME ALTERATIONS OF CONTRACTILE FUNCTION AND ((Neeraj Misra and P. K. Rath)) Physics Department, Lucknow University, Lucknow EXCITATION-CONTRACTION COUPLING IN VENTRICULAR 226 007, India (Spon. by L. Amende) MYOCYTES ISOLATED FROM HYPERTROPHIED RABBIT Changing the nonlinear element in a relaxation oscillator may be associated with the emergence HEARTS. ((R.U. Naqvi. D. Tweedie. K.T. MlacLeod.)) N\HLI. Imperial College of complex periodic behaviour. Under physiologic conditions, the sino - atrial (SA) node, the School of MNedicine. Dovehouse Street. London SWV3 6LY. UK. normal cardiac pacemaker and the atrio - ventricular (AV) junction, a subsidiary pacemaker, can be modeled as a pair of relaxation oscillators. The faster intrinsic firing frequency of the SA Cardiac myocyte hvpertrophv was produced by constriction of the ascending aorta node appears to entrain the slower subsidiary pacemaker which results in 1:1 phase locking of in rabbits. Animals were kept for 6 weeks following which the heart weight:body these oscillators. Studies of model systems have indicated that perturbation of such a network of sveight ratio was 59% greater (p<0.0001) in the hypertrophv (H. n=8) group of ail- entrained non - linear oscillators may alter the interaction such that a new cooperative, dynamic imals compared with sham-operated controls (C. n=7). Left ventricular myocytes state emerges. Analogous alterations in the interaction of relaxation oscillators might occur in were isolated by enzxmatic dissociation and a portion loaded wvith ;3pM indo-1 and the cardiac electro physiologic system, possibly induced by pathology in SA, atrial and AV field-stimulated (0.5. 0.2 Hz: 2mM1 Ca. 22°C). Cell membrane capacitance was in- junctional pacemaker activity. One example of this kind of global alteration is the cardiac creased by 29% in H (p<0.05). Twitch time-course was prolonged in H compared conduction system is the so called "sick sinus syndrome" which may reflect a wide variety of with C animals. Time-to-peak contraction in C = 0.66+0.06s vs H = 1.14+0.13s. structural and metabolic disturbances. It is hypothesised that "pathologic" coupling of p<0.0001. The associated indo-1 Ca signal was also slowed: time-to-peak fluores- pacemaker relaxation oscillators in patients with sick sinus syndrome will be associated with cence in C = 0.35+0.02s vs H = 0.48+0.06s. p<0.001. Time-to-90%c relaxation periodic behaviour similar to that observed in a variety of physical systems. Using computer of the twitch was also slower = 0.74+0.1s vs H = simulation techniques, we have mimicked bidirectional SA - AV node interaction with (R90) (C 1.26+0.2s. p<0.001) as was the indo-1 Ca = 0.69+0.06s vs H = 1.1+0.1s. computer model of coupled non - linear oscillators. The circuit includes two varactor diodes - signal (C p<0.001). Action electronic components with the same type of non -linear V- I relationship found in potential duration was increased by 38% in H (p<0.05). Both Ca current and its physiological pacemakers. current-voltage relationship were unchanged. SR Ca content. assessed using caf- feine after a train of 20 voltage-clamp pulses (-80 to 0 m%'. 200ms. 0.3 Hz). scas also unchanged.

CARDIAC MUSCLE: ENERGY METABOLISM Th-PosS6 Th-Pos57 RAPID ACTIVATION OF CPP32 CYSTEINE PROTEASE AND NORMAL INHIBITION OF PROTEIN KINASE C AND PHOSPHATASES BLOCKS MITOCHONDRIAL FUNCTION DURING EARLY APOPTOSIS IN NEONATAL RAT K-OPIOID RECEPTOR MEDIATED CARDIOPROTECTION. ((W.G. Pyle CARDIAC MYOCYTES ((J.A. Wojtezak, L.E. Rubin, V.K. Sharma, J.A. Olshowka, and P.A. Hofmann)) Dept of Physiol, U of Tennessee, Memphis, TN 38163. S.S. Sheu)) University of Rochester, Rochester, NY 14642 We have shown that the The temporal relationship between morphological and biochemical events during previously Kc-opioid receptor agonist UB-50,488 (UB) apoptosis has not been well investigated. In this study we have performed a temporal improves post-ischemic contractile function in the rat heart, concomitant with a analysis ofthe events associated with staurosporine in neonatal reduction in both Ca'-dependent actomyosin ± Pre-ischemia * p<0.05 vs CON (STS)-induced apoptosis UB rat cardiac myocytes. We have found that after exposure to 2pM STS, cellular shrinkage, MgATPase activity and maximum 140 UPost-ischemia ttp

Th-Pos60 Th-Pos6l RUTHENIUM RED INHIBITS MITOCHONDRIAL CALCIUM TRANSIENTS IN RECIPROCAL SIGNALING BETWEEN CARDIAC MYOCYTES AND VASCULAR ADULT RABBIT CARDIAC MYOCYTES ENDOTHELLAL CELLS. ((S. Winegrad, D. Henrion**, JL. Sinuel#, L. Rppapo#)). ((Donna R. Trollinger, Wayne E.Casciot and John J. Lemasters)) Departments of Cell Biology Dept of Physiology, School of Medicine, University of Penna., Phila. Pa. & & Anatomy and tMedicine, University of North Carolina at USA. INSERM Chapel Hill, U127# & U141a, Hopilal Lariboisiere, Pars. INTRODUCTION: Using laser scanning contocal microscopy, we showed mitochondrial free Ca2 transients in adult rabbit cardiac myocytes after electrical field stimulation (FEBS Coronary vascular endothelia cells (EC) release separa substnces tht can increase ordecrease Letters 382, 31-36; BBRC 236, 738-742). The goal of the present work was to determine the contrctility or produce a rigor-like state in cardiac myocytes (CM). The rate at which these mechanism of mitochondrial Ca2+ uptake after stimulation. METHODS: Adult rabbit cardiac substances are secreted is sensitive to tissue oxygen tension. The oxygen sensitivity of the myocytes were loaded with the acetoxymethyl (AM) ester of the Ca2+ sensitive dye, Rhod-2, for secretion of the cardloactive substances requires the presence ofnon-endothelial cells. Using a I h at 4°C and then incubated 3-6 h at 37'C. Cold of Rhod-2 facilitated cascade systen in which the venous effluent from an isolated perfused heart is assayed on the loading mitochondrial corhaclty of a mabecula isolated frn another heart we found that oxygen sensitivity remained accumulation, whereas subsequent warm incubation allowed leakage of cytosolic Rhod-2, and conactility ofthe trabecula changed even after EC in the perused heart had been disnrptedL leaving behind only mitochondrial fluorophore. To monitor cytosolic Ca2+ transients, myocytes The EC of the Irabecula had to be intact however. From these data, we proposed that there is an were loaded with Rhod-2 AM or Fluo-3 AM for h at 37'C. Br-A23187 (20 iM), a Ca2+ oxygen sensor in CM, which release a substance or substances that cause EC to release ionophore, was used to verify localization of fluorophores in myocytes. Red (Rhod-2) and green cardioregulatory agents. To test this hypothesis we isolated adult rat CM and incubated them in (Fluo-3) fluorescence was imnaged by laser scanning confocal microscopy. RESULTS: Cold a solution equilibrated with 5, 10, or 20 % 02. The CM were removed from the medium after loading of Rhod-2 with subsequent warm incubation for several hours resulted in nearly 4 hrs. The culture solution was equilibrated with 100% 02 and used to bathe isolated aortic exclusive mitochondrial loading. After stimulation, Rhod-2 fluorescence increased in rings. Medium incubated with 10% 02 caused a rise and then a fali in tension of the ring. mitochondria and then rapidly decayed to baseline. Ruthenium red (RR, 10 pM), an inhibitor of Endothelin receptor A (ER) blockade inhibited the rse but not the fail in tension. Diarupion of mitochondrial Ca2+ uniport. blocked Rhod-2 fluorescent transients, but did not block EC in the aertic ring inhibited both changes in tension. These data suggest that CM produce contractions. Br-A23 187 confirmed localization of Rhod-2 to mitochondria. In other two substances that stimulate EC to secrete respectively endothelin and a vasodilator. When experiments, Fluo-3 or Rhod-2 was loaded at 37'C. Under this loading condition, mitochondria 20% 02 was used, only only a rise in tension was produced, and the rise was completely were poorly labeled with fluorophore. Field stimulation of warm loaded myocytes revealed 2 inhibited by ER blockade. CM incubated in 5% 02 did not produce any increase but did reduce strong cytosolic Ca transients that were not blocked by 10 pM RR. CONCLUSION: tension. These results show that tension in the of the CM can be Mitochondrial free Ca2+ transients occurred in isolated 02 vicinity stabilized by a during excitation-contraction coupling combination of 02 sensing by CM, signals from the CM to EC, and EC secretion of adult rabbit cardiac myocytes. Selective labeling of mitochondria with Rhod-2 eliminated the vasoactive substances. (Sup. from & possible artifact of bleed-through of fluorescence from cytosolic to mitochondrial pixels during by Fogarty Fellowship NIH, INSERM) confocal imaging. Ruthenium red inhibited initochondrial but not cytosolic Ca + transients, indicating that mitochondrial Ca2+ uptake occurred via the mitochondrial Ca2+ uniporter.

PHOSPHOLAMBAN Th-Pos62 Th-Pos63 COMPARISON OF THE STOICHIOMETRY OF PHOSPHOLAMBAN TO 3 DIMENSIONAL STRUCTURE OF THE PHOSPHOLAMBAN-CALCIUM SERCA2a IN CARDIAC AND SKELETAL MUSCLE PUMP COMPLEX BY CRYOELECTRON MICROSCOPY ((H.S. Youngt, L.R. Jones2, D.L. Stokest.)) IStructural Biology Program, Skirball Institute and Dept. of Cell ((Deborah A. Femngton, Patricia L. Moewe, Qing Yao and Diana J. Biology, New York University Medical Center, NY 10016 2Krannert Institute of Bigelow)) Department of Biochemistry, University of Kansas, Lawrence, KS Cardiology, Department of Medicine, Indiana Univeristy School of Medicine, IN 46202 In cardiac muscle, phospholamban (PLB) regulates the magnitude of calcium transients bv Phospholamban (PLB) and the SERCA2a isoform of the Ca-ATPase directly interacting with the CaATPase in sarcoplasmic reticulum (SR). In response to 3- are co-expressed in both cardiac and slow-twitch skeletal muscle. adrenergic stimulation. cAMP-dependent protein kinase phosphorvlates PLB thus relieving Quantitation of the in vivo stoichiometry of these two proteins is essential for its inhibitory effects on the calcium affinity of CaATPase. The resulting increase in calcium defining functionally relevant experimental models. Thus we have measured transport into the SR increases the rate of relaxation as well as the rate of subsequent con- the concentration of these proteins in SR preparations from rat cardiac tractions. Recent evidence suggests that PLB and CaATPase interact in both cytoplasmic muscle and from two fiber-type distinct skeletal muscles, by comparing their and transmembrane domains, the latter being responsible for inhibition of calcium trans- antibody immunoreaction on slot-blots with the reaction of standard (affinity- port. In addition, the monomeric form of PLB appears to be the active form, whereas the purified) SERCA2a and recombinant PLB expressed and purified from E pentameric state of PLB may represent a storage form for inactive (phosphorylated) PLB. coli. We find that the SR from soleus skeletal muscle, which is composed Co-reconstitution and co-crystallization studies in our laboratory have yielded tubular co- of crystals of recombinant PLB with skeletal muscle CaATPase. Cryoelectron microscopy of predominantly slow-twitch musde fibers, has a PLB to SERCA2a ratio of these tubular crystals has yielded a preliminary reconstruction at 14A resolution (average 1.1 to 1. Identical PLB:SERCA2a stoichiometries were found in SR from of 5 image pairs). This map is somewhat noisier than previous reconstructions of native SR, extensor digitorum longus (EDL) muscle in which the slow-twitch fiber is a probably due to the inherent instability of these reconstituted tubular co-crystals. Despite minor component. In contrast, in cardiac SR, we find that PLB is expressed this difficulty, our map offers the first glimpse of the structural basis for PLB regulation: in excess of SERCA2a with molar ratios ranging from 5-8 PLB for each there is evidence of a clockwise twist of the CaATPase transmembrane domain, and extra SERCA2a. This large molar excess of PLB to SERCA2a in the heart transmembrane density which mav be attributable to PLB. However, this interpretation compared to the equimolar expression of these two proteins in skeletal is preliminary and efforts are underway in our laboratory to improve the quality of the muscle suggest mechanistic differences in the regulation of the calcium reconstruction.[Supported by NIH grants HL48807 (to DLS). HL06308 (to LRJ), NIH post- pump when expressed in different tissues. doctoral fellowship GM18281 (to HSY)] PHOSPHOLAMBANPHOSPHOLAMBAN A357 Th-Pos64 Th-Pos65 THE USE OF SYPRO ORANGE AS AN ALTERNATIVE TO WESTERN ARTIFACTS ASSOCIATED WITH THE USE OF SDS-PAGE TO BLOTS FOR VISUALIZING PHOSPHOLAMBAN OLIGOMERS ON SDS- MEASURE THE SELF-ASSOCIATION OF PHOSPHOLAMBAN ((Qing PAGE. ((Swee Yong Goh, Q. Yao and Diana J. Bigelow)) Dept. of Yao, Swee Yong Goh, Colin L. Taylor, and Diana J. Bigelow)) Department Biochemistry, Univ. of Kansas, Lawrence, KS 66045. of Biochemistry, University of Kansas, Lawrence, KS 66045. SDS-PAGE is routinely used to detect the self-association behavior of Much effort is currently aimed towards identification of the oligomeric both wild type and mutant phospholamban (PLB) as a means of species of phospholamban (PLB) that may be functionally relevant in the examining the functional role of PLB's oligomeric structure. However, regulation of the cardiac Ca-ATPase in SR membranes. An important tool because of the low and unequal sensitivity of Coomassie blue to each in these studies is the use of SDS-PAGE to screen mutant and derivatized oligomeric species, Westem immunoblots are more often employed as PLB proteins for their self-association behavior in detergent micelles a means to visualize PLB oligomers especially when protein is in low which has recently been shown to reflect, under some conditions, the abundance. Disadvantages of this technique include that it requires association of PLB in lipid bilayers (Comea et al., 1997 Biochemistry 36, additional and lengthy (>8hrs) steps after the original electrophoretic 2960-7). However, this use of SDS-PAGE is subject to a number of separation and is subject to artifacts relating to differences in efficiency artifacts related to sample history, detergent concentration, and of electroblotting. Therefore, we have investigated the use of the temperature that may render misleading results. For example, we find fluorescent dye SYPRO Orange as an alternative method to visualize that recombinant PLB expressed in E. coli and purified in detergent does purified PLB oligomers. We find that SYPRO Orange has approximately not show the preferential association of pentameric and monomeric 10-fold greater sensitivity than either Coomassie blue or Western blots, species on SDS-PAGE that native PLB from heart does. However, requiring protein loads of only 0.5 micrograms. In addition, SYPRO chromatography of native PLB in detergent results in the loss of Orange reports self-association behavior in good agreement with preferential association of PLB. Subsequent reconstitution of either Westem blots for a number of mutant PLB species. We suggest the use native or recombinant PLB into lipids restores the native-like preference of SYPRO Orange as a rapid and sensitive means to screen purified for pentameric and monomeric species, suggesting that detergent PLB for their self-association behavior on SDS-PAGE. micelles prevent free exchange of PLB between oligomeric complexes.

Th-Pos66 Th-Pos67 THE CYTOSOLIC DOMAIN OF PHOSPHOLAMBAN REMAINS ASSOCIATED WITH SUBCELLULAR CHARACTERIZATION OF A FOUR-FOLD PHOSPHOLAMBAN THE Ca-ATPase FOLLOWING PHOSPHORYLATION BY PKA. ((S. Negash, H. Sun, Q. OVEREXPRESSION MOUSE MODEL. ((R. Dash*, V. J. Kadambi*, J. M. Harrer', R. G. Yao, S. Y. Goh, D. J. Bigelow and T. C. Squier.)) Dept. of Biochemistry, Univ. of KS, Johnson Jr.", and Evangelia G. Kranias*.)) *Univ. of Cincinnati, Cincinnati, OH; "Merck Lawrence, KS 66045. Research Laboratories, West Point PA. (Spon. by G. Chu) We have used fluorescence spectroscopy to investigate possible alterations in the association Phospholamban (PLB) isa 52 amino acid protein located in Mhe cardiac sarcoplasmic reticulum betweenthe cytosoicdomanof ospholnaban (PLB)andthe Ca-ATPasethat resultfrom the (SR) membrane. Dephosphorylated PLB inhibits the Ca'2 affinity of the SR calcium ATPase phosphorylationofPLBby PKA. Followingthe walentattachmentofdansyl-chloride to Lys3 protein (SERCA), whereas phosphorylation of PLB relieves that inhibition. SERCA represents inPLB, we have coreconstituted PLB withthepurified skeletal SR Ca-ATPase. The calcium- the primary means of calcium sequestration during diastole. Thus, phospholamban's regulation dependent ATPaae activity of the reconstituted Ca-ATPase is analogous to that obseeved in of SERCA is a major determinant of diasotoic and systoic function in cardiac musde. native cardiac SR membranes both before and after the phosphorylation of PLB when the Transgenic mouse line, expressing twice the PLB levels (2xPLB-OE) found in wild-type hearts, stoichiometry of PLB incorporation is similar to the native membranes, indicating the exhibit a decreased affinity of SERCA for Ca', with consequent decreases in contractile physiological relevance of our reconstituted preparation. Upon phosphorylation of PLB parametersand caldum transient hinetica. The 2xPLB-OE mice were mated to 'homozygosityf reconstituted with the Ca-ATPase we observe a decrease inthe average fluorescence lifetimc in an effort toachieve maximal inhibition of SERCA by increasing PLBtvls. The homozygous ofdansyl-chloride. Incontnrs is insensitivetophosphorylationwith PKA when PLB genotypes were veriied by PCR and Southern blotting of tail DNA. Western blot analysis isonly with lipids, and is analogoustothatobservedin the samplecoreconstituted with the Ca- revealed that the PLB tevels in the hormozygous overexprssion mouse hearts were 425 ± ATPase followingphosphorylation. These results indicate thatthere are stucuralchanges in 26.2% (4xPLB-OE) compared to wild-types (100%). Neitr SERCA nor calsequestrin tevels thevicinity ofLys3uponphoWhorylationofSer,6 thatcoespondto alterations inthestructur changed significantly in these hearts. Preliminary dataindicated that the ECsovalue of the SR features of PLB with respect to the Ca-ATPase. Complementary measurements of the calcium uptake for calcium was 0.59 ± 0.021rM (n=3) for the 4xPLB-OE mouse hearts as fluorescence anisotrpy ofdansyl-chloride permit the resolution ofpossible alterations in the compared to 0.27 ± 0.01irM in wild-types, while the V,, levels appeared to be unchanged interacionbetweenPLBand the Ca-ATPase. TherotationaldynanuicsofPLB are muchslower between the two groups. Interestingly, the cardiac tevels of the Ne'/Ca" exchanger protein in the presence of the Ca-ATPase than that observed for PLB in lipids only, consistent with were 51.3 ± 5.5% higher in the 4xPLB-OE mice than in the wild-types (100%). Pathologic previous measuwmts that indicate a direct stuctural interaction between PLB and the Ca- examination of these mice indicated no gross or histologic abnormalities. Simlarty, there was ATPase. However, the phosphorylation of PLB by PKA does not significantly alter the no difference in heart-to-body weight ratios and no apparent cardiac hypertrophy as determined rotational dynamics of PLB, indicating that PLB renains tightly associated with the Ca- by echocardiography of the hearts. These findings indicate that a four-fold increase in PLB ATPase subsequent to its phosphorylation and the corresponding activation of the transport expression, in vivo, is assoiated wih: a) a higher degree of SERCA inhibiton compared to activity ofthe Ca-ATPase. two-fold PLB overexpreasion; and b) upregulation of the Na'/Ca9 exchanger which may be an important compensatory response to the increased inhibiion of SERCA

Th-Pos68 Th-Pos69 EPR OF SPIN-LABELED PHOSPHOLAMBAN (PLB) AND MUTANTS: EVALUATION OF CYSTEINE RESIDUES IN THE STABILITY OF THE IMPLICATIONS FOR PROTEIN-PROTEIN INTERACTIONS INVOLVED TRANSMEMBRANE DOMAIN OF PHOSPHOLAMBAN PENTAMER. IN CARDIAC CALCIUM TRANSPORT REGULATION. ((Christine B. Karim, Henriette A. Remmer*, Christopher G. Marquardt, Gregg ((John D. Stamm, Christine B. Karim, Tara L. Kirby, Joseph M. Autry*, Larry R. B. Fields*and David D. Thomas)) Department of Biochemistry and *Biomedical Jones, and David D. Thomas)) Department of Biochemistry, University of Engineering Center, University of Minnesota Medical School, Minneapolis, MN Minnesota Medical School, Minneapolis, MN 55455, and Krannert Institute for 55455. Cardiology, Indiana University School of Medicine, Indianapolis, IN 46202. The specific residues responsible for stabilizing the pentameric form of We used EPR of spin-labeled recombinant PLB and mutants to investigate the phospholamban have been identified by mutational analysis. For interactions involved in both PLB example, mutation of any of the three Cys (36, 41 and 46; all found in the protein-protein oligomerization and regulation transmembrane of the cardiac SR Ca-ATPase. PLB migrates as a pentamer on SDS-PAGE, but domain) to Ser, Ala or Phe diminishes the stability of the neither the nature of the oligomer in the membrane nor the role of PLB pentamer, even in the presence of DTT, suggesting that the oligomerization in cardiac SR Ca-ATPase regulation is completely known. We transmembrane cysteine residues are important for pentamer formation have shown that EPR of MTSSL labeled PLB and mutants is sensitive to even they are not disulfide-bonded. The critical properties of the Cys oligomerization. We now use EPR to study PLB's oligomeric state in the residues for pentamer formation could be either steric packing or membrane before and after phosphorylation and in the presence of the Ca- hydrogen bond formation with other groups within the protein. In order ATPase. Several PLB mutants, each containing a single cysteine, have been to test this hypothesis, we have replaced the Cys residues with alpha- labeled with EPR probes in order to study the local environment of PLB as amino-n-butyric acid (Abu), which is essentially isosteric with Cys and affected by protein-protein interactions. EPR of these singly-labeled PLB mutants has similar polarity, but has no hydrogen-bonding capability. Fmoc solid has been used to investigate sites of interaction between PLB monomers and phase synthesis was used to prepare the transmembrane domain between PLB and the Ca-ATPase in the membrane. PLB(26-52) and [Abu32641]-PLB(26-52). The properties of the Abu containing peptide analogs have been assayed by mobility on SDS- PAGE. Replace of C36 and C41 with Abu maintained pentamer formation, suggesting that the thiol groups on these residues are not critical for pentamer stability. Studies on other Abu derivatives will be reported. A358 PHOSPHOLAMBAN Th-Pos70 Th-Pos7l PHOSPHOLAMBAN PHOSPHORYLATION IS INCREASED IN CARDIAC SPECIC TARGETTING OF PHOSPHATASE 1 (PP1) TO PHOSPHOLAMBAN (PLB) IN HYPERTROPHY: A POSSIBLE ROLE OF ENDOGENOUS CARDIAC CARDIAC TISSUE ((Berrebi-Bertrand I., Camelin J.C, Souchet M. and A. Bril.)) PHOSPHATASE IN THE MYOCARDIUM. SmithKline Beecham Laboratoires Pharmaceutiques, Saint Gregoire, France. S.Y. Boateng, A-M. Seymour, M.J. Dunn, M.H. Yacoub and K.R. Boheler Cardiothoracic Surgery, Imperial College at NHLI, London, SW3 6LY, UK. Recent evidence indicates that a novel class of proteins, known as targetting subunits (Tsub), mediate the binding of the PPI catalytic subunit (PPt) to a specific subcellular Phospholamban (PLB) regulates the activity of the sarcoplasmic reticulum (SR) location (Tloc). In skeletal muscle, a protein called G plays a dual role in targetting PPI CaATPase in cardiac muscle via its ability to be phosphorylated. Studying native PLB to both glycogen and the plasma membranes. In the heart, this protein called PPIG is the phosphorylation in tissue has proved difficult because of the presence of endogenous major phosphatase. PPIG is an heterodimer composed of a 37 kDa PPI complexed to a degenerative enzymes. Using mobility shifts on SDS PAGE gels we have much larger subunit Tsub which is responsible for the association of PP1 with SR. demonstrated that significant dephosphorylation of PLB occurs during tissue However there is no clear evidence for a role of PPIG in the dephosphorylation of PLB homogenisation in the absence of phosphatase inhibitors even at low temperatures. and the regulation of cardiac contractility. The aim of the present study was to Endogenous kinases do not appear to alter PLB phosphorylation. Tissue investigate how PPI is targetted to SR. Molecular cloning and expression of both human homogenisation in increasing doses of NaF (a phosphatase inhibitor) from ImM to PLB and Tsub -associated protein phosphatase were carried out. PLB and Tsub were 50mM resulted in increased PLB phosphorylation. When 10mM NaF (a concentration successfully expressed using an "in vitro" transcription/translation system based on which should inhibit the CaATPase activity) was used in the preparation of crude SR rabbit reticulocyte lysate. PLB appears to be produced in both its monomeric (5-6 Kd) homogenates, CaATPase activity (measured by oxalate stimulated calcium uptake) and its multimeric form ( >25 Kd) whereas Tsub was detected as a 160 Kd protein. was paradoxically stimulated almost 2 fold, p<0.01. Increased CaATPase activity in Coimmunoprecipitation study in the presence of monoclonal antibodies raised against NaF was associated with increased PLB phosphorylation. Phosphatase and kinase PLB revealed both sense proteins on autoradiography which demonstrate for the first inhibitors were used in tissue homogenisation to determine PLB phosphorylation in time the interaction between the Tsub and PLB with an approximate ratio of Tsub normal hearts and in cardiac hypertrophy induced by abdominal aortic constriction. In /PLB=25/1 taken together. This study demonstrates that cardiac PPI is targetted to PLB 50mM NaF which completely inhibits endogenous phosphatases, PLB from through a specific protein called Tsub. Therefore, intervention on this molecular hypertrophied hearts had a slower mobility compared with normal hearts. This association may provide lusitropic actions. Agents of such action may improve cardiac suggests that PLB was more highly phosphorylated in cardiac hypertrophy. Increased relaxation and potentially provide beneficial effects in disease such as congestive heart PLB phosphorylation following cardiac hypertrophy may enable the myocardium to failure. compensate functionally in the early stages of adaptation.

Th-Pos72 Th-Pos73 OVEREXPRESSION OF A PHOSPHOLAMBAN PHOSPHORYLATION MUTANT IN STRONG INHIBITION OF THE CARDIAC CALCIUM PUMP BY TRANSGENIC MICE. ((A.G. Brittsan, V.J. Kadambi, B.D. Hoit, and E.G. Kranias)) PENTAMERIC PHOSPHOLAMBAN. ((U. Kirchhefer, J.M. Autry, J. University of Cincinnati, Cincinnati, OH 45267. (Sponsored by E.G. Kranias) Neumann* and L.R. Jones)) Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN 46202. *Institut fur Phospholamban (PLB), in its dephosphorylated state, inhibits the cardiac sarcoplasmic reticulum (SR) Pharmakologie und Toxikologie, Westfalische Wilhelms-Universitat, Munster, Ca2-ATPase (SERCA) by lowering the affinity of this enzyme for Ca2>. When PLB is phosphorylated Germany. at Ser16 and Thr17 by P-adrenergic stimulation, this inhibition is removed and the pump's affinity for Ca2> is increased, thereby enhancing cardiac relaxation. To assess the functional significance of PLB Phospholamban (PLB) is an integral membrane protein of cardiac sarcoplasmic phosphorylation in vivo, PCR site-directed mutagenesis was used to mutate Sert6 and Thr'7 to Ala16 reticulum which regulates Ca2+-ATPase activity. It has been suggested that the and Ala'7 in PLB, and transgenic mice expressing the mutant PLB were generated. Quantitative cytoplasmic and intramembrane domains of PLB are important for direct immunoblotting indicated a 4.6 ± 0.3 fold increase in the cardiac PLB protein levels compared to wild- interaction with the Ca2 pump. To evaluate the role of the putative hinge domain of PLB (amino acids Gln26 to Leu3'), we performed scanning-alanine type (WT) controls. To determine whether the mutant form of PLB could functionally inhibit SERCA, PLB alanine transgenic mice were analyzed at the biochemical and physiological levels. In vitro phosphorylation mutagenesis in this region. All mutated molecules substituted with formed stable pentamers on SDS-PAGE. Mutant constructs were co-expressed studies, using either the catalytic subunit of cAMP-dependent protein kinase or Ca2-calmodulin- with SERCA2a in Sf21 cell membranes and analyzed for functional activity by dependent protein kinase, followed by Westem blot analysis, using PLB phosphorylation specffic Ca" uptake assay. Wild-type (WT) PLB reduced the apparent calcium affinity of antibodies, indicated that the there was no phosphorylation of the overexpressed mutant PLB form. the calcium pump by 2-fold, whereas N27A-PLB decreased the calcium affinity SR Ca2-uptake assays in cardiac homogenates revealed that the ECso value for Ca2> was 0.62 + by 5-fold. Q26A-, Q29A-, and N30A-PLB also produced stronger inhibition of 0.01 iM (n=4) in the PLB-mutant hearts compared to 0.29 + 0.01 g1M (n=3) in WT hearts, suggesting the calcium pump than did WT-PLB. Changing residues Asn27 or Gln" to that the mutant form of PLB was capable of inhibiting SERCA. Two-dimensional targeted M-mode phenylalanine, however, produced mutant subunits (N27F-PLB, Q29F-PLB) echocardiography showed depressed velocity of circumferential fiber shortening (Vcf,) in PLB mutant that were no more effective than WT-PLB. Our results demonstrate that some mice, compared to WT littermates (PLB-MU: 3.87 ± 0.11 circ-1; WT: 5.67 ± 0.11 circ-1 ). In addition, pentameric mutants of PLB inhibit the calcium pump as strongly as the most administration of the 5-agonist dobutamine i.p. (12.5 mg/kg) resulted in a 62% increase in Vcf, in PLB potent monomeric mutants of PLB (e.g., L37A-PLB). If the PLB monomer is mutant mice, while this parameter increased by 110% in WT mice. These findings suggest that responsible for binding to and inhibiting SERCA2a in the membrane, then overexpression of the phosphorylation mutant form of PLB in transgenic mice results in a depression monomers from PLB mutated in the putative hinge region (i.e., N27A-PLB) of the affinity of SERCA for Ca2+ and contractile function in vivo. must bind to SERCA2a with great avidity. Experiments are currently in progress to determine if the monomeric or pentameric form of N27A-PLB is primarily responsible for SERCA2a inhibition.

SR PROTEINS Th-Pos74 Th-Pos75 FUNCTIONAL PROPERTIES OF TRIADIN EXPRESSED IN Characterization of the C. elegans calsequestrin, a calcium binding EUKARYOTIC CELLS ((Qianhui Zhang. A.H. Caswell. and N.R. Brandt.)) protein expressed in body-wall muscle ((Gve Won Park. Young Soo Oh. Kyu Department of Pharmacology. University of Miami School of Mledicine. P.O. Box Yeong Chweo, Do Han Kim, NVoo Jin Park, and Joohong Ahnn.)) Department 016189. Miami. FL 33101 of Life Science, Kwangju Institute ofScience and Technology Kwangju, 506-712, Korea Triaditi has been transiently expressed in Cos and CHO cells and stably trans- fected in ('HO cells. Triadin in reducing gels is detected as a prominent upper Calsequestrin(CS) is a high capacity and moderate affinity calcium binding pro- band of Mr 94K and a less intense one of 91 K. The 91K band predominates after tein. which was identified and characterized from vertebrate sarcoplasmic reticulum. treatnment ssith glycosidase. The protein is present largely in the monomeric form In order to study functions of CS genetically, we attempted to identify homologues ssith a loxs concentration of disulfide linked dimers and higher polymers. A mutant of calsequestrin in genetic model organisms. A putative homologue of calsequestrin hasing only one cysteine is present onl1 as monomer and dimer. This low concen- gene (csq-1) was found in the nematode C. elegans. The csq-1 encodes a 417 amino tration of polymers contrasts wsith the largely polymeric form of triadin in skeletal acid-long protein showing the most similarity (50% similarity, 30% identity) to the muscle. Expressed triadin is detected by immunofluorescence using a C terminal rabbit skeletal calsequestrin. The C. elegans CS expressed in E. coli demonstrated mAb in cells fixed wsith formaldehyde and either saponin or Triton X-100. Equal calcium binding activity. Antibodies raised against C. elegans CS recognized a single signals sere observed wcith either detergent. Since saponin does not dissolve the protein band of 64-kDa from C. elegans extract. Immunostaining revealed that the membranes of the ER. this indicates that the epitope is cvtoplasmic. Transient CS protein is expressed in body-wall muscle. which corresdonds to skeletal muscle co-expressed triadin and dihvdropyridine receptor (DHPr) have been visualized by of vertebrate. Striated pattern of staining suggested that CS may be localized along immunofluorescence microscopy and also by construction of a fusion protein of the with myofibrils. Northern analysis and in situ hybridization data showed the csq-1 DHPr containing an HN terminal green fluorescent protein. In each case the two was expressed during bodv-wall muscle development in embryos and the expres- proteins are observed by confocal microscopy to colocalize in the cell. However. the sion was maintained through the adult stage in muscle cells. Genome sequencing DHPr is distributed largely wvithin the cell rather than being confined to the surface project and Southern analysis have suggested that this gene might be a single copy even sshen both a and b subunits are co-expressed. In CHO cells expressing both gene of calsequestrin in C. elegans. Chromosomal deficiency mutants which delete calcium release channel and triadin treatment with 5 mMI caffeine causes a regular calsequestrin gene region suggest that calsequestrin is not essential for initiation train of increases and decreases in cytoplasmic calcium indicative of calcium release of muscle formation. This is the first report of calsequestrin gene being found in and reuptake.(Supported by NIH and Am Ht Assoc) invertebrate muscle. SR PROTEINS A359 SR PROTEINS A359~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Th-Pos76 Th-Pos77 CARDIAC AND SKELETAL CALSEQUESTRINS SHOW DISTINCT ANNEXIN VI, A MAJOR PROTEIN OF SKELETAL MUSCLE EXPRESSION PATTERNS DURING EARLY MOUSE TRANSVERSE TUBULES, BINDS TO TRIADIN IN TERMINAL EMBRYOGENESIS ((K.W. Park, J.H. Goo, D.H. Kim and W.J. Park.)) CISTERNAE. ((N.R. Brandt and A.H. Caswell.)) Department of Pharmacology, Department of Life Science, Kwangju Institute of Science and Technology, University of Miami School of Medicine, P.O. Box 016189, Miami FL 33101 Kwangju 506-712, Korea. The major 72 KDa protein of the skeletal muscle triad junction has been isolated by Calsequestrin (CS) is a moderate-affinity and high-capacity calcium binding pro- DEAE and phenyl Sepharose chromatography and identified by peptide sequencing tein in the sarcoplasmic reticulum (SR). Previous studies have shown that CS is as annexin VI. A polyclonal antibody against purified rabbit annexin VI bound only associated with triadin, junctin and ryanodine receptor and probably involved in to the terminal cisternae/triad fraction of muscle and to the T tubule fraction when the regulation of calcium release from SR. In the present study, we have cloned the triads were disrupted in a French press and centrifuged on an isopycnic gradient. and sequenced mouse cardiac and skeletal CS cDNAs. The deduced amino acid Annexin can be extracted from the T tubules by EGTA. When [125I] annexin VI was sequences are highly homologous to those of other mammalian CSs (more than 90% overlaid onto blots from terminal cisternae it bound to monomeric triadin from a re- identity). As expected, the cardiac and skeletal CSs are expressed specifically and ducing gel and to polymeric triadin from a non-reducing gel matching the position of exclusively in adult hearts and skeletal muscles, respectively. In situ hybridization triadin as indicated by an antibody. Annexin VI overlays blotted triadin-GST fusion was performed to examine the expression pattern of the CSs in the developing mouse proteins and binds to triadin-GST-glutathione sepharose columns in physiological embryos. The cardiac CS transcripts were detected in the cardiac primordial cells saline. Strongest binding occurred to the fusion protein derived from the putative of 8 and 9 days old embryos whereas the skeletal CS transcripts were found in my- cytoplasmic domain of triadin which did not appear to be Ca2+ dependent. Since otomes of 10 days old embryos. This suggests that the cardiac and skeletal CSs are annexin VI is reported to bind to plasma membrane lipids and to subsarcolemma mutually exclusive in their expression and probably in their function as well even proteins, these findings further support topological models where internal domains at the early stages of development. Our data rule out the possibility that the two of triadin are in the muscle cytoplasm.(Supported by NIH) CSs may function redundantly in the developing organisms. We have also isolated a genomic clone containing at least three exons of the mouse cardiac calsequestrin gene which can eventually be used for producing cardiac CS transgenic mice.

Th-Pos78 TRIADIN-l IS PARTIALLY GLYCOSYLATED AND IS THE PREDOMINANT ISOFORM EXPRESSED IN CARDIAC TISSUE. ((Yvonne M. Kobayashi and Larry R. Jones)) Krannert Institute of Cardiology, Dept. of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202. Triadin is an integral membrane protein of the junctional sarcoplasmic reticulum (JSR) shown to form a quaternary complex with calsequestrin, junctin, and the ryanodine receptor and suggested to play a role in excitation- contraction coupling. Molecular cloning and immunological studies have shown triadin to have three isoforrms in rabbit heart of 35-, 40-, and 92-kDa. To further characterize cardiac triadin, the cDNA corresponding to canine cardiac triadin-l was cloned. A generic antibody that recognizes all three isoforms, and a specific antibody that recognizes the unique C-terminus of canine cardiac triadin-1, were raised in rabbits. Both of these antibodies detected two prominent triadin bands of 35-kDa and 40-kDa on immunoblots of canine cardiac SR vesicles. Endo H treatment of cardiac SR vesicles completely converted the 40-kDa triadin band into the 35-kDa triadin band. Moreover, overexpression of canine cardiac triadin-t in transgenic mouse hearts gave two prominent triadin bands of 35-kDa and 38-kDa, and endo H treatment converted the 38-kDa form to the 35-kDa form. The same two endo H-convertible bands were detected in cardiac SR vesicles isolated from rabbits, mice, rats, humans, and guinea pigs-using either the triadin-1, C-terninal specific antibody or using the generic triadin antibody. We conclude that cardiac triadin-l is the predominant isoform expressed in heart, which appears as a doublet on SDS-PAGE due to the incomplete glycosylation of the protein.

SR CA2+ ATPASE Th-Pos79 Th-Pos80 CLONING AND CHARACTERIZATION OF HUMAN SERCA3. FLUORESCENT LABELING OF RECOMBINANT SERCA2A ((J. Lytton, E. Poch, S. Leach, S. Lloyd, R. Winkfein, W. Yang)) EXPRESSED IN SF21 INSECT CELLS. ((R. L. Cornea. J. M. Autrv. D. D. Dept. Med. Biochem., University of Calgary, Calgary, AB, Canada, T2N 4N1. Thomas*, L. R. Jones.)) Indiana Ulniversity School of Medicine and Krannert Calcium-ATPase pumps of the sarco/endoplasmic reticulum (SERCA) are Institute of Cardiology. Indianapolis, IN 46202, and *Universitv of Minnesota encoded by three genes. The rat SERCA3 gene product has distinct properties Medical School. Department of Biochemistry. Minneapolis. MN 55455 and a unique pattern of expression when compared to SERCAl or 2. Altered SERCA3 expression has also been implicated in cellular calcium dys-regulation It has been previously shown that the cardiac sarcoplasmic reticulum Ca-pump associated with both hypertension and diabetes. We now report the isolation of (SERCA2a) can be covalently labeled with spectroscopic probes at a unique reactive cDNA and genomic clones encoding human SERCA3. Analysis of tissue RNA lysine in the ATP binding cleft. This has proven a sensitive tool in spectroscopic by Northern blotting indicates that human SERCA3 has a very similar pattern of studies of the structure and molecular dvnamics of the Ca-pump. W1'e have previ- expression to the rat enzyme. Evidence based on the sequence of our clones, and ously expressed recombinant SERCA2a in Sf21 insect cell membranes. in amounts on PCR products from Jurkat T-cell RNA, suggests that human SERCA3 similar to those found in cardiac SR vesicles. Here. we show that recombinant transcripts are alternatively spliced to generate protein products that differ at their SERCA2a is specifically labeled with fluorescein isothiocyanate (FITC) in Sf21 mi- carboxyl-termini. cDNA clones corresponding to the different alternatively crosomes. On SDS-PAGE, all spliced products were expressed in HEK cells. Analysis of the transfected cells detectable FITC fluorescence is found only in the by immunoblotting demonstrated that the monoclonal antibody PLJM 430 protein band corresponding to the Ca-pump, suggesting a high labeling specificitv. recognized the human SERCA3 clones (but not rat SERCA3) with very high similar to the cardiac SR controls. We found that the fluorescence intensity of specificity. Each human SERCA3 clone displayed the same low apparent affinity FITC-labeled recombinant Ca-pump depends on the concentration of ionized Ca for calcium as rat SERCA3, which was shifted rightward -3-fold compared to (measuring the E1/E2 conformational equilibrium) in a fashion similar to skeletal rat SERCAl. The subcellular localization of human SERCA3 was compared to and cardiac SR. Labeling of this recombinant P-type ion pump specifically with that of human SERCA2b, both in transfected HEK cells and in Jurkat cells, by FITC will allow direct structure-function studies without the need for solubiliza- inimunofluorescence using the antibodies PL/IM 430 and 11D8. Both antibodies tion. purification, and reconstitution, and opens the way for planned mutagenesis revealed an indistinguishable perinuclear and reticular expression pattern. studies on SERCA2a structure-function correlations. Experiments are in progress in Analysis of the human SERCA3 gene demonstrates a high conservation of exon which FITC and related phosphorescent probes are used to further characterize the boundaries in comparison to rabbit SERCAI and 2 genes. Studies to map the recombinant Ca-pump expressed by itself and in the presence of phospholamban, its transcriptional start site and to define the properties of the human SERCA3 native intramembrane regulator. promoter are currently underway. til PAOUUA anCD t-PALIrV A,X. IFLIATPArpAor,R. Th-Pos8l Th-Pos82 ERYTHROSIN ISOTHIOCYANATE SELECTIVELY LABELS LYSINE464 PROTEOLYSIS OF THE CARDIAC SARCOPLASMIC RETICULUM Ca2l WITHIN THE PUTATIVE REGULATORY SITE OF THE Ca-ATPASE IN ATPASE BY CALPAIN. ((N. W. Seidler, B. A. Cucchetti)) University of Health SKELETAL SARCOPLASMIC RETICULUM. ((S. Huang, S. Negash, and T. Sciences, Kansas City, MO C. Squier)) Dept. ofBiochemistry, Univ. ofKansas, Lawrence, KS 66045. The removal of ineffective, chemically-modified structural and catalytic We have identified a specific labeling site oferythrosin isothiocyanate (Er-ITC) proteins prevents the accumulation of cellular debris and ensures the in the nucleotide binding domain optimal ofthe sarcoplasmic reticulum (SR) Ca-ATPase. functioning oftissues. The process of protein degredation is especially important The major labeling site for Er-ITC has been identified using reversed-phase in cardiac tissue which exhibits HPLC and positive FAB mass spectrometry subsequent to exhaustive tryptic high tumover rates (degradation and synthesis) of digestion of the Er-ITC modified Ca-ATPase. An ATP-protectable peptide cellular proteins as well as high metabolic activity. The cardiac sarcoplasmic corresponding to M452NVFNTEVRNLSK^4VER,67 is modified by Er-ITC, reticulum (CSR), an organelle central to the contractile process of the heart, whose average mass is 2830.1 ± 0.3 Da. The exclusive modification oflysine contains Ca2l release channels (ryanodine receptor) and Ca2" transport proteins residues by Er-ITC indicates Lys464 is the labeling site. Derivatization with Er- (Ca2e ATPase) both ofwhich regulate the force-generating calcium fluxes. Calpain, ITC results in selective inhibition ofthe ATP-dependent secondary activation of the Ca2"-activated neutral protease, plays a significant role in the protein-degrading the Ca-ATPase and ATP stimulation of phosphoenzyme decay rate. However, processes in the myocardium. We were interested in examining whether chemical modification of the Ca-ATPase with Er-ITC has no effect on i) the apparent modification ofthe CSR Ca2+-ATPase would increase the likelihood that the protein affinity of ATP in the catalytic site, ii) the maximal extent of phosphoenzyme would act as a substrate for calpain. The literature contains evidence that the formation by ATP, and iii) ATP utilization in the catalytic site in either native SR ryanodine receptor is the only physiologically-relevant substrate in SR from membranes or subsequent to detergent solubilization. The preservation ofthe skeletal muscle. We observed however that calpain degraded the CSR Ca2- secondary activation ofthe detergent solubilized Ca-ATPase indicates that the ATPase to 75.8% i 2.8 ofcontrols at 3.4 units calpain/ml and 0.8 mg CSR/ml and putative regulatory site and the catalytic site are within a single Ca-ATPase to 29.9% ± 3.4 ofcontrols at 10 units calpain/ml and 2.0 mg CSR/ml after 30 min polypeptide. It is concluded that Er-ITC either blocks nucleotide occupancy at incubations at 30°C. This finding will allow for further studies examining whether a regulatory site proximal to the catalytic site or interferes with ATP occupancy within the catalytic site ofthe phosphoenzyme. or not the rates of calpain-catalyzed degradation of the CSR Ca2" ATPase can by altered by chemical modificaton.

Th-Pos83 FUNCTIONAL REGULATION AND MOLECULAR DYNAMICS OF PURIFIED SKELETAL SARCOPLASMIC RETICULUM Ca-ATPase CORECONSTITUTED WITH PHOSPHOLAMBAN ((Laxma G. Reddy, Gregory W. Hunter, Joseph M. Autry*, Larry R. Jones* and David D. Thomas)) Dept. of Biochemistry, Univ. of Minnesota, Minneapolis, MN 55455, and Krannert Institute of Cardiology, Indiana Univ., Indianapolis, IN 46202. Phosphorylation under adrenergic stimulation of the transmembrane peptide phospholamban (PLB) regulates the sarcoplasmic reticulum (SR) Ca-pump in cardiac muscle. PLB was also shown to regulate the skeletal SR Ca-pump in a reconstituted system. Models that have been proposed for the regulatory interaction of PLB and the Ca-pump include the modulation of PLB's oligomeric structure. Previously, we have shown that PLB aggregates and inhibits the cardiac SR Ca-ATPase, and the phosphorylation of PLB relieves this inhibition by disaggregating the Ca-pump. In a reconstituted system using purified skeletal SR Ca-ATPase and recombinant PLB, we show that a monomeric PLB mutant (L37A) is more potent than the pentameric WT-PLB, and a tetrameric mutant (C41 L) has little or no regulatory effect on the Ca-ATPase. Fluorescence energy transfer studies of donor and acceptor labeled PLB indicate an increase in the fraction of monomeric PLB when coreconstituted with Ca-ATPase in DOPC vesicles. Molecular dynamics of phosphorescent Ca-ATPase show that PLB aggregates the Ca-pump even in a coreconstituted system. These results indicate that the oligomenc state of PLB is important for its function and PLB may be inhibiting the Ca-ATPase by inducing the aggregation of the pump.

MUSCLE ELECTROPHYSIOLOGY Th-Pos84 Th-Pos85 REDUCED EXPRESSION OF CLC- PARALLELS THE DECREASE IN Insulin potentiates depolarization-induced paralysis in human SARCOLEMMAL CHLORIDE CONDUCTANCE IN AGED RAT MUSCLE hypokalemic periodic paralysis by reducing inward rectifier K channel ((S. Pierno, A. DeLuca, C. L. Beck, A. L. George, Jr., D. Conte-Camerino)) conductance. ((Robert L. Ruff. M.D.. Ph.D.)) Neurology Service 1271-\W University of Bari, Italy, and Vanderbilt University, Nashville, TN USA. Cleveland VAMC 10701 East Blvd Cleveland. OH 44106 The principal Cl- channel of mammalian skeletal muscle (ClC-I) is believed Hypokalemic periodic paralysis (HOPP) is an autosomal dominant muscle disease responsible for the high resting membrane chloride conductance (gc,) that is genetically linked to a mutation in a skeletal muscle L-type Ca channel. H- characteristic of this tissue. A reduction of ga was found in skeletal muscle fibers pokalemia triggers a persistent inward current which causes depolarization-induced of aged rats. We used Northern blot analysis to test whether the reduction of gc1 paralysis. Insulin potentiates paralysis and with insulin. paralvsis occurs at higher observed in aged skeletal muscle is due to a reduction in ClC-1 mRNA levels. serum levels of K. The relationship of the Ca channel mutation to the persistent Total RNA (10 pg) samples isolated from the tibialis anterior muscle of aged inward current (24-29 mo. old, n=12) and normal adult (3-4 mo. old, n=8) rats were and the mechanism of insulin's potentiation of the depolarization electrophoresed on denaturing agarose gels, transferred to nylon membranes, and are not yet known. I studied single fiber membrane currents in intercostal muscle hybridized with a radiolabeled rat CIC- I probe (nt 298-638). In parallel, resting fibers from two patients with HOPP and from controls at 37 C using a 3-electrode membrane gc, was measured from extensor digitorum longus muscle fibers from voltage clamp. Reducing extracellular K from 4 mM to 1 mM depolarized HOPP each adult and aged rat in vitro using a two intracellular microelectrode fibers by 12 mV, but hyperpolarized control fibers by 14 mV. Adding 12 mU/ml technique. Aged rats exhibit a mean 45% decrease in gcl values as compared zinc-free insulin to 1 mM K bathing fluid increased the depolarization of HOPP with adult rats (mean gc,=2985 ± 130 1LS/cm2). Expression of CIC-1 mRNA fibers 24 mV and increased the normal hyperpolarization to 18 mV. In 4 mM K, normalized to the level of 18S ribosomal RNA exhibited a similar mean 41.5% insulin depolarized HOPP fibers by 12 mV and hyperpolarized control fibers by 4 reduction in aged rat muscle. Because of interindividual variability in the mV. HOPP fibers were inexcitable in 1 mM K or in 4 mM K with insulin. Blocking measured decrease in gc,, we also tested whether gc, and CIC-1 mRNA were Na channels with TTX and L-type Ca channels with nitrendipine did not prevent correlated, and found that expression of CIC- I mRNA is linearly correlated with depolarization. Insulin reduced the conductance of the inward rectifier K channel muscle gc0 in aged rats (Pearson's correlation coefficient = 0.78, p < 0.01). These over the range of membrane potentials where the channel conducts inward current. results suggest that the primary mechanism of gc, decrease observed during aging which occurs near the resting membrane potential. Inward rectifier K conductance may be reduced Cl- channel expression in skeletal muscle, and provide further was not changed over the range of potentials that the current is outward. Insulin evidence that CIC- I is the main determinant of sarcolemmal did not alter chloride conductance. Supported bv the Office of Medical Research of gc,. the Department of Veterans Affairs. -~ ~~~ ~~~~~~~~YOKLTNA6CYTOSKELETON A361 Th-Pos86 Th-Pos87 THE MECHANISM OF CELL ALIGNMENT CHANGE ELICITED Optical rheology of cytoskeletal protein networks ((Denis %Virtz*, Jingyuan BY CYCLIC STRETCH. ((K. Kada. K. Yasui, K. Naruse' and J. Toyama.)) Xu*, Scot C. Kuo**, Andre Palmer*.)) *Chemical Engineering Department, Johns Dept. of Circulation, Res. Inst. Environ. Med.. Nagoya Univ.. Nagoya 464-01 and Hopkins University Baltimore, MD 21218 ** Biomedical Engineering Department, 'Dept. of Physiology, Nagoya Univ. School of NMed., Nagoya 466. Japan Johns Hopkins University Baltimore, MD 21205

We investigated the effect of cyclic stretch stimulation on morphology and orienta- Filamentous actin, one of the main constituents of the cytoskeleton, is believed to tion of cultured cardiocytes. Embryonic rat (17-day-p.c.) cardiomvocytes cultured be the most important participant to the motion and mechanical integrity of non- on silicone dishes were cyclically stretched tol20% in length at a frequency of 30 muscle cells. Traditionally, the viscoelastic moduli have been meassired by imposing cycles/min. When the cardiocvtes were stretched in the initial stage of cultivation a small mechanical strain and quantifying the resulting stress. For pure F-actini inet- (within 12 hr), both cells and intracellular myofibrils oriented parallel to the stretch works, the amplitude of the elastic modulus, its concentration dependence of and the direction. When the cells were stretched only in the later stage (after 24 hr of cul- extent of the linear regime have been subject of debate. This paper helps resolve the tivation), thev tended to orient perpendicular to the stretch. Next we examined discrepancy betweern the values of the F-actin networksl elastic modulus reported in the effects of chemical compounds on these phase-related changes in myofibril ori- the literature, establishes tle extent of the linear reginie of actin networks l rheology, entation. None of the drugs tested (H-7. herbimycin A. gadolinium. and EGTA) probes for the first time the high-frequency behavior of actin networks, artd by usiIlg blocked the parallel orientation induced by the initial stage stretch. By contrast. these iiew nseasuirements, tests recent models of actin gel rheology. lo achieve these either H-7 or herbimycin A did inhibit almost completely the perpendicular orien- objectives, we have developed a novel, light-scattering based technique. diffusiiig tation induced by the late stage stretch but gadolinium nor EGTA did not. Our wave spectroscopy (DWS), which probes the linear rheological properties of actin results indicate that the alignment change induced by cyclic stretch depends on the networks non-invasively. Because it avoids the limitations of meclsanical rlseoiletry, stage of cultivation; with stretch in the initial stage (within 12 hr). cells and ms- our optical assay generates measurements of the mechanical properties of actin gels ofibrils orient parallel to the stretch: with stretch in the later stage (after 24 hr). that are less ainbiguous than mechanical measurements. We observe that the elastic they orient perpendicular to the stretch. The effect of stretch in the later stage is modulus has a small magnitude as well as a weak concentration dependence. There- likely mediated by protein kinase C and tyrosine kinase pathways. fore, actin alone is unsufficient to generate the elastic modulus necessary to sustain the strucural rigidity of the cell and/or consolidate a new cellular protruision. We also show that, the viscoelastic behavior of actin solutions is frequenlcy dependernt, i.e. solid-like at small excitation frequencies and liquid-like at large frequencies. WVe compare our in-vitro measurements with in-vivo measurements of the rheology of cytoplasm.

Th-Pos88 Th-Pos89 MICROSCOPIC VISCOELASTICITY IN ACTIN NETWORKS: A Changes in the organization and dynamics of the actin cytoskeleton in MODEL SYSTEM FOR SEMIFLEXIBLE POLYMERS. ((F. Gittes'. B. mammalian cells monitored with a GFP-actin fusion protein ((Axel Schnurr', "X. MIoehlerl. P.D. Olmsted2, J.V. Shah3. MI. Osterfield3. P.A. ' Choidas, Andreas Jungbluth, Antonio Sechi, John Murphy. Axel Ullrich and Janmev3. F.C. MacKintosh'. C.F. Schmidt'.)) University of MIichigan. Dept. Gerard Marriott.)) Max Planck Institute for Biochemistry. 82132 Martinsried. Phys. & Biophys. Res. Div., Ann Arbor. MI1 48109 2 University of Leeds. Dept. Germany Phys.. Leeds LS2 9JT. UK 3 Harvard Mledical School. Dept. Biomed. Biol. Sci.. Boston. MIA 02115 A GFP-actin fusion protein, permanently or transiently transfected in diverse mammalian cell lines, was shown to be a faithful intrinsic probe of both the organi- W\e hase developed a new microscopic technique to measure viscoelastic param- eters in soft by thermal fluctuations of embedded zation and dvnamics of the actin cytoskeleton. For example in Swiss 3T3 and NIH polymer materials, monitoring 3T3 the fusion probe particles laser interferometry in a Different modes of cells, protein was found to accumulate in lamellipodia. filopodia, using light microscope. focal contacts interferometrv allow us to obtain either 1D or 2D-displacement information. from and stress fibers and in Listeria infected HeLa cells. the GFP-actin which we calculate power spectra from 0.1 Hz to 20 kHz. Detection in 2D uses a associated dynamically with F-actin rich comet tails that formed at the base of quadrant photodiode sensing the angular distribution in scattered and transmitted motile bacteria. The expression behavior of GFP-actin in polyclonal transfected laser light. Using linear response theory, we determined the frequency-dependent cells was found to be cell line specific: while GFP-actin was constitutively expressed loss and storage shear moduli up to frequencies on the order of kHz. Our technique in PC12 and HEK-293 cells, only a minor proportion of NBT-II epithelia and NIH measures local values of the viscoelastic response, swithout actively straining the sys- 3T3 and Swiss 3T3 fibroblasts expressed the fusion protein. Interestingly. the ex- tem. In the macroscopic limit we find shear moduli at 0.1 Hz of G' = 0.11 ±0.03 Pa pression level of GFP-actin increased during the growth factor mediated epithelial and 0.17 ± 0.07 Pa for 1 and 2 mg/ml actin solutions, close to the onset of the to mesenchyme transformation of NBT-II cells. GFP-actin fluorescence was used to elastic plateau, and scaling behavior consistent with G'(.) _ "/4 at higher fre- investigate the changes in the organization and dynamics of the actin cytoskeleton quencies. For polyacrylamide that served as control, we measured plateau moduli during the transformation. Thus, in NBT-II epithelia cells GFP-actin was enriched of 2.0, 24, 100 and 280 Pa for crosslinked gels of 2. 2.5, 3 and 5% concentration in the cortex at sites of cell-cell contact and in actin fibers. In motile. mesenchyme (weight/volume) respectively, in agreement to within a factor of two with values cells the fusion protein integrated into actin fibers at the basal membrane. at sites obtained on identical samples from conventional rheology. XVe compare actin prepa- of membrane ruffling at the leading edge, and in focal contacts in the lamella and rations from different laboratories and prepared with different buffers to investigate in stress fibers. The difference in the organization and dynamics of actin at the the persistent discrepancies reported from macroscopic experiments. Supported by leading edge of NBT-II mesenchyme cells, compared to motile NIH 3T3 fibroblast the Whitaker Foundation. NSF (BIR-9512699) and the Petroleum Resarch Founda- cells, suggests that membrane ruffling plays an important role in the protrusion of tion. the leading edge

Th-Pos9O LJSTERLA MOTILITY IN CELLULAR EXTRACTS: IMPLICATIONS FOR MECHANISMS OF FORCE TRANSDUCTION. ((D. J. Olbris and J. Herzfeld)) Dept. of Chemistry (MS #015) and Keck Institute for Cellular Visualization, Brandeis University, Waltham, MA 02254-9110 The motility of LAsteria moncytogenes has been observed in cytoplasm and cellular extracts. The differing properties of those two media allows the underlying mechanism of motility to be probed. First, if extract has no cytoskeleton to which Listeria's actin tail can be anchored, what keeps the tail from being propelled backward? We present a simple model for bacterium- tail motion which suggests that the tail's architecture serves to minimize the tail's movement. Second, the low viscosity of extracts compared to cytoplasm greatly magnifies the effects of diffusion, allowing discrimination between motility models that rely on independent diffusion of the bacterium and those in which the bacterium is attached to its tail. We estimate the consequences of increased diffusion on Listeria motility and identify the conditions under which attachment of the bacterium to its tail may be definitively tested. We also comment on the implications of these calculations for understanding polymerization-based motility in crawling cells. A362 MUSCLE MECHANICSMEECHANICS AND ULTRASTRUCTURE IIH Th-Pos9l Th-Pos92 EFFECT OF MgADP ON FORCE GENERATION IN SKINNED CARDIAC THIN FILAMENT ACTIVATION IN THE PRESENCE OF MgITP. CARDIAC TRABECULAE. ((Hunter Martin, G. Ellis-Davies & R.J. ((Stephen H. Smith and Franklin Fuchs)) Dept. Cell Biology and Barsotti)) Department of Physiology, Allegheny University of the Health Physiology, University of Pittsburgh Sch. Med., Pittsburgh, PA 15261. Sciences, Phila., PA 19146 Recent work in this laboratory has focused on the interplay of cross-bridges We have reported that MgADP-bound cross-bridges alter relaxation from rigor and Ca2+ regulatory proteins in the mechanical modulation of myocardial initiated by photolysis of caged-ATP (Martin & Barsotti, Biophys. J. 66:1115-28 force generation. Since the apparent affinity of myosin for actin is much and 67:1933-41, 1994). To determine the role of these cross-bridges in steady- higher in the presence of MgITP than in the presence of MgATP (Moos, 1.05 , state force and force development, we monitored CSH Symp. 37:137, 1972) we have undertaken a study of Ca2'+-mediated ADP ""i' the changes in tension associated with photolysis and cross-bridge mediated activation of the thin filament in skinned bovine of caged-ADP in fully activated trabeculae (pCa ventricular muscle in the presence of MgITP. With 5mM MgITP there was 4.5, 20°C, I = 200mM, pH 7.1) and the effects of substantial force generation in the absence of Ca2+; at pCa 8.0 the [ / added MgADP on force development initiated by developed force was 40-70% of that at pCa 5.0. Neither the Ca2+- of independent nor Ca2+-dependent force was inhibited by phosphate (10mM) -1.00 :, photolysis caged-calcium (NP-EGTA). or vanadate (1mM). Both were partially inhibited by AlFx (2mM). The Photolytic production ofvarying [MgADP] relative ineffectiveness of and in 200msc caused a sustained rise in whose phosphate phosphate analogs inhibiting force, amplitude force suggests that the M.IDP-P complex is much less stable than the was to the released n = 6 proportional [MgADP] (PADP/Po=1.05 i 0.01, at M-ADP.P complex. The ratio of Ca2+-independent force to total force but whose rate was constant 6.0 ± 0.3 n = -500i.M MgADP) (kADp= sec- , 6). increased as the sarcomere length was increased from 1.9pm to added to 2.3gm. Similarly MgADP (<375 mM) relaxed fibers equilibrated with caged- Thus in the absence of Ca2+ increase in length produced a graded increase calcium caused a slow increase in pre-flash force but did not alter the rate of in the number of thin filament regulatory units in the "open' configuration. force development after calcium release. These results are consistent with a The MgITP-activated fiber may be a useful system for studying the model in which MgADP detains cross-bridges in a state near the end of the interactions between strong-binding cross-bridges and the Ca2+ regulatory power stroke that bears but does not generate force, resulting in a built-up in the proteins in the modulation of myocardial contraction. number of force generating cross-bridges. Supported by HL 40953 to RIB

Th-Pos93 Th-Pos94 EFFECT OF IONIC STRENGTH ON LENGTH-DEPENDENCE OF Ca2' SARCOMERE LENGTH (SL) AND MgATP STUDY: IS THE THIN SENSITIVITY IN SKINNED CARDIAC MUSCLE. ((Stephen H. Smith and FILAMENT COMPLIANCE SIGNIFICANT ENOUGH TO ALTER THE Franklin Fuchs)) Dept. Cell Biology and Physiology, University of Pittsburgh CROSS-BRIDGE KINETICS? Sch. Med., PA 15261. ((M. Kawai, G. Wang, and W. Ding)) Dept. of Pittsburgh, Anatomy and Cell Biology, The University ofIowa, Iowa City, IA 52242. Full activation of the thin filament involves a cooperative effect of Ca2+ binding to troponin C and strong-binding cross-bridge attachment to actin. The role of thin filament compliance in elementary steps ofthe cross-bridge cycle Recent studies on the effect of sarcomere length and interfilament spacing was examined. This study is based on the fact that, in rabbit psoas, the same num- indicate that length dependence of Ca2+ sensitivity in cardiac muscle is ber of cross-bridges is involved in force generation when SL is increased from 2.1 based on variation in the number of strong binding cross-bridges, but the to 2.4 gtm, but extra series compliance is introduced in the I-band (Higuchi, et. al., step in the cross-bridge cycle which is length dependent is not known. Biophys. J 69: 1000-1010, 1995). The elementary steps ofthe cross-bridge cycle Preliminary evidence from this laboratory (Smith, et al, Biophys. J. 72:A55, were assessed by sinusoidal analysis at pCa 4.66, pH 7.0, 200 mM ionic strength, 1997) indicates that Ca2+ activation is more length-dependent than cross- 8 mMphosphate and at 0-5 mM MgATP (20'C). Our results are consistent with bridge activation, raising the possibility of a role for a weak-binding actin- Step la Step lb Step 2 the cross-bridge scheme at myosin intermediate. Reducing the ionic strength at pCa 8.0 favors the left, where A=actin, M= formation of the weak-binding 'closed" state of the thin filament (Head, et al, K10k2 Eur. J. Biochem. 227:694, We obtained force-pCa curves with AMAM AMAM +Skl -AM'SAM --- A+MSA+MS myosin, and 1995). kS kS Our results showS=MgATp2-.that, when skinned bovine ventricular muscle at sarcomere lengths 1.7-1.8,um and 2.3- S k-lb k-2 SL was increased from 2.1 to and at ionic -0.17 and -0.06. At the ionic 2.4gm strengths higher strength 2.4 the apparent rate constant 27c remained the same, and the apparent rate the was 0.3-0.4 At low ptm, pCa50 highly length-dependent (ApCaw, pCa units). constants 2sd increased. These results are ionic there was normal Ca2+ but the length dependence contrary to the expectation based on strength regulation increased series et. of pCa50 was eliminated. These results suggest that sarcomere length may compliance (Luo, al., Am, J. Physiol.265:C279-C288, 1993). determine the number of thin filament regulatory units in the "closed" state. Our results further indicate that the change in the kinetic constants of steps I a-2 is small (<25%), except that klb (ATP-isomerization step) increases by 76%. These results demonstrate that the presence ofthe thin filament compliance does not interfere with our ability to detect the elementary steps using sinusoidal analysis.

Th-Pos95 Th-Pos96 CONTRIBUTION OF THIN FILAMENTS TO THE ELASTIC PROPERTIES OF RELAXED TENSION RESPONSE OF RABBIT PSOAS SKINNED MUSCLE FIBERS TO QUICK SKELETAL MYOFIBRILS. ((M. Ivemeyer, J.C. Ruegg and W.A. Linke)) Institute of STRETCHES AT LOW Pi CONCENTRATION ((Perry L. Sun, Jody A. Dantzig, Physiology II, Univ. of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany Martin R. Webb*, Henry Shuman, and Yale E. Goldman)) Penna. Muscle Institute, The passive length-tension relation of vertebrate striated muscle is mainly determined by forces Univ. of Penna., Phila., PA, 19104 and *NIMR, Mill Hill, London, NW7 IAA. developing upon stretch of I-band titin. However, other sarcomeric proteins can modulate the elasticity of relaxed muscle: we have previously shown that thin filament removal from cardiac The second component of tension recovery (phase 2s,1w), within 50 msof a quick (<180 myofibrils results in a pronounced stiffness decrease. In the present study, the effect of thin ts) stretch of active muscle fibers, is accelerated by increasing [Pi] from 0.5 - 25 mM, filament removal on passive tension and stiffness was studied in skeletal muscle. that detachment or reversal ofthe power stroke this phase isolated from rat suggesting cross-bridge during Myofibrils psoas and soleus muscles were suspended between a piezoelectric et J. If motor and a sensitive force transducer. Passive tension stretch was recorded before and accompanies Pi binding (Dantzig al., Biophys. 70:A126, 1996). phase 2s, during of then the rate of the tension after addition of a used to remove the thin requires binding Pi , recovery should be markedly slowed Ca2'-independent gelsolin fragment selectively when is decreased further. filaments. Also, stiffness was determined from the force response to small sinusoidal length [Pi] We studied tension transients in the presence of a "P oscillations (20Hz). Additionally, the positions oftwo nebutin epitopes (N-terminus, C-terminus) super-mop", consisting of purine nucleoside phosphorylase (500 units/ml) and 7- were measured by immunofluorescence microscopy in gelsolin-treated and control myofibrils. methylguanosine (MEG, I mM; Webb, PNAS 89:4884, 1992), phosphodeoxyribose Upon incubation of myofibrils with gelsolin at a constant sarcomere length (SL) above slack, we mutase (50 IiM), glucose-1,6-bisphosphate (50 gM) and Mn + (45 laM). These linked generally found a significant reduction of passive stiffness after 45min. In another set of enzymes remove Pi by converting MEG to a- 260 experiments, myofibrils were allowed to slacken between repetitive cycles of stretch and release. ribose- I-phosphate and then ribose-5- _ Under these conditions, acin extraction often resulted in increased passive tension and stiffness phosphate. [P] in the fiber interior was E L while slack SL decreased slightly. During actin extraction, the nebulin N-terminus moved estimated to be 0.5- 0.7 mM in the absence of z + P,super-mop -0. Ipm towardsthe Z-disc, whereas the C-terminal nebulin epitope remained unaffected. mop (Dantzig et al., J. Physiol. 451:247 L _ Ts Our results show several contributions of thin filaments to passive tension in skeletal muscle. 1992), and <10 tM in the presence of the 170 SPesuper-mop Actin may maintain an ordered I-band filament array by preventing titin from aggregating with super-mop. The rate of phase 2sisw of the 3 neighboring titin or nebulin filaments in unstretched sarcomeres, which would lead to increased tension recovery after a 3 nm/half-sarcomere nm/h.s.L . 80 Length passive tension/stiffness. The observed stiffness decrease during actin extraction from stretched stretch was 62.5 ± 8.6 s (mean ± S.E.M., n = myofibrils may be explained by the loss of weak interactions between titin and the thin filament. -10 ionic Such interactions with actin be for the 5; 'C, pCa2+4.5, strength 195 mM) in the absence and 19.9 +9.2 s-I in the may expected highly charged, relatively long PEVK ofthe These results indicate that most to region of titin in skeletal muscle. Since we found that, unlike in cardiac the of presence super-mop. cross-bridges contributing muscle, position of the mechanical transients a stretch first bind and then the T12 titin epitope near the Z-line was unaffected by treatment, we conclude that the phase 2siow following Pi reverse gelsolin their force transition or NIH Z-disc flanking titin region may remain bound to actin and not contribute to a stiffness change. generating detach.Supported by grant AR42333. MIVMUSCLE5-I MitAMECHANICSNC ANDflImyAsULTRASTRUCTURETIs -DvTA % AT HAl PJ A163 Th-Pos97 Th-Pos98 ACTIVE AND RIGOR STIFFNESS IN SKINNED FIBERS OF RABBIT PSOAS BIREFRINGENCE OF THE A-BANDS OF STRIATED MUSCLE FIBERS MUSCLE ((F. Vanzi, J.A. Dantzig, U.A. van der Heide and Y.E. Goldman)) MEASURED WITH A NOVEL INTERFERENCE MICROSCOPE. ((G.N. Pennsylvania Muscle Institute, Univ. of Pennsylvania, Philadelphia, PA, 19104 Vishnyakovi, G.G. Levini, L.K. Srebnitskaya2, O.A. Andreev- , D.S. Ushakov3 & Mechanical stiffness has been used to estimate the fraction of cross-bridges attached J. Borejdo3)) 'VNIOFI, Moscow, Russia, 2ITEB, Pushchino, Russia & 3Dept. of during full activation. We measured stiffness in single glycerol-extracted fibers as the Biochem. & Mol. Biol., The University of North Texas, Fort Worth, TX 76107. slope of the T, curve (Huxley and Simmons, Nature 233:533-538, 1971) obtained by applying quick (< 180 ps) length changes to the fibers in rigor and full activation (200 Muscle contraction involves the cyclical interaction between myosin heads and mM ionic strength, 10 °C, active pCa -4.5). Active stiffness was 61 i 4 % (mean ± actin filaments. Myosin heads are thought to generate a contractile force by S.E.M., n=14) of that in rigor. This ratio should be corrected for filament compliance their orientation with to actin. The a and single-headed attachments to estimate the fraction of cross-bridges attached during changing respect birefringence of myosin contraction. Previous studies (Thomas & Cooke, Biophys. J. 32:891-906, 1980; Cooke head is a convenient measure of this orientation. Birefringence was measured by & Franks, Biochemistry 19:2265-2269, 1980; Lovell et al., Nature 293:664-666, 1981) a novel transmission microscope based on Mach-Zender interferometric principle. have shown that virtually all cross-bridges are attached in rigor. However, do they all The source of illumination is Ar-Kr laser. The reference channel consists of a half contribute to stiffness? If the distribution of strains among the cross-bridges is wide wave plate and a piezzo mirror to change the optical path length of the reference enough, a fraction of them may be slack. To test this possibility we used two beam. The object channel consists of the high NA objective and a sample. The approaches: I) The fiber was put into rigor and then stretched 40-200 nm per half- reference and object beams are recombined and projected into a CCD camera. The sarcomere in the presence of either Mg-pyrophosphate (Mg-PP, , 2 mM) or Mg- AMPPNP (2 mM) so that the most highly strained bridges would detach and reattach at experiment consists ofchanging the optical path of the reference beam (by varying positions with lower strain. This procedure should narrow the strain distribution. 2) The the position of a piezzo mirror) in 4 steps giving rise to 4 interferograms. The fiber was put into rigor in the presence of BDM (50 mM), to eliminate shortening and image is reconstructed from the interferograms by a four-frame-phase-shifting again give a narrow strain distribution. Stiffness was then measured after removal of algorithm (Tang, Proc. SPIE 2860:34-44, 1996). Muscle is successively Mg-PP,, Mg-AMPPNP or BDM. None of these procedures significantly changed the illuminated with the light polarized parallel and perpendicular to the fiber axis stiffness compared to the original rigor value. Also, all of the rigor force-extension 2 Phase retardation curves were more linear than expected on the slack-bridge hypothesis. These results giving orthogonal images. (and birefringence) is proportional suggest either that stiffness measurements in rigor represent -100% of the cross-bridges to the arithmetical difference between these 2 images. The high NA objective or that our procedures do not narrow the strain distribution. Supported by NIH grant makes it possible to visualize individual sarcomeres in a whole muscle fiber. The AR42333. birefringence of the A-band of rigor muscle was 2.2x10-3. Supported by NIAMS.

Th-Pos99 Th-PoslOO ANALYSIS OF RABBIT CARDIAC MUSCLE THICK FILAMENTS. UNDERLYING SIMILARITIES IN DISPARATE ((R. W. Kensler.)) Department of Anatomy, University of Puerto Rico Medical SPECTROSCOPIES: APPLICATION OF EPR AND School. San Juan, PR 00936-5067 FLUORESCENCE POLARIZATION TO MUSCLE In spite of its significance. little detailed information is available on the structure of CONTRACTION ((Diane S. Eschliman, David W. Hayden and the cardiac muscle thick filament. We have developed a procedure for the isolation of David D. Thomas)) Dept. of Biochemistry, University of Minnesota rabbit cardiac muscle thick filaments, which preserves their native helical symmetry. Medical School, Minneapolis, MN 55455. The isolated filaments appear highly periodic with a near-helical repeat every 3rd crossbridge level. Computed Fourier transforms extend out to the 10th or 11th layer methods such as electron resonance line of a 43 nm near-helical repeat. Forbidden meridional reflections, not expected Spectroscopic paramagnetic from ideal helical symmetry (Huxley and Brown. 1963. J. Mol. Biol. 30:383), are (EPR) and fluorescence polarization (FP) provide valuable relatively strong, indicating a significant regular perturbation of the crossbridges insights into the mechanism of muscle contraction. However, from ideal helical symmetry. Both analysis of phases for reflections on the first correlating the spectroscopic data obtained from the different layer line and filtered images are consistent with a three-stranded arrangement of methods has proven difficult. A method of comparing different the myosin crossbridges, as previously shown for vertebrate skeletal muscle thick spectroscopic data for various models is needed. Mathematical filaments. The results suggest that the weakness of layer lines in the X-ray diffraction of fiber data and data to patterns of cardiac muscle (Matsubara et al. 1989. J. Physiol. 417:555) is not due fitting EPR-scallop FP-rabbit fiber the to an inherent disorder in the myosin crossbridge arrangement. The results also same model parameters was performed. The point of these fits demonstrate that the cardiac filaments may differ from skeletal muscle filaments in is to explore the possibility that the EPR and FP data are their strong tendency to remain attached to actin under relaxing conditions. This consistent with similar molecular models. Given EPR data, can investigation was supported by a Research Centers in Minority Institutions award the FP values be predicted, and are they consistent with the RR-03051, from the National Center for Research Resources (NIH), and a Grant- observed results? Our results show that EPR and FP results are in-Aid Award from the American Heart Association, Puerto Rico Affiliate. both consistent with a model in which muscle contraction corresponds to a shift in the distribution between two oriented populations of myosin heads.

Th-PoslOl Th-PoslO2 LARGE ROTATION OF THE REGULATORY DOMAIN OF MYOSIN STRUCTURAL CHANGES IN RIGOR (NUCLEOTIDE-FREE) CROSS-BRIDGES UPON MUSCLE CONTRACTION DETECTED BY SPIN LABEL EPR. INDUCED BY THE ADDITION OF MgADP. ((S. Xu, J. Gu, S. Frisbie and LC. Yu)) ((Leslie E.W. LaConte, Ingrid Brust-Mascher, Josh E. Baker, David D. NIAMS, NIH, Bethesda, MD 20892. Thomas)) University of Minnesota, Minneapolis, MN 55455. Whitaker et al. (Nature, 1995) reported structral changes in the acto-S 1 complex induced The orientation of the myosin light-chain binding domain was studied by MgADP. However, the observation was liniited to smooth myosin S1 and brush border with electron paramagnetic resonance (EPR). We replaced the myosin I. Similarly, Yagi et al. (J. Mus. Res. CellMoth. 1996) observed an increase in the regulatory light chain (RLC) in scallop adductor muscle fiber bundles with intensity of the meridional 144 A reflection in the X-ray diffraction pattem from frog spin-labeled chicken gizzard RLC. Calcium regulation of force and sartorius upon addition of MgADP. However, in the frog muscle such an increase is also ATPase activity showed that this exchange was functional. The spin seen when muscle is relaxed by MgATP. Our previous studies on radial elasticity of probe was oriented and immobilized, because spectra of minced fibers attached cross-bridges suggested that the molecular structure ofattached cross-bridges with were not affected by ATP or Ca. Spectra of oriented fibers were recorded bound MgADP differed from those in rigor (Xu et al., J. PhysioL 1993). To clarify the in the physiological states of rigor, relaxation and contraction. Spectral effect ofMgADP on skeletal muscle, X-ray diffraction pattems from skinned rabbit psoas simulations show that all spectra could be resolved into a linear muscle were recorded at 4 IC and 170 mM ionic strength in the presence 2 mM MgADP combination of two oriented components with well-defined central angles and in the absence ofany nucleotide (rigor). The intensities of 144 A and 72 A meridional axially separated by 36°. The distribution between these components reflections increased approximately 50% while the first actin layer line and the equatorial varies with the physiological state of the muscle, demonstrating that 20% reflections remained unchanged. The 6t actin layer lines increased slightly. The results of the heads rotate by 360 upon muscle contraction. Addition of Ca cannot be explained in terms of relaxation by MgATP, since HPLC profiles of the to rigor had no significant effect, showing that the spectral change upon MgADP-containing solution used showed .0.03% MgATP (0.6 pM). Furthermore, in contraction is not due to a change in the probe environment. Partially skinned rabbit psoas muscle at 4 °C, relaxation would decrease the intensity ofthe 144 A extracted fibers are active in the absence of calcium and have a meridinal reflection (Xu, et al., Biophys. J. 1997). Preliminary modeling ofthe actomyosin distribution similar to contraction, confirming that the rotation is structures in rigor (coordinates provided by R. Milligan) and in MgADP suggests that the associated with force generation. These distinct orientations cannot be results are consistent with a small but significant axial movement in the a-helical part of assigned to a single biochemical state, since trapping some intermediate the myosin head (about 15') or a smaller axial movement (about 8°) ifthe motor domain states with nucleotide analogs has no effect on the distribution. is involved. The results suggest that there is more than one conformation of the various strongly bound cross-bridge states. A-A tA2AULTTQ('IMlI.NVl. U' MECHANICSMV(_pATJTU'Q ANIANB ITITaACUTRSTUCTUETUTJTTaU' n7 Th-PoslO3 Th-Pos104 A MAXIMUM ENTROPY ANALYSIS OF THE ORIENTATION OF REGULATORY MILRINONE FAVORS SHIFTS OF SKELETAL MUSCLE LIGHT CHAINS IN SKELETAL MUSCLE FIBERS ((U.A. van der Heide, S.C. Hop- CROSSBRIDGES FROM HIGH TO LOW FORCE STATES. ((C. Y. kins and Y.E. Goldman)) Penna. Muscle Inst., Univ. of Penna., Phila., PA 19104 Seow, L. Morishita and B. H. Bressler.)) Dept. Anatomy and Pharmacology/Therapeutics, Univ. of British Columbia, Vancouver, BC, Canada, Recently, the orientation of the myosin regulatory light chain RLC V6T 1Z3 (RLC) was investigated in muscle fibers using three mutant - each containing a pair of cysteines at positions chosen Direct action of the cardiotonic bipyridine milrinone on the crossbridges of single RLCs, skinned rabbit skeletal muscle fibers was investigated. from the x-ray structure, and labeled with a bifunctional rhodam- At 10 C and pH 7.0, milrinone J. reduced isometric force in a logarithmically concentration-dependent manner, with a ine, to create a 3-dimensional framework of probes (Biophys. 55% reduction at 0.6 mM. Milrinone also reduced calcium sensitivity of the skinned 72:M-AM-A2, M-Pos-150-153, 1997). The labeled mutant RLCs fibers in terms of force production; the shift in the force-pCa curve indicated a change were exchanged for the endogenous subunits in glycerol-extracted in the value of the pCa50% (pCa value at 50% maximum force) from 6.10 to 5.94. single fibers from rabbit psoas muscle. Fluorescence polarization The unloaded velocity of shortening was reduced by 18% in the presence of 0.6 mM measurements were carried out separately for each mutant, m. milrinone. These findings suggest that the positive inotropic effect of milrinone on Order parameters (P2.) and (P4) were extracted from the data, providing information cardiac muscle is not due to the direct action of the drug on the muscle crossbridges. on the tilt fland twist yof the RLC region because the orientation of the probes relative Tension transient measurements indicated that milrinone increased relative stiffness to the RLC is known. Here we use the principle that thermodynamic entropy is a meas- of the fibers, i.e., reduced force per bridge, suggesting that the bipyridine detained ure of uncertainty (Shannon 1948, Bell Sys. Tech. J. 27:379; Jaynes 1957, Phys. Rev. crossbridges in a low force state. The internal load produced by these detained 106:620) to derive the distribution function f(,y)for the orientation of the RLCs with bridges could account for the decrease in shortening velocity. The time course maximum entropy: f(8,y)= exp(-Z[A2P2. (I,3,) + A4.P4,,(,y)]). The set of 6 or- of the rapid force recovery following a length step was not significantly altered by der parameters obtained from 3 mutants can be mapped uniquely onto the 6 A's defin- milrinone. The specific and reversible action of the bipyridine on muscle crossbridges ing the distribution function. Because no additional assumptions are made about the makes it a potentially useful tool for probing the chemomechanics of the crossbridge distribution's shape, this function provides an unbiased representation of the informa- cycle. tion available about fiand yand their interdependence. Thus maximum entropy analysis helps to elucidate the motions of the cross bridge that lead to force generation and fila- ment sliding. Supported by NIH Grant AR26846 to the PMI.

Th-PoslO5 Th-Pos106 CORRELATION BETWEEN PHYSIOLOGICAL FUNCTIONS AND PARVALBUMIN CONTENTS, MYOSIN HEAVY CHAIN DISTRIBUTION WATER STRUCTURE IN SKINNED SKELETAL MUSCLE AND AND CONTRACTILE PROPERTIES OF MOUSE SKELETAL MUSCLES. CORNEAS. ((S. Takemori, M. Yamaguchi, S. Toshima, K. Ohno. M. Hiramatsu. ((Ph. Gailly, G. Beckers-Bleukx and M. Colson)) Y. Umazume.)) Dept. Physiol.(I) and Dept. Ophthalmol., Jikei Univ. Sch. Med. Catholic University of Louvain, Brussels, Belgium. Minato-ku Tokyo 105, Japan. We compared the mechanical characteristics of three mouse Physiological tissue functions naturally involve water. Water molecules in biological hind limb muscles in relation with their myosin heavy tissues are known to be more or less restricted to form a local water structure. chain (MHC) composition and their contents in parvalbumin Skeletal muscle and the cornea both composed of uniform filament lattices of arrayed (PA). Extensor digitorum longus (EDL), soleus (SOL) and proteins have an advantage to define milieu for water molecules in these tissues. flexor digitorum brevis (FDB) essentially contained two MHC isoforms : 2B and 2X for EDL, 1 and 2A for SOL and 2A Using CPMG method on 300 MHz NMR equipment (varian Gemini 2000 BB). and 2X for FDB. EDL had a higher unloaded shortening we observed transverse relaxation of proton spin to estimate the state of water in velocity than SOL and FDB. No difference was observed isolated skinned fibers of frog skeletal muscle (120-200 micron in diameter and 15 between FDB and SOL, suggesting that fibres containing mm in length) and in dissected pieces (5 mm x 5mm) of rabbit corneas. Relaxation MHC 2X isoform were not faster than fibres containing MHC process of nuclear spin to the thermal equilibrium is considered to involve magnetic 2A, but that both were slower than fibres containing MHC interaction between neighboring molecules, and therefore reflect restriction imposed 2B. High amounts of PA were found in EDL and FDB. The on their movement. In relaxed skinned fibers, about 80% of water protons relaxed half relaxation time of the twitch contraction as well as significantly faster than in rigor fibers and in corneas. Considering static functional the initial phase of relaxation of the isometric tetanus states of the cornea and rigor muscle, we propose that contractile proteins in relaxed (t55) were smaller in EDL and FDB than in SOL; the t5j muscle set water molecules in the myofilament lattice free to get readv to such a also diminished in function of the duration of the dynamic process as contraction. A well-known evidence that tumors. compared with tetanus at a rate compatible with the dissociation rate normal tissue, are more dynamic in its function as well as in its state of water is in of the PA-magnesium complex. We conclude that FDB, a muscle very rich in MHC 2X, contracts as slowly as SOL, a line with the present proposal in the sense that relatively unrestrained water is a muscle containing MHC 1 and 2A but has a faster initial requisite for dynamic function of biological tissue. phase of relaxation probably due to its high contents in PA.

Th-PoslO7 Th-Pos108 CAFFEINE REDUCES MAXIMUM CROSSBRIDGE CYCLING IN EFFECT OF CA ACTIVATION LEVEL ON RATES OF FORCE SKINNED RAT SOLEUS FIBERS. ((Philip A. Wahr and Joseph M. DEVELOPMENT AND RELAXATION FOLLOWING PHOTOLYSIS OF NP- Metzger)) Dept. Of Physiology, Univ. of Michigan, Ann Arbor, MI 48109 EGTA AND DIAZO-2. ((Philp A. Wahr and Joseph M. Metzger)) Dept. of Physiology, University of Michigan, Ann Arbor, MI 48109. Caffeine has been shown to increase the Ca2e sensitivity of the contractile proteins, apparently in the absence of changes in the Ca?' binding to TnC. Flash photolysis of 2 mM of the caged compounds NP-EGTA and diazo-2 was The mechanism of this effect is currently unknown. We investigated the used to rapidly change the Ca?' concentration in skinned rat soleus fibers and cardiac myocytes at 15 "C. The Call level prior to photolysis and the energy possibility that this shift in Ca2? sensitivity is mediated by a direct effect of level of the flash were selected to give a series of thin filament Ca?' activation caffeine on crossbridge kinetics which subsequently influences the ability of steps between fully relaxed and fully activated. In this way any dependence of the myosin to activate the thin filament. We examined the effects of 0-30 mM rates of force development or relaxation on Ca2+ activation of the thin filament caffeine on the kinetics and Ca2e sensitivity of contraction at 15° C in skinned would be revealed. In addition, the rates of force development and relaxation were rat soleus fibers. The addition of 30 mM caffeine produced a substantial examined in the presence of either 8 mM MgATP or MgCTP. The rate of force decrease in the maximum force to 0.59 of the caffeine-free generated force. development following the photolysis of NP-EGTA in soleus fibers was found to This reduction in force was accompanied by a shift in the midpoint of the depend linearly on the thin filament activation level with a slope of 4.4 s-"/relative force-pCa relation from 5.88±0.02 in the absence of caffeine to 6.19±0.02 force and y-intercept of 1.6 s- (r = 0.675) in the presence of 8 mM MgATP. A in the presence of 30 mM caffeine with no change in the Hill coefficient. change in the nucleotide to 8 mM MgCTP resulted in an increase in the rate of The rate of tension redevelopment following a quick release and re-stretch force development at all force levels, with linear slope 8.1sI'/rel. force and y- (k,) was reduced from 3.28±0.12 s-5 to 2.45±0.15 s' with the addition of 30 intercept 3.5 s t (r = 0.860). In contrast, the diazo-2 induced relaxation rate in mM caffeine. Additionally, V.,, was determined by fitting Hill's equation to cardiac myocytes had an inverse relation with Ca2e activation. With ATP, the of relative force vs shortening velocity per fiber length. The addition of relaxation was fit linearly with a slope of -8.9 s-'/rel. force and y-intercept 16.6 s-a plots (r=0.690), and with CTP the slope was -20.8 s-1/rel. force, y-intercept 20.0 s-' 30 mM caffeine also led to a reduction in Vmax from 1.17 ML/s to 0.64 (r=0.602). Thus, the thin filament Ca2e activation level exerts significant effects ML/s. These results indicate that caffeine has significant direct effects on the on the rates of force development and relaxation. rates of crossbridge cycling which may, in turn, underlie changes in thin filament activation that lead to a shift in Ca2e sensitivity. MUTSCLEIVAaI MECHANICSA A.,hIANW AND ULTRASTRUCTURETIhtDA%IIA VIPIs'III L t% A-M-9 Th-Posl09 Th-PosllO TENSION REDEVELOPMENT RATE (ktr) IN SKINNED FIBERS FROM RATES OF MYOFIBRILLAR TENSION DEVELOPMENT ARE DEPRESSED IN CANINE RABBIT PSOAS MUSCLE IS ALTERED BY REPLACEMENT OF NATIVE EXPERIMENTAL HEART FAILURE. ((1W. R. Wolff, J.R Torrealba, J. R. Patel)) TROPONIN C (TnC) WITH CHICKEN SKELETAL RECOMBINANT TnC. Departments ofMedicine and Physiology, University of Wisconsin, Madison WI 53792. ((M. Regnier. P.B. Chase, L.B. Smillie. MNIA. Sorenson.)) Dept. of Bioengineering and Depts. of Radiology and Physiology/ Biophysics, University of WNashington, Seattle, WA Depression ofthe maximal rate of left ventricular pressure rise (dP/dt,,,) is a prominent 98195. NIRC Group in Protein Structure and Function, Dept. of Biochemistrv, Universitv feature ofthe systolic dysfunction accompanying dilated cardiomyopathy (DCM), yet the of Alberta, Edmnonton, Canada; Depto. Bioquimica Mledica, Universidade Federal do Rio subcellular mechanisms accounting for this dysfunction are unknown. We examined the de Janeiro, Rio de Janeiro, Brazil. potential role ofaltered dynamic myofibrillar function in the systolic dysfunction seen in a canine model ofDCM produced by chronic rapid ventricular pacing (200-250 beats/min for 33 ± 7 days, n = 4). Compared to sham-operated controls (n = 5), DCM was characterized by At maximal Ca2+ activation, kt, depends on the rate of cross-bridge cycling. In contrast, during submaximal [Ca'+] activations, it depends on the dynamics of individual regulatory reduced dP/dt,,,, (625 ± 111 vs 1017 ± 73 mm Hg/sec) and increased LV end-diastolic tinits on the thin filament, shich are influenced by the properties of TnC. We have further pressures (22.0 ± 5.3 vs 4.6 ± 2.3 mm Hg). Rates of myofibrillar tension development (kc.) explored the role of TnC iti regulating the rate of tension development by replacing native were examined in smal (width 48 ± 18 smn) permeabilized left ventricular myocardial rabbit TnC ivith chick recombinant TnC, either wild-type (rTnC) or a mutant (NHdel) with preparations following photolysis of NP-EGTA, a high affinity Ca2e chelator that is rapidly decreased Ca2+ affinity and an increased Ca2+ off-rate (Chandra et al., 1994 J Biol Chem converted to a low affinity forn after exposure to UV light, 269:14988; Rennie et al.. 1996 Biophys J 72:A282). Despite marked differences in Ca2+ resulting in a pulse increase in [Ca2+] and an increase in UVfbsh sensitivity of steady-state tension (pCa 50 = 5.34±0.02 for NHdel vs 6.01±0.01 for rTnC developed tension. We observed faster rates oftension oSham 0 atid 6.12±0.01 for native TnC at 15°C), both recombinant proteins cause surprisingly similar development (1 mM NP-EGTA, [Ca2l = 375-500 pM, sarcomere changes iti the kt,-tension relationship: the highly curvilinear kt,-tension relationship seen length 2.30, 15° C) with increasing levels of activation in both ° / wsitli natise TnC is maintained, such that at low levels of Ca +-activated tension kt, is similar failing (n = 6) and non-failing (n = 5) preparations. However, or it to at only slightly elevated by the recombinants, but increases maximal values lower maximal rates of myofibrillar tension development were tension levels than in native is the same for fibers. Mlaximal klr native TnC and rTnC, but in niay be reduced slightly (10-25%) wvith NHdel. Thus a dramatic change in Ca2+ sensitivity significantly slower failing compared to non-failing ' of steady-state tension (NHdel) is associated wsith only minor differences in the ktr-tension preparations (tio = 0.41 ± 0.15 vs 0.22 ± 0.01 sec, p = 0.035). relationship. superimposed on a substantial effect due to reconstitution with a different TnC Differences in kc. between failing and non-failing preparations were even more apparent species. Support:HL52558/51277(US-NIH); MIRC(Canada), CNPq/FINEP(Brazil). following UV flashes resulting in sub-maximal activations. These data suggest that myofibriBlar dysfunction likely contributes to the systolic dysfunction and reduced dP/dt,,., in DCM.

PROTEIN-LIGAND INTERACTIONS Th-Poslll Th-Posll2 CRYSTAL STRUCTURE OF BPI, THE HUMAN TRANSFERREDNOESY NMR STRUCTURES OF PROTEIN-BOUND BACTERICIDAL/PERMEABILITY-INCREASING PROTEIN ((Lesa PEPTIDE LIGANDS: A BIOTIN MIMETIC PEPTIDE. J. Beamer. Stephen F. Carroll. and David Eisenberg.)) Mlolecular Biology FSHPQNT-STREPTAVIDIN MODEL COMPLEX ((D. Gizachew and E. A. Institute. UCLA. PO Box 9.51570 Los Angeles. CA 90095-1570. and XOANIA Dratz.)) Department of Chemistry and Biochemistry, Montana state University, Bozeman, Corporation. 2910 7th Street. Berkeley. CA 94710. and Nlolecular Biology MT 39717 Institute. UCLA. PO Box 93157-0 Los Angeles. CA 90095-1370 To improve the ability of transferredNOESY (TrNOESY) NMR to provide accurate 3D structures of peptide ligands bound to protein receptors. we have studied a biotinmimetic Bactericidal/permeabilitx increasing protein (BPI) is a potent antimicrobial pro- peptide, FSHPQNT. bound to streptavidin as a model svstem. The x-ray crystal structure of tein from polvmorphonuclear neutrophils swhich swhich binds to. clears. and neutral- biotin-mimetic peptides bound to streptavidin have recentlv been obtained to high resolution izes lipopolvsaccharides (LPS) from the outer membrane of Gram-negatise bacteria. by Katz (Biochem., 34:15421,1995). The FSHPQNT peptide has nearly as favorable an WN hen present in the mammalian bloodstream. bacteria and their LPS triggers an enthalpy of binding to streptavidin as biotin, but because of its flexible nature in solution it has an unfavorable entropy of binding (Weber et al,. Biochem., 31:9350,1992) which leads inflammatory response swhich can lead to septic shock. A bioactive N-terminal BPI to a Kd ca. 100uM that is suitable for TrNOESY NMR analvsis. We have studied the protein is currently under clinical investigation for the treatment of Gram-negative offrate of the FSHPQNT peptide from streptavidin as a function of pH and temperature, bacterial infections and their complications. The crystal structure of human BPI at using CF3CO-aminolabeled and paraflurophenylalanine labeled peptide and F-19 and H- 2.4 A resolution revealed an unusual boomerang-shaped molecule formed by two sim- 1 NMR, monitoring line widths and chemical shifts and the spinlock power dependence ilar domains. The electron density maps indicated two ligands bound in apolar pock- of the Tlrho relaxation. After sufficiently fast exchange conditions were established, we ets of the protein. Tandem mass spectrometric analvsis showed that these ligands used a spinecho filter to remove broad NOESY cross peaks between the protein and ligand asere phosphatidslcholine. The acyl carbon chains of the phospholipid are buried in protons. A combination of TrNOESY and TrROESY cross peak intensities are used to the core of the and the zwvitterionic head is to correct for indirect effects of proteinmediated ligand proton cross relaxation (which may be hvdrophobic protein. group exposed quite substantial for certain proton pairs) and for spin diffusion effects within the bound solvent. The structure of BPI provides a model for the related lipopolysaccharide- ligand. which provides more accurate distance constraints within the bound peptide. For binding protein LBP and the plasma lipid transfer proteins. CETP and PLTP. cross peaks between residues TOCSY interference is not present in the TrROESY. The NMR structure of the proteinbound peptide is compared to the xray crystal structure of the bound peptide to provide benchmarks for refinement of proteinbound peptide structures. (WVe thank Dr. S. Busse for assistance with NMR methods, supported by NIH and NSF)

Th-Posll3 Th-Posll4 INTERACTION OF TERMINAL COMPLEMENT PROTEINS: A FORMATION OF E. COLI PHOSPHOFRUCTOKINASE TETRAMERS OF THERMODYNAMIC ANALYSIS. ((Cristian Saez and Alfred F. Esser)) MIXED SUBUNIT COMPOSITION ((J. L. Johnson. N. D. Lasagna. and G. D. School of of Kansas MO 64110. Reinhart.)) Department of Chemistry. Southwestern Oklahoma State Universitv. Biological Sciences, University Missouri, City, WVeatherford. OK 73096. and Department of Biochemistrv and Biophysics. Texas A&M University. College Station, TX 77843-2128 Exposure of cryptic binding sites on terminal complement proteins is thought to Inherent to analyses of the interactions between the substrates and effectors on the al- be required for assembly of the membrane attack complex, C5b-9, or MAC. losteric. tetrameric enzyme phosphofructokinase (PFK) from E. colt is the problem of dis- This conclusion is baacd mainly on the inhibitory effect of charged polycations tinguishing betseen the relative contributions from each of the four identical active and and polyanions, such as protamine, heparin and the drug Suramin, on MAC- effector sites. We have generated a series of charge-tagged mutants to allow for separa- mediated hemolysis. Earlier results also demonstrated that these proteins interact tion. via anion-exchange chromatography, of mutant vs wild-type protein and all possible with each other at low ionic strength but the identity of the interaction sites on hvbrid combinations of subunits. The charge-tagged mutants were: (1) K1,2E, in which the native proteins in unknown. To gain information on the role ofthese interac- lvsine 1 and 2 were mutated to glutamates; (2) E195,199K; and (3) K304E. All mutated tion sites we have determined the affinity between C8 and C9 and effect of residues are solvent-exposed and not involved in binding sites or subunit interfaces. Upon Suramin using analytical ultracentrifugation. The two proteins formed a com- expression and purification, each of the three mutants exhibited wild-type activitv and an plex at physiological ionic strength and the temperature dependence of the altered chromatographic response on an FPLC Mono-Q column. At low concentrations of association was examined from 8 to 37°C. The formation of the complex (Kd = KSCN. complete subunit exchange was induced between E195.199K and wild-type E. colt 0.6 WtM at 20°C) was very sensitive to temperature (Kd = 3 FM at 37°C) and PFK. When the mixture was eluted from the Mono-Q column, a peak corresponding to van't Hoff analysis indicated a negative change in enthalpy (-12.9 kcal/mol) and each possible combination was observed. Moreover, the percentage of the total area asso- a ciated with each peak roughly corresponds to the ratio theoreticallv predicted based solely entropy (-15.9 cal/moledeg), suggesting that electrostatic forces play promi- upon combinatorial degeneracy. Each hybrid enzyme form can be stabilized for at least a nent role in the interaction of C8 with C9. Low concentrations of charged week without redistribution into other hybrid combinations if stored in the presence of the ligands like heparin (M,5 000) and Suramin inhibited the interaction. In the case substrate. fructose 6-phosphate. By introducing an additional W311F or W311Y mutation of Suramin it was shown that it induced trimerization of C8 (Kd = 0.1 gM at into the charge-modified subunits, this procedure has been utilized to manipulate the tryp- 20°C) and dimerization of C9 (Kd = 0.86 p.M at 20°C). The van't Hoffplot for tophan composition of the mixed tetramers so that only the wild-tvpe subunits contain a Suramin-induced C8 trimerization was biphasic with a sharp break at 20°C. single tryptophan. Supported by grant GM 33216 from NIH. However, DSC data of native C8 with or without Suramin are linear at this temperature although the drug lowers the main transition temperature and decreases the enthalpy of unfolding. (Supported by NIH grant ROI AI-19478) A366 Al"XE IdRPJU)TEI-IIGANDmlJLPROTEIN-LIGAND INTERACTIONSI.T ATIN Th-Posll5 Th-Posll6 TRANSITION-STATE THEORY OF DIFFUSION-CONTROLLED TRANSITION-STATE THEORY OF DIFFUSION-CONTROLLED PROTEIN-PROTEIN ASSOCIATION: APPLICATION TO PROTEIN-PROTEIN ASSOCIATION: TEST OF ACCURACY ((AI. BARNASE-BARSTAR COMPLEX ((H.-X. Zhou, M. Vijayakumar. K.-Y. Vijayakumar. K.-Y. NVong. H.-X. Zhou.)) Department of Biochemistry. Hong Kong Wong.)) Department of Biochemistry, Hong Kong University of Science and University of Science and Technology. Clear WNater Bav. Kowloon. Hong Kong Technology, Clear Water Bav, Kowloon, Hong Kong The orientational constraints for forming a stereospecific protein complex severely The electrostatic enhancement of the association rate of barnase and barstar is cal- restrict the rate of protein-protein association. Electrostatic interactions are knowcn culated using a transition-state theory like expression and atomic detail modeling of experimentally to compensate for such restriction and enhance the rate by as much the protein molecules. This expression predicts that the rate enhancement is simply as four orders of magnitude. However. prediction of the electrostatic rate enhance- the average Boltzmann facter in the region of configurational space where associa- ment by Brownian dynamics (BD) simulations is a formidable task because it is very tion occurs instantaneously in the diffusion-controlled limit. Based on experimental time-consuming to calculate the forces and torques on the associating proteins. In evidence, this "transition state" is defined by configurations in which, relative to the the past these vere calculated by treating one of the proteins as a set of test charges. stereospecifically bound complex, the two proteins are shifted apart by 8 A (so a This leads to significant underestimation of the rate enhancement. The underesti- layer of water can be accommodated in the interface) and the two binding surfaces mation is decreased slightly by replacing the test-charge model by an effective-charge are rotated away hy < 3°. The values of the average Boltzmann factor, calculated model. Recently we found that the rate enhancement can be predicted by the elec- by solving the Poisson-Boltzmann equation, for the wild-type complex and 16 com- trostatic free energy of the transition state. wchere association occurs instantaneously plexes with single mutations are found to correlate well with experimental results in the diffusion-controlled limit. for forming the stereospecific protein complex. This for the electrostatic rate enhancement. The predicted rate enhancement is found reduces a kinetic problem to a thermodynamic problem and eliminates the need for to be somewhat insensitive to the precise definition of the transition state. due to BD simulation. Here we illustrate the accuracy of this transition-state theory on the long-range nature of electrostatic interactions. The experimental ionic strength the barnase-barstar complex by comparing against BD simulation. The results from dependence of the rate enhancement is also reasonablv reproduced. the two approaches agree to within 10%. Though the comparison is based on using the effective-charge model (necessitated by the BD simulation). the transition-state theory is also expected to be accurate even wshen the electrostatic interactions are treated rigorouslv.

Th-Posll7 Th-Posll8 Molecular surface complementarity at protein-protein interfaces ((Nlurad SELECTIVITY OF LIGAND COPRECIPITATION FOR SINGLE N\asal. Boris Klebanskv. Richard Fine. and Barry Honig.)) Department of PROTEINS. (Rex Loyrien, Daumantas Matulis, Charles Wu), Univ. of Biochemistrs and Mlolecular Biophysics. Columbia Universitv. N\esw York. .Newc Minnesota, St. Paul. 'York 10032 Matrix ligand coprecipitating agents are able to capture proteins from Protein-protein recognition plays a central role throughout biology. Predicting the dilute (0.001-0.02%) mixtures. The process is reversible and protective. It correct association mode of two proteins treated as rigid bodies amounts to a search 'pulls' (lowers chemical potential of the product, stabilizing the coprecipitate), in of freedom of the two while an versus 'pushing' (atempting to raise chemical potential of proteins in solution). rotational/translational degrees proteins using ap- In some cases - four of out of propriate model of the binding energy to find the optimal binding mode. While this examples coprecipitating lectins, nearly pure has documented to crudes will be shown - matrix coprecipitation is surprisingly selective. A approach successes. it tends be computationallv expensive. A question is why. prerequisite to effective protein-protein recognition is geometrical complementaritv Mutual structural, binding, stacking, associative forces of proteins and between the surfaces of the two proteins at the interaction site. Hence. the search ligands are important, but probably not the sole origin, maybe not even main for the correct binding mode can be reduced to a search on the protein surface for origin for selectivity. The overall reaction: Protein(s) (mixed solution) + surface patches of complementary shapes capable of forming a favorable interface. Matrix Ligand(s) -- Selected, coprecipitated protein, is a multireaction of about Here we extend previous work done by the Olson group that used volumetric tech- 10 subreactions. Some are linked, and cooperative. A few may be niques to represent the protein surface where the molecular surface is defined as independently measured. The profile of the overall coprecipitative free energy, an isocontour of a protein density function. This representation allows a degree of dependent on degree of progression through the network of subreactions, may control over the level of structural details depicted on the molecular surface. Thus. be sharp and deep. Only one protein fitting the profile becomes selected. Thus it allows the exploration of the extent of protein-protein surface complementaritv at selectivity may be dependent on the multireactions nature of the net reaction, various structural resolution levels. In this work the protein-protein surface comple- besides dependent on structures of optimum ligands and target proteins. Ref.: mentaritv as a function of effective length scale used to define the molecular surface J. Molecular Recog. 2, 433-443 (1996). is investigated in a sample of protein complexes of known structure. Subsequently. the efficacy of a sparse surface representation based on the characterization of foci of optimal surface complementaritv (geometrical and physico-chemical) in rigid body docking of proteins is tested.

Th-Posll9 Th-Posl20 PROTON LINKAGE OF THE BINDING OF OVOMUCOID PH DEPENDENCE OF ANTIBODY-HAPTEN ASSOCIATION THIRD DOMAIN TO PANCREATIC ELASTASE IN ACID ((D.R.Livesay, S.D. Linthicum, and S. Subramaniam.)) Department of Chemistry. SOLUTION. ((Stephen P. Edgcomb, Brian M. Baker and Kenneth University of Illinois, Urbana, IL; Department of Veterinary Pathobiology. Texas P. Murphy)) Department of Biochemistry, University of Iowa, Iowa A&M University, College Station, TX: and Beckman Institute. Unversity of City, IA 52242 Illinois, Urbana, IL The affinity of serine proteases for protons changes upon binding of Monoclonal antibody NC6.8 is specific for the superpotent sweetener. N-(p- protease inhibitors. This change of affinity links inhibitor binding to cyanophenyl)-N'-(diphenylmethyl)-guanidiniumacetic acid. The structure of the na- solution pH and regulates protease function. Recently, we have used tive and complexed NC6.8 Fab fragments have been obtain from x-ray crystallog- isothermal titration calorimetry to characterize the proton linkage of raphy by Edmundson and coworkers. The complex reveals that the association is the inhibitor, turkey ovomucoid third domain (OMTKY3) binding to mediated by complementary charged residues on the antibody and groups on the the elastase in solutions near hapten. As a result, these interactions are influenced by factors such as solvent pH protease, porcine pancreatic (PPE) and ionic strength. We have used continuum electrostatics methods to neutral We now use isothermal titration and differential investigate pH. the pH dependence of the association energetics. The computed titration behavior scanning calorimetry to extend this characterization to include provides the influence matrix of titrating groups near the key salt-link residues in binding in acid solution. At low pH the binding of OMTKY3 by the interface. Several mutant fragments were constructed to investigate the im- PPE is considerably weaker than in solutions near neutral pH. The portance of these proximal residues. The computational results are compared with weakened interaction cannot be accounted for with only the known experimental data on the antibody-hapten binding (This research was supported by intrinsic binding constant and the known proton linkage of binding an NIH R01 grant). in near-neutral solutions. The additional proton linkage may be a consequence of protonation events within the binding region of the two proteins as well as a pH induced conformational change in PPE. PROTEIN-LIGANDlAA'W1NJJANTb INTERACTIONSa ACTAWLN A2U7AWi Th-Posl21 Th-Posl22 MECHANISMS OF PHOTOINACTIVATION OF ENZYMES INVESTIGATION OF THE BINDING SPECIFICITIES OF THREE PORPHYRIN MEDIATED BY TRIARYLMETHANE DYES ((Ketan Amin, Mauricio PHOTOSENSITIZERS TO HUMAN SERUM ALBUMIN (HSA)PROTEIN USING S. Baptista and Guilherme L. Indig.)) School of Pharmacy, University of CIRCULAR DICHROISM (CD) AND UV-VIS ABSORPTION SPECTROSCOPIC Wisconsin, Madison, WI 53706 TECHNIQUES. ((John Belletti, Rama subbu*, Ravi Pandey+, Thomas Dougherty+ and Thamarapu Srikrishnan )) Departments ofBiophysics and Radiation Biology+, Roswell Park The use of high-intensity pulsed laser irradiation to photoactivate exogenous Cancer Institute, Department ofOral Biology*, SUNYAB School of Dentistry, Buffalo. molecules designed to perform specific tasks in biological systems is a subject of increasing interest. Triarylmethane (TAM) dyes have been successfully used as a Photodynamic therapy (PDT) is a treatment of choice for many carcinomas. The in several of laser inactivation of mechanism of action of PDT is based on the incorporation and retention of the photosensitizer applications dye-assisted enzymes. in a solid tumor. One of the most remarkable of this rests on the strat- photosensitizer Human serum albumin (HSA) is a protein found abundantly strengths general technique in blood plasma. Binding to HSA extends the lifetime of and decreases their of antibodies - rather than the to be inactivated drugs egy labeling anti-enzyme enzymes concentration. Investigation on various alkyl ether analogs ofpyrophrophorbide-a showed themselves - with the since it photosensitizer, permits the achievement of a high that there was in increase in vivo efficacy by increasing level of 3 OR selectivity with regard to the destruction of target biopolymers (Beermann the length of the carbon atom, with a maximum found and Jay, Methods Cell. Biol. 1994, 44, 715). TAM dyes have also been considered CH in the hexyl and heptyl ether derivatives. (see figure for HH2c for employment as photosensitizers in photodynamic therapy of neoplastic diseases. the drugs used in this study). Binding studies ofthese NH N The mechanisms of TAM-promoted photoinactivation of enzymes are not well under- three drugs to HSAwere carried out. HPPH binds to both H 7 stood. Here the mechanisms by which inactivation takes place is investigated with site I and II where as hexenyl binds to site I and heptyl to H3CH3 the employment of non-covalent complexes of TAM dyes with three model biological site II. For the very frst time, we used CD and UV-VIS targets, horseradish peroxidase, hexokinase, and bovine serum albumin. As accessed spectroscopic techniques to analyze the binding specificities JO° through large scale photoreactions using BSA as the host biopolymer, the details of ofHSA to several drugs used in PDT. CD and UV-VIS COOH the mechanisms to be conserved photosensitization appear highly among the ethyl spectra were run on HSA and the three drugs at the 5pM R =3Hexyl 2. R = Heptyl violet, crystal violet, and malachite green dye series. Because the quantum efficiency concentration range. The most marked changes are in the 3. R =Hexesyl of photobleaching of these TAM dyes non-covalently bound to proteins increases in Soret band region, around 400 nm, indicating binding to the aromatic residues in HSA. the absence of oxygen, they can be seen as photosensitizers suited for employment These changes were in three distinct regions, 315, 365 and 400nm for HPPH; at 350 and in anaerobic environments, including hypoxic or poorly perfused tumor areas 405nm for the Heptyl and 350 and 400nm for the HexenyL. Our results show that the sensitizing abilities of various drugs can be predicted on the basis oftheir CD spectra and it can be run routinely for the new drugs before they go for preclinical trials.

Th-Posl23 Th-Posl24 COMPARISON OF THE STRUCTURE/ENERGETIC RELATIONSHIP FOR REEXAMINING THE INTERACTION OF MYOGLOBIN WITH ANESTHETICS THE BINDING OF TWO INHIBITORS TO A SERINE PROTEASE. ((James ((Jonathan W. Tanner*, Jonas S. Johansson*+, Paul A. Liebmans, R. Horn, Weston Waterbeck, and Kenneth P. Murphy)) Departnent of and Roderic G. Eckenhoff*)) of Iowa IA 52242 Depatnments of *Anesthesia, ePhysiology, and 'Biochemistry and Biophysics, Biochemistry, University Iowa, City, University ofPennsylvania Medical Center, Philadelphia, PA 19104. To fully understand the structural basis for molecular recognition between The best structural inforniation to date about the interaction between inhaled anesthetics and a proteins, explicit knowledge of the energetics is required. Here we compare the lipid-free protein comes from crystallographic studies of (sperm whale) myoglobin binding measured energetics of inhibitors binding to a serine protease to energetics xenon, cyclopropane, and dichloromethane (reviewed by Eckenhoff and Johansson in calculated from structure. These protein-protein interactions display proton Anesthesia: Biologic Foundations, Lippincott-Raven, 1997). Assuming that folded protein linkage effects, which have also been studied. Previous research has examined stnicture is necessary for these binding sites, and they are stronger than less specific (e.g., the of the ovomucoid third domain to hydrophobic) interactions between these agents and the rest of the protein, we predicted that binding inhibitor, turkey (OMTKY3), the these agents would stabilize the folded structure of myoglobin, the way halothane stabilizes protease, porcine pancreatic elastase (PPE). Intrinsic binding energetics were serum albumin (Tanner et al., Biophys. J. 72:A417). To test this prediction, we obtained determined experimentally and found to be in good agreement with values myoglobin (from horse skeletal muscle) from Sigma in powdered form, and dissolved it in calculated from changes in polar and apolar accessible surface area upon aqueous 20 mM KH2PO4, 150 mM NaCI (pH 7). Stability was assessed by differential scanning binding. We hope to expand these structure-energetic correlations by calorimetry (measuring heat capacity versus temperature from 35 to 95 'C), circular dichroism different structural environments. The effects of different structural (following loss of ellipticity at 220 nm from 60 to 95 'C), and hydrogen-tritium exchange examnining the rate for environments is an evaluation of the of (detecting slowly exchanging protein hydrogens at 45 'C). To some extent 40 mM, being quantified through enthalpy and to a greater extent 200 mM, dichloromethane decreased the temperature at peak heat as a for the binding function of solution conditions protease, subtilisin capacity, the ellipticity transition midpoint temperature, and the protection factor for H Carlsberg, and two different protease inhibitors, OMTKY3 and eglin c. Initial exchange. Thus, using all three ofthese techniques, we found that, contrasy to our expectation, results of the interaction between both OMTKY3 and eglin c with subtilisin dichloromethane lowersthe free energy change for unfolding of myoglobin. This suggests that Carlsberg indicate different degrees of proton linkage. Understanding these the non-specific interactions between anesthetics and the unfolded protein actually have more differences will be necessary to generalize structure-energetic correlations. effect on the protein than binding at sites identified crystallographically in the native myoglobin structure, at least for determining the equilibrium between the folded and unfolded states. It underscores the importance of studying low-affinity ligands with solution techniques besides crystallography. Supported by N.I.H. and the Foundation for Anesthesia Education & Research.

Th-Posl25 Th-Posl26 On the mechanism of binding of the thyroid hormone to its receptor. MOLECULAR DYNAMICS SIMULATIONS OF CELLULAR RETINOL BINDING PRO- ((Dorina Kosztin', Sergei Izrailev and Klaus Schulten.)) Beckman Institute, TEIN 11 (APO AND HOLO) ((Michael Tychko, and Thomas B. Woolf)) Depts. Physiol. Department of Physics and Chemistry, University of Illinois at Urbana-Champaign and Biophys., Johns Hopkins Univ., Balfimore, MD 21205. Knowledge of the mechanism by which the thyroid hormone binding induces con- formational changes in the thyroid hormone receptor protein is critical for under- Cellular retinol binding proteins (CRBPs) are ten-stranded "cIam shell structures standing the functional role of the hormone. The hormone binding pathways were that selectively bind retinoids in an intemal cavity. Our investigations into the molec- studied by the method of steered molecular dynamics which emplovs external per- ular basis of their discrimination could aid efforts for rational drug design and increase turbations to drive the system out of equilibrium. The simulations emploved the understanding of lipid:protein interactions. Two simula- crystal structure of the thyroid hormone receptor ligand binding domain and placed tions of retinol binding protein 11(one holo and one apo) will different types of ligands in the binding site. By applying an external force to the be discussed. These simulations use the CHARMm pro- to facilitate its from ligand unbinding the protein, pathways of unbinding were in- with a water solvation model. The latest and residues that an role in the gram droplet vestigated play important binding process were results can be with our identified. Mutations that are likely to affect the binding affinity of the hormone to usefully compared previous simu- the receptor are suggested. lations of intestinal, human muscle and adipocyte lipid binding proteins. Particular attention was paid to the inter- action energies between interior amino acid residues, water, and the ligand. These trajectories form the basis for linear response type relative free energy calculations that can aid site-directed mutagenesis experiments. In partic- ular, experimental work has highlighted the Q109/R106 (CRBP-IIA-FABP) site [below ligand in figure] (Jakoby et al., Biochemistry (1993) 32:872-878) as forming an elec- trostatic switch that can convert ligand preference between retinol and fatty acid. A368A1_vo PIUTIPROTEIN-LIGANDN-TA;lAl_.ItAN1 .,,,-INTERACTIONSA-A-A- NS Th-Posl27 Th-Posl28 Calcium binding properties of Androcam, a testis-specific Binding of caffeine and urate to haemocyanin studied by ITC (( calmodulin-related protein from Drosophila. ((S.R.Mlartin A.A.Lu 2 N.Hellmann'. XIAXlenze§. H.Decker'. MI.K.Grieshaber§ )) K.M.Beckingham 2. P.NI.Bayley 1.)) 'Div. of Physical Biochemistr%. N.I.M.R.. §Institute of Zoophysiology. Heinrich-Heine-tUniversity Dusseldorf MIill Hill, London NW7 IAA and 2Dept.Biochemistry and Cell Biology. Rice 'Institute for !Molecular Biophysics. University of Mlainz Universitv. Houston. TX 77005. We investigated the binding of urate and the derivative caffeine to 12-meric haemocsaniii The encodes a 66% identity to calmodulin of the lobster. Homarus vulgaris. Both substances wsere found to act as allosteric effectors Drosophila genome protein swith (CaM) of the oxygen binding properties of this respiratory protein [1]. In order to determine the that shows distinctive changes in the sequence analogous to calcium binding site binding constants of urate and caffeine wse employed Isothermal Titration Calorimetrs II of CaMN. The gene is expressed exclusively in the testis. and swe have named the (ITC). Binding isotherms swere determined for fully oxygenated haemocyanin at 20 C and protein Androcam (ACaM). Direct calcium binding assavs with dibromo-BA.PTA pH 8.0. Urate and caffeine stabilizes a conformation of haemocyanin with high affinity for and Quin-2 indicators shosc that ACaM binds 2 Ca ions scith 60-fold higher aserage oxygen. This is in accordance swith our effector binding curves. which did not shoss any affinity than the high affinity sites III and IX' of CaM: any other sites bind only sign of positive cooperativity. Four binding sites per 12-meric haemocyanin for caffeine weakly. The calcium dissociation rate observed bh Trp fluorescence in stopped flos with a binding constant of about 20-30 pSM were found. Comparison of caffeine-binding in measurements is 15-fold sloswer than svith CaM. Far UV'CD indicates a significanlt TRIS and HEPES-buffer indicates. that binding of coffein in accompanied bh an uptake of Ca-induced increase in for overall helical protons. The urate-binding curves could only be analyzed assuming a binding alpha-helix ACaM. although content (botih stoichiometry derived from caffeine-binding. Under these prerequisites the binding +/ Ca) is only 80% of that of CaM. ACaM shosvs lower thermostability than CaM. constant for urate amounted 50-100 pM. In addition. there is a wveak. presumably svith Tm 45uC which is Ca-independent. and attributed to the -N-domain. ACaM unspecific binding site for urate wehich cannot be saturated seith urate. This weak binding shosws a 1000x reduced affinity (Kd 1pM) for Trp-containing peptide derivatives site has also been reported in earlier binding studies. which employed equilibrium dialysis of the target sequence of skeletal mvosin light chain kinase. and their interactions experiments [2]. The binding behaviour of the twso effectors in dependence on temperature. with the (putative) -N-terminal lobe of ACaM differ significantly from those seen buffer composition and haemocyanin concentration ivill be discussed. svith CaM itself. These results suggest a testis-specific role for ACaM involsing This work was supported by the N\aturwissenschaftlich-.tledizinisches Forschungs:cntrum calcium binding and target interactions that are distinctly different from those of .tainis. and Fonds der Chemzschen Industrie (.K.KG) References of its domains [1] Morris S. Bridges CR. Grieshaber MIK (1983) J.exp.Zool. 235:135-139 CaMN. In calmodulin. the differential affinity tsvo allowvs the possibilits [2] Nies A. Zeis B. Bridges CR. Grieshaber XIK (1992) J,Exp.Bio. 168:111-124 of Ca regulation by one domain specifically. The stronglv reduced Ca-affinitv of the -N-domain of ACaM might suggest that this protein lacks normal regulators properties and. although apparently containing one normal and one abnormal EF- hand sequences. this domain mav have an as set unknowvn and distinctive function.

Th-Posl29 Th-Posl30 GLU161 AND ARG162 ARE NOT REQUIRED FOR THE PHOSPHO(ENOL)PYRUVATE HAS LITTLE EFFECT ON THE INHIBITION BY PHOSPHO(ENOL)PYRUVATE OF CONFORMATIONAL CHANGE INDUCED BY FRUCTOSE PHOSPHOFRUCTOKINASE FROM B. STEAROTHERMOPHILUS 6-PHOSPHATE IN PHOSPHOFRUCTOKINASE FROM E. COLI ((T. ((J. L. Kimmel and G. D. Reinhart.)) Department of Biochemistry and C. Pham and G. D. Reinhart.)) Department of Biochemistry and Biophysics. Biophysics. Texas A&-M University. College Station. TX 77843-2128. Texas A & NMlUniversity. College Station. TX 77843-2128. Phospliofructokinase from Bacillus stearothermophilus (BsPFK) is a homotetramer The unique tryptophan per subunit of phosphofructokinase (PFK) provides a con- swhich is subject to both K-type allosteric inhibition and activation. The basis of venient spectroscopic probe to monitor ligand binding. PEK contains a common the regulation stems from the ability of MgADP and phospho(enol)pyruvate (PEP) binding site per homotetrameric subunit for both the allosteric inhibitor. phospho- to enhance or diminish. respectively, the ability of the enzvme to bind the substrate enolpyruvate (PEP). and the activator. MgADP. These ligands act by modifying fructose 6-phosphate (Fru-6-P). BsPFK contains only one allosteric site to sshich the affinit-vwith which the substrate fructose 6-phosphate (Fru-6-P) binds to PFK. both MlgADP and PEP bind. While the allosteric regulation of BsPFK has been The binding of Fru-6-P has been shown to be well modeled by a two-step process: extensively studied. the molecular basis of this regulation is still unclear. Compar- the formation on an initial binary cormplex followed by an induced conformational isons of crystal structures with either Fru-6-P or phosphoglycolate. an analog of change (Auzat et al. (1995) J. Mol. Biol. 249, 478-492). We have monitored the PEP. bound have shown that the positively charged Argl62 residue resides within binding of Fru-6-P as a function of PEP concentration by following the change in in- the active site when Fru-6-P is bound. Upon binding of phosphoglvcolate. Argl62 trinsic fluorescence using stopped-flow. At low concentrations of PEP the apparent virtually swvitches places with the negatively charged Glul6l. It has been proposed. rate of Fru-6-P binding decreases significantly. as observed by Auzat et al. This ob- therefore. that an electrostatic repulsion between Glu161 and the negatively charged servation is consistent with a slow PEP dissociation proceeding prior to the binding Fru-6-P explains the mechanism of inhibition (Schirmer. T. and Evans. P. R. (1990) of Fru-6-P to free enzyme to produce the binary PFK-F6P complex. As PEP con- Nsature .343. 140-145). Using site directed mutagenesis, three mutants of BsPFK have centration is increased, however, Fru-6-P begins to bind directly to the PEP-PFK been produced which substitute an alanine residue for either Glu161 or Argl62 or form to generate the PEP-PFK-F6P ternary complex. At very high concentrations both Glu161 and Argl62 simultaneously. Steady-state kinetics experiments suggest of PEP (> 50mM) this latter reaction dominates. and the system can once again be that Argl62 substantially facilitates the binding of Fru-6-P. However. replacing ei- analyzed with the two-step induced fit model used in the absence of PEP. The results ther Gl1f61 or Argl62 with an alanine does not abolish the abilitv of PEP to inhibit indicate that almost all of the inhibition of Fru-6-P binding at high concentrations the binding of substrate as predicted bh the above mechanism. On the contrary of PEP is attributable to the first binding step. The magnitudes of the first order PEP binds more tightly, and the coupling free energv between Fru-6-P and PEP rate constants that describe the induced conformational change after Fru-6-P binds for the E161A mutant is comparable in magnitude to that for wild-type BsPFK. to form the ternary complex are comparable to the corresponding rate constants Supported by grants GM33216 from N'IH and A1368 from the Welch Foundation. when Fru-6-P binds to free enzyme. Supported by grant GM33216 from NIH.

Th-Posl31 Th-Posl32 FUNCTIONAL LINKAGE IN THE ESCHERICHIM COLI REPRESSOR OF STRUCTURAL PROPERTIES OF TRYPTOPHANYL t-RNA BIOTIN BIOSYNTHESIS, ((Emily D. Streaker, Shreyesh Ruparelia and SYNTHETASE MUTANTS OF B.subtilis AND B.stearothermophilus Dorothy Beckett)), Department of Chemistry and Biochemistry, University of ((B.Rajendrant. A. G. Szaboi. and G. Guillemette2.)) l Dept. of Chemistry and Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250 Biochemistry, University of Windsor. XX indsor. Ontario. Canada X\9B 3P4. 2 Dept. of Chemistrv. University of Waterloo. Waterloo. Ontario. Canada .N2L 3G1 The Escherichiacoli repressor of biotin biosynthesis is a 35.3 kD winged helix-turn-helix protein that binds site-specifically to the forty basepair biotin Tryptophanyl-tRNA\'A synthetase (TrpRS) is the key enzyme responsible for the operator site. The stoichiometry of the protein-DNA complex has been shown to aminoacylation of t-RNATCP with tryptophan (Trp). Previously. in our lab. be 2 protein monomers per target site and these two monomers bind cooperatively to the two half-sites of the nearly perfect inverted palindromic target. Weak B.Subtilis TrpRS wild type and its Trp analogue incorporated mutants w-ere dimerization ofthe protein has been demonstrated to be thermodynamically prepared"2. Their characteristics and the structural similarities w-ere studied. To coupled to binding of the corepressor, biotinyl-5'-AMP, to BirA. The repressor determine whether W92 can be replaced. other amino acid mutants were pre- also functions as the enzyme that catalyzes covalent linkage of biotin to acetyl- pared where W92 was substituted by Tyrosine. This substitution was suggested CoA carboxylase via this adenylate intermediate. A high resolution 3-dimensional by the sequence of the M.genitalium TrpRS. which has a tyrosine in position structure of the apo-repressor has been determined. However, since the 92. Thus, we have prepared B.Subtilis TrpRS mutants W92Y and W92YI46F. unliganded protein is monomeric no information is provided in the structural and examined some of their properties. The relative activity and the structural model about the location of the monomer-monomer interface. We have cloned and differences of these mutants will be discussed. The work on TrpRS has been purified two single site mutants of the repressor, AA 147 and D197Y, which, extended to B.Stearothermophilus TrpRS, which has three Trp residues. The based on their phenotypes, hypothetically define the interface. Results of initial B.Stearothermophilus mutants, W91Y. W48Y. W48YW290Y. W48YW91Y and measurements of binding of the purified proteins to the biotin operator performed W48YW91YWV290Y were prepared and their activities and structural properties using the DNaseI footprint titration method indicate that the affinities of both were measured using different optical spectroscopic techniques such as circular proteins for the operator site are significantly reduced relative to that ofthe wild dichroism. fluorescence and absorbance. The complex TrpRS.Trp-adenylate of the type repressor. Results of measurements of small ligand binding and catalytic above enzymes have been isolated using different Trp analogues and ATP. In addi- activities of the mutants indicate, however, that the functional defects in the tion, the analogue 7-azatryptophan (7AW) was incorporated biosynthetically into proteins extend beyond their site-specific DNA binding properties. The combined selected mutants and structural features of these special mutant enzymes were re- results indicate the existence of a more complex pathway oflinkage in the system vealed. References: ' Hogue. C.W.. and et al.. J. Mol. Biol. (1996) 260, 446-466 than originally anticipated. 2 Hogue. C. WV., and Szabo, A.G., Biophysical Chemistry. (1993) 48, 159-169 P14YSICALPHVA 7A-. CHEMISTRY% I4 WMIi TWa-OF PROTEINSA JTI1IN,a A 3si lu Qs- A369 Th-Posl33 Th-Posl34 Bliophysical Characterization Of A D)esigned TMV Coat Protein Mutant, R46G, That Elicits A Moderate Hypersensitivity Response In N. srlvestris. *((John M. Toedt, Emory H. Bra.swell, Todd M. Schuster, and David A. Yphlianis)) Dept. of Molecular and Cell Biology and the Nattionial Analytical Ultracentrifugation Facility Uniiversity of Connecticut 06269((Zenobia F. Taraporewala .ud Ja,nes N. Culver)) Biotcchno1ogy Inslituic College Park, MD 20742

Coalt protein) froin U1 tobacco mosaic virus (TMV), a kniowni tion-elicitor, and coat proteini from the moderate hypersensitive response (HR) elicit(or R46G were examined to determine variations in the structural or hydrodynamic properties that would allow N. svlvestris to recognize the invading virus. The coat proteins were exainiiied via circular dichroism, velocity sedimentation, and kinetic viral reconstitution studies. Far UV CD specira reveal no significant secondary WITHDRAWN structural differenlces betweecn the Ut wild type anid R46G mutant coat proteins. Furthermore, near UV CD spectra also disphty ito siginificauit (dilercitces suggesting no measurable differenices in tertiary structure niear the aromatic regioits. Sedlitnetitationi velocity studies, however, do show a difference in the conlcentration dependence of the weight averaged sedimentation coefficient (w) at 4°C. Viral reconstitution kinetic results show a decreased reconstitution lag time for the reconstitutions with mutant coat proteilt that ittitially lack the 20S aggregate compared to that performed with wild type. However, experimenits performed with 20S initially present reveal no detectable differences indicating that the mechanism of assembly of virus is similar for the two viral species. Therefore, an increased rate of f(rmation of 20S in R46G compared to that of the Ut coat protein may be the determining factor tor the difference in lag time. The inferred increased rate of formation of 20S is verified by direct mea-surements of the 20S boundary as a function of time usitig velocity sedimentation analysis. Il addition, this velocity study reveals no distitict stable intermediate aggregate fonnation during the 20S assembly for the mutant coat protein compaired to diat of the wild type. The results are consistent with the interpretation that there may be an altered distribution of the sm,all coaxt protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus, whichi in tuni leads to the HR and subsequent limitation of infectioti. * Supported by grants from the Natiotial Science Foundation: grant # BIR9318373 to the NAUF and grsust # MCB95t(X149

Th-Posl35 Th-Posl36 EXPERIMENTALLY TESTING COMPUTATIONAL MODELS FOR IONIZATION DISORDER AT METAL SITES IN PROTEINS: A HIGH FREQUENCY EQUILIBRIA IN TURKEY OVOMUCOID THIRD DOMAIN. ((William R. Forsyth," EMR STUDY. Michael K. Gilson,5 Jan Antosiewicz,t Olav R. Jaren,t and Andrew D. Robertsont)) tDept. of ( B.J. Gaffney , T. J. Morgan, Jr. and B.C. Maguire)) National High Biochem., Univ. of Iowa, Iowa City, IA 52242. 'CARB, NIST, Rockville, MD 20850. Magnetic Field Lab and Institute for Molecular Biophysics, Florida State tDept. of Biophys., Univ. of Warsaw, 02-089, Warsaw, Poland. University, Tallahassee, FL 32310. (Spon. by B.J. Gaffney) The accurate prediction of experimentally accessible properties for any given protein Electron Magnetic Resonance (EMR) spectra of metailoproteins in is the ultimate test of our understanding of the interactions that govern protein function and crystals or frozen solutions are very sensitive to small structural changes stability. Such comparisons test the validity of the physical model as well as facilitate near the metal site. Multiple sites, or a distribution of them, are evident in interpretation of the experimental values. One property amenable to both theory and CW-EMR spectra obtained at low temperature. The proteins experimental observation is ionization euilibria. To this end, 2D-NMR experiments were transferrinilactoferrin and lipoxygenase have been examined at high (94 used to investigate the pH dependence of H chemical shifts for 15 of the 16 ionizing residues GHz, W-band) and low (9 GHz, X-band) EMR frequencies to evaluate the in turkey ovomucoid third domain (OMTKY3) and compared to values calculated as contributions of relaxation, unresolved nuclear splitting, geometric factors, described by Antosiewicz et al., [(1996) Biochemistry 35, 7819-7833]. The observed pKas of and other contributions to the spectra. For the transferrins, metals 7 groups are shifted more than I pH unit from the model compound values. Interestingly, the including iron, copper and chromium have been studied. The iron center in pK,s of the basic groups in OMTKY3 are generally elevated with respect to the model lipoxygenase has been studied in several spin states. compound values and are surprisingly insensitive to salt even though the groups are solvent exposed. While the calculations properly predict the direction of many of the shifts, there is a tendency to overestimate the shifts and their salt dependence. The calculated values are in better agreement than the null model, in which the protein structure has no influence on ionization equilibria, and are nearly as accurate as the new null model which predicts the observed pK. of a group to be the average of measured pK,s for that group in proteins. Site- directed mutagenesis experiments are underway to test hypotheses regarding the molecular origins of the pK. shifts in OMTKY3, such as the role of hydrogen bonding and the influence of long range (> 5A) interactions. This work was supported by the NIH (Grant 46869)

Th-Posl37 Th-Posl38 EFFECT OF COBALT BIN'DING ON TH4E ACTIVITY AND THERMAL PARTITION AND PERMEATION OF ANIONIC DENDRIMER IN CHARGED DENATURATION OF a-AMYLASE ((A. A. Sabouryl, M U. Dahot2 and A A Moosavi- POLYACRYLAMIDE GEL. ((L.A. Mark, P. Dubin and J.C. Williams)) Indiana Movahedi I)) IInstitute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran University-Purdue University at Indianapolis, Indianapolis, IN 46202. and 'Department of Biochemistrv, University of Sind, Jamshoro, Pakistan Experiments were performed to investigate restricted passage of charged molecules through like-charged matrices. Spherical, carboxy-terminal, cascade a-Amylase (a-1,4 glucan4-glucanohydrolase, EC 3 2 11) catalyzes the hydrolysis ofa-1,4 polymer (dendrimer) and charged gels were chosen as a system of well-character- glycosidic linkages of starch components and glycogen The binding of cobalt divalent cation ized components with which to carry out partition and permeation experiments. dendrimer (-20A radius, 108 carboxyl groups) and 10% poly- on the Bacil/its amyostefcienm a-Amylase was studied by equilibrium dialysis at pH=7 Generation-3 (G-3) 0, acrylamide gel (PAG), modified for -lOOmEq/L fixed anionic charge by addition Tris-HCI 50 mM, and 300 K The binding of cobalt is non-cooperative The number of of 2-acrylamido-2-methylpropane-sulfonic acid (AMPS) or acrylic acid (AA) be- binding sites and association equilibrium constant are 25 and K=10 mM-t, respectively. A fore polymerization, were used. Experiments were carried out in buffered, 160 mM new equation with a useful graphical method (very similar to the Scatchard plot in the ligand solution pH 2.0, 4.5 and 7.0. Partition of G-3 (0.48+0.03) was the same as that for an binding studies) was introduced Q/[Co2+1=K(AH-Q) Using an isothermal titration uncharged species (20A radius dextran, 0.49+0.02) in uncharged PAG at pH 7.0. G-3 partition was reduced in both charged gels at pH 7.0 (0.39+0.03 in AMPS microcalorimeter, the heat of reaction between a-Amylase and Co2+ (Q) in different fixed and AA). However, G-3 partition increased in all gels at lower pH and in several concentrations of cobalt ([Co2+]) can be obtained The molar enthalpy of binding has been groups was near or above 1.0 (1.37_0.13 in PAG and 1.49+0.17 in AA, pH 2.0; in remained -4.0 obtained from the slope of linear plot of versus Q; AH=-18 5 kJ molet1, and the 0.96_0.15 AMPS, pH 4.5). Dextran permeability xIO-6 cm/sec Q/[Co2'] regardless of drastic changes in buffer pH or gel type. However, G-3 permeability value of K was aforementioned above gave the surprising result of dependence on buffer pH in PAG, where no charge- charge interactions should exist. Permeability of G-3 was higher than dextran for The a-amylase activity is significantly increased with increasing Co2+1, however, the all groups. Results for G-3 in AMPS gel (23.1±4.2 x10-6, pH 7.0; 78.2±2.0 xIO-6, melting point of the enzvme (Tm) is decreased (see table) The values of Tm were obtained pH 4.5; 112±3.8 xIO-6 cm/sec, pH 2.0) were similar to AA and PAG. G-3 must from the change of optical density of enzyme due to the increase of temperature have an attractive interaction with the gel at pH 4.5 in AMPS and PAG and at pH 2.0 in AA and PAG to account for partition coefficients > 1.0. Such an interaction would also account for the high dendrimer permeabilities in this system. Thus, G-3 r[Co lmmM 6 o30 60 65) does not act as a charged, hard sphere in polyacrylamide gels as would be expected i % Acnivtyj 100 125 131 135 129 by steric considerations alone. It may be that G-3 does not gain the high negative T / K 336 l3281! 323 321 1 320 charge predicted, and it is possible that it G-3 aggregates in the hydrophobic environment of the gel, which would mask the repulsive charge effects expected. AJIVA 179 PuveiVrnXOA%-A9LJUAT . V.n.eTQTIVLIJMVAMUDIJOLI Vras rA%JlrLNOUInTWThW Th-Pos139 Th-Posl40 ONE POINT MUTATION: DRAMATIC CONFORMATIONAL CHANGES STABILITY CURVES OF LACTATE DEHYDROGENASES FROM COW, PIG, OF THE RIBONUCLEASE P2 FROM Sulfolobus Sofataricus UNDER CHICKEN, AND BACILLUSST'EAROTHERAJOPHILUS. ((Edward Cook, PRESSURE AND TEMPERATURE EVIDENCED BY TIME RESOLVED Mrudula Penta and B. Mark Britt)) Dqeament of Chemistry, Baylor University, FLUORESCENCE SPECTROSCOPY STUDIES POBox 97348, Waco, TX 76798. Stability curves - plots ofthe fiee energy of deatuain (AG&) vs. T - have been ((IP. Tauc, tP. Tortora, aiR. Lange and J.C. Brochon)) tUniversit&. Paris- obtained for lactate dehydrogenases firom several or s for the purpose of Sud, Bat 433, 91405 Orsay cedex, France. tUniversita degli di Milano, via determnning the relatioship between the d ional temperature (..), the opfinml Celoria 26, 20133 Milano, Italy. VINSERM U128, BP5051, 34033 Montpeliier, temperatre ofthe organiml source ofthe enzyme (T,,), and the teperatne of France. maximum stability ofthe enzyme (rmA5.. StabiLity curves were obtaied fron results ofheat and cold da inal experiments as monitored by absorbance spectroopy Pressurized were excited with a laser and and from fluorescence ttration by GuHCI at 24°C. The resulig stabilty curves samples Ti-sapphire sub-picosecond the Tq is fluorescence were in terms of distribution and display following features: 1) TMAX Tp,; 2) positioned roughyhalfway polarized decays analyzed lifetimne between T, and TmAx. This finding is significant as it challenges the assumpt in rotational correlation time distribution by the quantified Maximnum Entropy modem protin folding prediction studies that the functional enzyme exists as the Method. This small single tryptophan containing protein (7Kd) always displays most stable enzyme. Instead, the physiological enzymes appear to have achieved a heterogeneous fluorescence kinetics corresponding up to four lifetimes classes. compromise between complete conformational fluidity (the denatured state) and The rotational dynamics indicate rather globular rigid molecules at 200C. The maximum stability which is achieved at a temperature consisttly well belowthe wild type, even at low pH, is stable up to 90°C, whereas the mutant F3 IA is physiological temperature. This tertiary structure characteristic is expected to be a destabilized around 650C. Similarly the pressure effect is much less detectable in manifestation ofeach enzyme's prmary structure and it is shown that the percent the WT compared to the mutant. In addition, measurements at low temperature, relative sequence similarity ofthe enzymes vanes linearlywith Tp,,. We interpret without cryosolvant, has been performed using pressure. The differences these results as a requirement ofthe fiinctional enzymeto possess a metastable observed in response to extremes of temperature, pressure and pH may be conformation at the temperature at which it has evolved to fimction. rationalized by an unfolding mechanism involving larger parts of the peripheral protein while the integrity ofthe hydrophobic core is maintained.

Th-Posl41 Th-Posl42 X-RAY SCATTERING REVEALS PERTURBATIONS IN SOLVENT Molecular Basis for Counterion Dependence in 2D Streptavidin STRUCTURE AT HIGH CONCENTRATIONS OF GLYCEROL AND Crystals ((Todd C. Edwards. Sandy Koppenol. Wkolfgang Frey. WN'illiam R. Schief UREA ((W. Chen, R. M. Glaeser.)) Dept. Molecular and Cell Biology, Jr., '7iola X`ogel. Ronald E. Stenkamp*, and Patrick S. Stayton.)) Department of Stanley/Donner ASU, University of California, Berkeley, CA 94720 Bioengineering. University of W"ashington, Seattle WKA 98195-7962 *Department of Biological Structure and Biomolecular Structure Center. University of WNashington. X-ray scattering measurements have been used to probe the changes in water struc- Seattle WA 98195-7742 ture that occur in aqueous solutions of glycerol and of urea, at high concentration. The broad diffraction maximum that occurs in the scattering intensity of pure water The two dimensional (2D) crystallization of streptavidin at functionalized lipid at a resolution of about 0.31 nm, which is characteristic of the structure of liquid interfaces is one of the best studied model systems for investigating molecular self- water, changes progressively as the solute concentration increases. The most con- assembly processes of proteins. This system can also be used to elucidate the role spicuous change is a shift of the peak of the diffraction maximum to smaller angle. of protein-protein molecular recognition in crystallization properties such as space The extent of the shift is proportional to solute concentration for both solutes, but group symmetry, morphology, and solution conditions. Here we show that a single the magnitude of the shift for glycerol is more than twice that for urea. The shift of electrostatic interaction at the streptavidin 2D crystal contact is responsible for the scattering intensity to smaller angles implies that the structural organization of the counterion dependence of crystallization and is a key determinant of the kinetic bar- liquid expands, presumably because the hydrogen bonding configurations become riers controlling macroscopic crystal morphology. Molecular modeling suggests that increasingly idealized as the solute concentration increases. The fact that both glyc- the side-chain amines of Iysine 132 interact with each other across a C222 symmetry erol and urea are, in this sense, "structure makers" does not, however, imply that the related contact in the wild-type crystal, and leucine is substituted at this position liquid structures formed in association with the two solutes are necessarily similar. to remove the need for the salt-bridge contact. Brewster angle microscopy is used to A deeper understanding of these changes in water structure may be made possible image 2D crystals of wild-type and K132L streptavidin formed beneath biotinylated by comparing Molecular Dynamics simulations and experimental measurements of lipid monolayers on NaCl containing buffer, however, unlike wild-type streptavidin, the perturbation of the water ring, an approach that has recently been successful in the K132L mutant will also crystallize on a pure water subphase. Electron diffrac- analyzing the hydration structure around individual amino acid side chain residues tion analysis demonstrates that the crystal retains C222 symmetry in the presence (T. Head-Gordon et al. 1997 Biophys. J., 73: 2106-2115). or absence of counterions. The mutant crystal also displays a rectangular crystal morphology while retaining the C222 contact, suggesting that the kinetic barriers associated with formation of this electrostatic interaction underlie the macroscopic crystal morphology. These results may aid in the design of rational strategies for promoting and controlling protein crystallization and assembly at interfaces.

Th-Pos143 Th-Posl44 FLUORESCENT MICROSCOPIC IMAGING OF ANTIFREEZE EFFECT OF GLUTATHIONE ON NEAR-SIGHTEDNESS DUE TO NUCLEAR PROTEIN ADSORPTION TO ICE. ((Zhi-Wu Yu. WNeiming Yu'. Arthur L. CATARACT. DeVries. Chi-Hing C. Cheng.)) Physiology and LFD/Physics. University of ((T. T. Wu)) Northwestern Univ., Evanston, IL 60208. Illinois at Urbana-Champaign. Urbana. IL 61801. The author's nuclear cataract and associated near-sight have been monitored by Cheryl Zarat, MD since 1983. The Antifreeze glycoproteins (AFGPs) or peptides (AFPs) from polar fishes prevent level of glutathione in nuclear cataract lenses is about organismal freezing by adsorbing to ice crystals they carry and inhibiting ice growth. 10% of the normal level. Since glutathione is available concentrations of AFs the of ice over the counter, the author started taking 500 mg of it Physiological depress m.p. colligatively (-0.01°C) daily. The time course of his near-sightedaness is shown but lower the temperature of macroscopic ice growth to -1.2°C. a phenomenon knowvn in the following graph. Upper and lower values indicate as thermal hysteresis. Different AF adsorbs to different ice crystal plane. but none astigmatism. The reduction in near-sightedness of the adsorbs to the basal and thus limited C-direction occurs within the left eye is unexpected. This single case report should plane. growth be confirmed by large scale studies. hysteresis gap. This results in hexagonal bipyramidal ice crystals in the case of the AFPs and the short AFGPs (4-5 repeats of a glycotripeptide monomer), and 0 RIGHT EYE hexagonal pits on the basal plane in the case of the long AFGPs (>10 monomeric LEFT EYE repeats). These pyramidal and pit structures have thus far been observed with 2-D I light microscopy. In this study. we employ confocal and two-photon fluorescence C -10- microscopy and fluorescently-labeled AFGPs to obtain high resolution 3-D images to to discern the ice try crystallographic details of AF adsorption. 0 o -20 .t S

-30 83 85 87 89 91 93 95 98 YEAR IMINInivulM1;RRD A.N.QTDRITVTFr, a I xu4- I uTlKZ A.311AI71 Th-Posl45 Th-Posl46 STRUCTURE AND DYNAMICS OF THE DOCOSAHEXAENOIC EFFECT OF PROPOFOL, A GENERAL ANESTHETIC, ON THE ACID CHAIN IN BILAYERS STUDIED BY NMR AND X-RAY LAMELLAR PHASE DOMAIN STRUCTURES OF MODEL DIFFRACTION. ((L.L.Holte', B.W.Koenig', H.H.Strey2, and K.Gawrisch'.)) MEMBRANES ((S.V. Balasubramanian and R.M. Straubinger.)) Department 'Laboratory of Membrane Biochemistry and Biophysics, NIAAA; 2Laboratory of of Pharmaceutics, SUNY at Buffalo, Amherst NY, 14260 Structural Biology, DCRT; NIH, Rockville, MD 20852 The molecular site of anesthetic action still remains ais open question. It is not clear We ask if the critical dependence of neural membranes on docosahexaenoic acid, whether anesthetics bind to specific target proteins directly, thereby altering bio- DHA or 22:6n3, is at least partially due to properties of the membrane imparted by logical function, or whether activity is the indirect consequence of anesthetic effect lipids with DHA chains. The rigidity of the six cis C=C segments of DHA as well elsewhere, such as the perturbation of membrane properties. We investigated the as some structures proposed by molecular modeling techniques have contributed to effect of propofol, an intravenous general anesthetic, on the lamellar phase domain a notion that the DHA chain is inflexible. This is at variance with our studies of structures of dipalmitoylphosphatidylcholine (DPPC) membranes using Laurdan (6- membrane lateral compressibility, which indicate an exceptionally high deformabil- dodecanoyl-2-dimethylamino naphthalene) fluorescence. The fluorescence spectral ity of DHA chains when under lateral tension. We have obtained direct experimental properties of Laurdan are sensitive to lamellar phase domain structures and can be data on conformation and/or mobility of the DHA chain in multilamellar vesicles of utilized to quantify the lamellar phases. Propofol was found to interact with lamel- (18:0)(22:6n3)PC via MAS BH- and "3C NMR. Carbon-proton NMR order param- lar phase domains of model membranes, increasing the relative fraction of the liquid eters have been assigned for many resonances in the docosahexaenoic acid chain in crystalline phase. An analysis of the excitation spectra of Laurdan-labeled liposomes lipid bilayers. We have also measured hydrocarbon chain length and area for mem- indicated that propofol perturbs the water concentration in the head group region of branes containing one or two DHA chains by x-rays. With this information, we now the bilayer. An interfacial location of propofol in the bilayer is further supported by have some constraints by which to examine conformations for DHA proposed by differential scanning calorimetric (DSC) studies, in which propofol lowers the highly molecular modeling studies to determine their correlation with experimental data. cooperative phase transition temperature of the bilayer. The present study suggests that potent effects on membrane lamellar domains can be produced by a general anesthetic, and this phenomenon merits investigation for its role in anesthesia.

Th-Posl47 Th-Posl48 EFFECT OF ETHANOL ON THE THERMOTROPIC PHASE BEHAVIOR OF INTERACTION OF LOCAL ANESTHETICS WITH PHOSPHOLIPIDS IN FULLY HYDRATED MIXED-CHAIN PHOSPHATIDYLGLYCEROLS LANGMUIR MONOLAYERS ((Suzanne Amador, Samuel D. Floyd, and ((Ramesh V. Durvasula and Ching-hsien Huang)) Department of Biochemistry, Madison A. Compton)) Physics Department, Haverford College, Haverford, PA School of Medicine, University of Virginia, Charlottesville, VA 22908. 19041. Previous studies in this lab have delineated the relationship between the acyl chain Epifluorescence microscopy was used to study the interactions of local anesthetics asymmetry of mixed-chain phosphatidylcholines and the effect of ethanol concentration tetracaine and dibucaine with Langmuir monolayers of the phospholipid dipalmi- on their melting behavior (Li et. al, Biophys J, 70:2784-94, 1996). This study extends toylphosphatidylcholine (DPPC). The results show that incorporation of either these findings to another phospholipid family by using differential scanning calorimetry local anesthetic causes significant changes in the ordering of DPPC in the liquid to characterize the effect of ethanol concentration on the main transition phase condensed as evidenced the of two isomers of mixed-chain (LC) phase, by very different growth behavior of LC temperature (Tm) positional phosphatidylglycerol (PG). domains in For a linear decrease in the was measured as the ethanol monolayers containing local anesthetics compared to DPPC-only con- C(18):C(14)PG, Tm trols. concentration was increased up to 120mg/ml; this effect is characteristic of highly At low pH, the major differences were very different faceting and signifi- asymmetric phospholipids. In contrast, a biphasic profile in the plot of Tm versus cantly less compact LC domain growth. To probe whether local anesthetics influ- [EtOH] was observed for C(14):C(18)PG. Specifically, when [EtOH]<34mg/ml, a ence the phospholipid ordering via electrostatic interactions with the headgroups, linear decrease was observed for C(14):C(18)PG; at [EtOH]>34mg/ml, the Tm of the we obtained results for pH values well above and below the pKa of the local an- lipid dispersion increased, indicating that the lipid bilayer went through an isothermal esthetics. For high pH, at which both tetracaine and dibucaine are electrically phase transition from the gel phase into the Lp1 phase at T

Th-Posl49 Th-Posl50 EFFECT OF LIPID ACYL CHAIN LENGTH AND TOWARDS UNDERSTANDING THE ELECTROSTATIC FREE ENERGY DEGREE OF UNSATURATION ON LATERAL DETERMINANTS OF LATERAL DOMAIN FORMATION IN MEMBRANES ((D. Murrayl 2, N. Ben-Tal3, D. Petrey2, B. Honig2, S. McLaughlin)) 'Dept. of Physiology and COMPRESSIBILITY OF MEMBRANES Biophysics, SUNY Stony Brook, Stony Brook, NY 11794; 2Dept. of Biochemistry and ((B.W. Koenig', RH. Strey#, and K. Gawrisch')) NIAAA, #NICHD, NIH Molecular Biophysics, Columbia University, New, York, NY 10032; 3Dept. of Bethesda, MD 20892 Biochemistry, Tel Aviv University,Ramat Aviv 69978 Israel. Changes of cross-sectional area per lipid molecule in multilamellar vesicles as The basic effector region of the myristoylated alanine-rich C kinase substrate, MARCKS(151-175), forms lateral domains when it binds to phospholipid vesicles a fimction of osmotic stress have been determined to calculate the isothermal containing the acidic lipid phosphatidylserine (PS) and the zwitterionic, electrically neutral lateral compressibility modulus, K,, of the lipid bilayer in the liquid crystalline lipid phosphatidylcholine (PC) (Glaser et al. 1996. J. Biol. Chem. 271:26187). These phase {Koenig et al. (1997) Biophys. J. 73, 1954-66). X-ray diffraction and 2H domains are enriched in PS and membrane-associated MARCKS(151-175). Pentalysine, a peptide that corresponds to the first 5 residues of MARCKS(151-175), also forms domains NMR order parameter measurements were applied to monitor the structural when it binds to PC:PS vesicles. Denisov et al. (Biophys. J. in press) present a model for response of lipid membranes to lateral stress. In mixed chain lipids 2H NMR is pentalysine domain formation based on Gouy-Chapman-Stem theory which assumes the on the and adsorbed are sensitive to the of charges lipids peptides smeared uniformly over the surface of the compressibility the deuterated acyl chain in the sn-I position, membrane. The qualitative agreement between their model and experimental results while x-ray diffraction data reflect the lateral compressibility of the entire suggests electrostatics plays an important role in the formation of lateral domains by basic membrane unit cell. The effect of the acyl chain length and of the degree of peptides. We are developing a more realistic model for lateral domain formation based on the application of the Poisson-Boltzmann equation to atomic models of phospholipid unsaturation on structure and lateral compressibility of phosphatidylcholine membranes and basic peptides. In the first stage of this model, we calculated the bilayers was systematically studied in two series of experiments. Additional electrostatic free energy required to concentrate PS into a lateral domain. Atomic models experiments were performed on phosphatidylethanolamine bilayers to test the of phospholipid membranes with nonuniform distributions of PS and PC lipids were constructed using a simple Monte Carlo-like sampling procedure and were included in the influence of the polar headgroup on lateral compressibility. Poisson-Boltzmann calculations. Our results are similar to the smeared charge, analytical result. We present this work and its extension to membranes containing multivalent acidic lipids and membranes with adsorbed basic peptides. A372 MEMBRANE STRUCTURE Th-Posl5l Th-PoslS2 MOVEMENT OF MAGNETIC PARTICLES ON CELL Docosahexaenoic acid (DHA) alters the organization of cholesterol-rich MEMBRANES INDUCED BY MAGNETIC FIELDS ((Christian domains on the surface of murine leukemia cells ((E. Eugene Williams, Dietrich, Bing Yang and Ken Jacobson.)) Dept. of Cell Biology and Anatomy, Laura J. Jenski, and William Stillwell.)) Department of Biology, Indiana University of North Carolina, Chapel, NC 27599 University / Purdue University at Indianapolis, Indianapolis, IN 46202

Antibody coated magnetic beads, 2.8 microns in diameter, were specifically bound The shedding, or exfoliation, of small (50-200 nm) membranous vesicles from the to membrane compounds of muscle cells and fibroblasts. Applying digital image surface of normal and transformed cells is a naturally occurring and continuous processing, we studied the effect of an external magnetic field on the movement of process. Though it seems likely that these vesicles arise from distinct, non-random these beads at the cell membrane. The cell preparation is placed between two coils regions of the plasma membrane, this has never been proven. The vesicles exfoli- with iron cores. The setup is fairly similar to an instrument previously published ated from a murine leukemia cell line (T27A) have the same fatty acid profile as but is tuned to a force field and allows the plasma membrane from which they originate, but have a lower ratio of phos- (') produce homogenous high magnification phatidylethanolamine to phosphatidylcholine (PE/PC), higher molecular and Maximal forces are in the of few and order, imaging (100x/1.3 oil). typically range pN, are much richer in cholesterol. Culturing the cells in medium enriched with DHA can easily be varied by the current through the coils. Beads were attached to lipid molecules and in the results in a 10-fold increase in the DHA content of the phospholipids in both the proteins plasma membrane. A high percentage (60%) stick plasma membrane and the exfoliated vesicles, indicating that the exfoliation of DHA to the cell surface and forces exerted with this induced no movement. tightly setup is proportional to its abundance in the plasma membrane. In both the plasma mem- The rest of the particles, which showed Brownian movement, responded to the brane and exfoliated vesicles, the total amount of phospholipid fatty acid remains magnetic field. Only in a few examples, displacements were unrestricted as expected constant when DHA is added; DHA displaces other unsaturated fatty acids while the in magnetophoresis. In the majority of observations, beads stalled after few microns, proportion of saturates remains unchanged. Exfoliated vesicles from DHA-rich cells and moved back to the region of their starting point, after the field was turned off. display a fatty acid profile different from that of the plasma membrane. They also Experiments suggest that the bead encounters barriers or is tethered by components have a PE/PC ratio and molecular order equal to those of the plasma membrane, in the extra cellular matrix. li) F. Ziemann, J. Radler, and E. Sackmann. 1994. and have greatly reduced levels of cholesterol, all indicating that DHA dramatically Local measurements of viscoelastic moduli of entangled actin. Biophys. J. 66:2210- changes the tendency of different lipids to be shed in vesicles. The transbilayer 2216. distribution of primary amine-containing phospholipids was assessed biochemically and the size distribution of vesicles formed in the absence and presence of exoge- nous DHA was determined using electron microscopy and gel chromatography. The combined data suggest that DHA alters the organization of cholesterol-rich domains on the surface of these cells.

Th-PoslS3 Th-Posl54 WATER DISTRIBUTION IN PHOSPHOLIPID MEMBRANES - A PLASMA DESORPTION MASS SPECTROMETRY OF LIPIDS NEUTRON DIFFRACTION STUDY ((T. Gutberlet. G. Klose, T. Haul3.)) ((Adam MI. Slickmani and David G. Rhodes2.)) 1. Department of Chemistry. Inst. f. Exp. Physik I, University of Leipzig, Linnestr. 5. 04103 Leipzig, Germany Trinity College. Hartford, CT 06106 2. Department of Pharmaceutical Sciences. BENSC, HMI, Glienicker Str. 100, 14109 Berlin, Germany Universitv of Connecticut. Storrs. CT 06269

The phase behaviour of phospholipids stongly depends on the water content of Plasma desorption mass spectrometry (PDMS) is a time of flight (TOF) method in the system. The biological relevant fluid phase of phospholipid membranes re- which the analyte is desorbed by ionizing radiation from a 252Cf source. PDMIS has quires a minimum amount of water present. hydrating the hvdrophilic portion of the been extensively used for peptide analysis. but there are no reports of PDMIS for lipid molecules and separating attached bilayers. The distribution of the water molecules analysis. A simple. rapid means for quantitative determination of lipid composition within the hydrophilic parts of phospholipid membranes and the mobility of is of would be of great value for biomimetic studies of membrane permeability. partition- certain interest to understand the structure and dynamic behaviour of phospho- ing, or structure. WVe find that for most lipids the expected molecular ion peak (mi) lipd membranes as general model of biological membranes. Molecular modelling, is evident and that some fragmentation also occurs. In many cases hoswever. one or neutron diffraction and NMR experiments have been used to describe the complex more additional peaks are obtained at m < m0 and in some cases. apparent aggre- interactions present between phospholipid and water molecules. In the study to be gate or polymer peaks are obtained at m > in. These artifacts are not obtained for presented the internal structure of DMPC bilayers hydrated at 100% relative hu- peptides. but are evident in hydrophobic samples such as cholesterol and alkanes. midity in the lamellar gel and fluid phase and DMPC bilavers modified by non-ionic The anomalous peaks also depend on the disposition of the sample on the substrate. surfactant C12E2 are investigated using neutron diffraction. Of the obtained diffrac- as evidenced by experiments with samples applied by Langmuir-Schaeffer deposi- tion patterns highly resolved scattering length density profiles have been obtained tion or other techniques. It appears that the desorption kinetics may contribute to and the position of the water molecules in the different model systems could be the overall TOF in such a way that hydrophobic analytes appear to travel faster localized. The results are discussed with respect to the water distribution in phos- than hvdrophilic analvtes. This work suported by Pfizer, Inc. (DGR). AXIS was the pholipid membranes, their effect on the dvnamic of the exchange of water molecules Pfizer Summer Research Fellow in Pharmaceutics at L'Conn. between membrane bound and free water and the forces interacting betwveen fluid phospholipid membranes.

Th-Posl55 Th-Posl56 IMAGING FRET DETECTS CLUSTERING OF GANGLIOSIDE GM, DEPOCYTTM, A LIPOSOMAL ANTICANCER FORMULATION, MOLECULES WITH ONE ANOTHER, BUT NOT WITH A UNDERGOES A BILAYER TO NON-BILAYER TRANSITION NEAR GPI-ANCHORED PROTEIN, 5' NT, ON THE APICAL SURFACE ROOM TEMPERATURE. ((J.F. Ellenas, R.Solis2, D.S. Cafisol, and M.B. OF MDCK CELLS. ((A. K. Kenworthy and M. Edidin.)) Department of Sankaram2.)) 'Chemistry Dept. and Biophysics Program, Universitv of Virginia, Biology, Johns Hopkins Baltimore, MD 21218. Charlottesville, VA 22903; 2DepoTech Corp., 10450 Science Center Dr., San Diego, CA UJniversity, 92121. Membrane microdomains ("lipid rafts") enriched in glycosylphosphatidylinositol DepoCytTM is a sustained-release formulation of DepoFoamTM-encapsulated cytarabine (GPI)-anchored proteins and glycosphingolipids (GSL) have been implicated in the used for treatment of neoplastic meningitis. DepoFoam is a liposomal drug delivery system polarized sorting of these molecules and in cell signaling events. However, the size typically containing DOPC, DPPG, triolein. and cholesterol in 0.9% (w/v) saline Depo- and composition of lipid rafts in intact cell membranes are not known. To probe Foam consists of large (ca. 10-20 pm) spherical vesicles with many internal, nonconcentric intact cells for rafts enriched in GPI anchored proteins and GSL, we used imag- chambers. We have examined the temperature dependence of 3ip NMR spectra of De- ing fluorescence resonance energy transfer (imaging FRET), a method that detects poCyt and related preparations having different phospholipid acyl chain content. We have molecular at of <100 A. Our markers for rafts at the also prepared variants of DepoCyt containing deuterated phospholipid acyl chains and have proximities lengthscales apical examined the of 2H NMR of these The membrane of cells were the temperature dependence spectra preparations. 31p polarized MDCK GPI-anchored protein 5' nucleotidase of at 3 C is a and cholera toxin spectrum DepoCyt 15 partially averaged anisotropic 31p powder pattern typi- (5' NT) B-subunit (CTX), which binds to ganglioside GM,. We cally seen for bilayers with a low degree of curvature. When the temperature is raised to 25 first used imaging FRET to see if CTX is clustered at the apical surface of MDCK C, one sees the appearance of an isotropic spectral component in addition to the partially cells. Energy transfer was measured between donor and acceptor-labeled CTX, that averaged powder pattern component. The relative intensity of this component increases as did not scale simply with the acceptor surface density. This result is consistent with the temperature is raised to 85 °C. When the DepoCyt is then cooled to 25 °C, the spec- a clustered, rather than a random, distribution of CTX. We then tried to detect tral lineshape obtained is very similar to that obtained before heating. Light scattering and CTX in proximity to antibody-labeled 5' NT. However, no energy transfer could microscopy were used to examine the sample used for NMR before and after heating. These be detected between donor-labeled 5' NT and two techniques revealed no structural modifications induced by heating. The optical results acceptor-labeled CTX. This suggests the that is not along with temperature reversibility of the NMR spectra suggest that the isotropic 31p GMi preferentially associated with 5' NT. Thus though we find evidence NMR not to for component is due the formation of small bilayer vesicles. The isotropic 3ip GSL-enriched membrane microdomains on the apical surface of polarized MDCK NMR component is likely due to a nonlamellar phase. Additional data will be presented cells, these domains do not appear to be enriched in a GPI-anchored protein under and implications for the mechanism of drug delivery via DepoFoam will be discussed. steady state conditions. Supported by NIH grant 5POlDK44375 to M.E. fflLMnDRLIFLllqrlM.MRZD ANR u.TDTTITTTDRa A BLUL, A iulum PA.3Alll10 Th-Pos157 Th-PoslS8 THE PHASE BEHAVIOR OF HYDROXY AND NON-HYDROXY FATTY LOW PERMEABILITY OF LIPOSOMAL MEMBRANES COMPOSED OF ACID CERAMIDES BIPOLAR TETRAETHER LIPIDS FROM THERMOACIDOPHILIC ((David J. Moore*, Mark E. Rerek* and Richard Mendelsohn)) *ISP, ARCHAEBACTERIUM SULFOLOBUSACIDOCALDARIUS Skin R&D, Wayne, NJ 07470 and bRutgers University, Department of ((Hiroaki Komatsu and Parkson Lee-Gau Chong)) Dept. of Biochemistry, Temple Chemistry, Newark, NJ 07102 Univ. School ofMedicine, Philadelphia, PA 19140. ( Spon. by Jimmy H. Collins ) The physical origin of the extremely high thermal stability of tetraether liposomes The barrer function of skin resides in the lipid bilayers of the stratum composed of the polar lipid fraction E (PLFE) from the thermoacidophilic comeum (SC). The unusual properties of the stacked lipid bilayers of the archaebacterium Sulfolobus acidocaldarius has been investigated. The leakage rate SC are provided, in large part, by ceramides. The current study utilizes of trapped 5,6-carboxyfluorescein (5(6)CF) and the proton permeability in PLFE Fourier transform infrared (FTIR) spectroscopy to characterize headgroup liposomes have been measured using fluorescence probe techniques in the and chain inter- and intramolecular interactions in hydroxy fatty acid temperature range of 25-85 °C. The results are compared with those obtained from (HFA) and non-hydroxy fatty acid (NFA) ceramides, the major ceramide non-archaebsctera liposomes. Egg yolk phosphatidylglycerol (eggPG) and PLFE species in SC. At physiological temperatures NFA ceramide is highly liposomes exhibit similar large negative zeta potentials (-31 mV to -34 mV) and conformationally ordered and packed in an orthorhombic subcell low permeability coefficients for 5(6)CF, indicating that membrane surface charge At 60 NFA ceramide a is responsible for the low leakage rate of5(6)CF in PLFE lipososnes. This assertion structure. °C, undergoes phase transition to a is confirmed by the observation of an increased leakage rate of 5(6)CF with conformationally ordered hexagonal phase. This is followed by a decreasing membrane surface negative charge via varying the content of egg yolk transition to a conformationally disordered non-bilayer phase at 80 "C. In phosphatidylcholine (eggPC) in eggPC/eggPG binary mixtures. Gel state additon, the headgroup amide I and 11 modes of NFA ceramide are each dipalmitoylphosphafidylcholine bilayers and PLFE liposomes exhibit similar split into two bands indicating strong intermolecular headgroup coupling permeability coefficients for 5(6)CF, suggesting that lipid packing also plays an between NFA ceramide molecules. In contrast, HFA ceramide undergoes important role in the low leakage rate of 5(6)CF. PLFE liposomes, especially those a single transition from a highly conformationally ordered bilayer phase to with -60 am in diameter, are remarkably thermal stable as regards proton a conformationally disordered phase at 76 *C. The headgroup amide I permeability, which increases by less than 2 x 10.10 cm/s from 25 to 82 °C. The and 11 modes of HFA ceramide are not split. The distinctive behavior of proton permeability comparison ofvarious liposomes reveals that the tight and rigid the HFA and NFA ceramides suggests different roles for each ceramide lipid packing is the major contributor of the extreme low proton permeation in in the structural of the SC barrier. PLFE liposomes; the inositol moiety and the branched methyl groups may also providing integrity lipid contribute, but to a much lesser extent. (Supported by NSF)

Th-Posl59 Th-Pos160 Membrane Thickness by X-ray Diffraction Using Dehydration STRUCTURAL STUDIES ON EQUIMOLAR SUSPENSIONS OF Method-Resolving the Question about Critical Swelling of Phospholipid PALMITIC ACID AND Bilayers ((H. W. Huang', F. Y. Chen2. W. C. Hung2.)) 'Physics Dept. Rice 1-LYSO-PALMITOYL-PHOSPHATIDYLCHOLINE ((Jesper Lemmich,1'2, Univ. Houston, TX 77005: 2Physics Dept. National Central University, Chung-Li. Frank Richter,3 Thomas H0nger Callisen,2, and Yeshayahu Talmon,4.)) Taiwan 'Condensed Matter Physics and Chemistry Department, Riso National Laboratory, DK-4000 Roskilde, Denmark, 2Department of Chemistry, The We reexamine the critical phenomenon of phospholipids in lamellar phases. The Technical University of Denmark, Building 206, DK-2800 Lyngby. Denmark, question is: Is the anomalous divergence in the repeat spacing near the main transi- 3EMBL Outstation at DESY. Building 25A, Notkestra3e 85, D-22603 Hamburg, tion the result of a divergence of the water layer or of the lipid bilayer? The key to Germany, 4Department of Chemical Engineering, Technion-Israel Institute of this problem is in the measurement of membrane thickness. Experiments measuring Technology, Haifa 32000, Israel changes of membrane thickness with various parameters are important to the studies of energetics of membranes and of membrane-protein interactions. The frequently An equimolar mixture of palmitic acid (PA) and 1-lvso-palmitoyl- used NMR method and gravimetric method have been critically reviewed by Nagle phosphatidylcholine (Lyso-PPC), corresponding to the hydrolysis products obtained recently. The dehydration method has been successfully applied to a number of from di-palmitoyl-phosphatidylcholine (DPPC), has been studied by small angle membrane-protein systems. Here we show that this method can even be used in and wide angle X-ray scattering (SAXS and WAXS, respectively) as well as cryo- the relatively difficult pretransitional regions of phospholipids. X-ray diffraction of transmission electron microscopy. The aim of the study is firstly to investigate the DLPC lamellae was measured in partially dehydrated conditions. Its critical be- rich phase behavior and the interesting slow kinetics, displayed by this mixture, havior is much more pronounced than the previously studied DMPC. The bilayer when it is temperature cycled between room temperature and 33 IC. We have in thickness was calculated from dehydrated conditions and then extrapolated to full connection with the X-ray experiments performed differential scanning calorimetry hydration. The results show that the anomalous divergence is primarily due to the (DSC) with similar thermal history for the same samples. We discuss our results in water layer expansion. relation to other experimental data, e.g. on structural and dynamical aspects of the phosholipase A2 (PLA2) hydrolysis time course.

Th-Posl61 Th-Posl62 MONTE CARLO SIMULATION OF TWO-COMPONENT GIANT LIPOSOMES WITH MULTIPLE MEMBRANE-LINKING BILAYERS: DMPC/DSPC MIXTURES ((I.P. Sugar+, T.E. Thompson*, PORES. ((K. Akashi, H. Miyata, H. Itoh, K. Kinosita, Jr.)) Department of K.K. Thompson*, and R.L. Biltonen'.)) +Departments of Biomathematical Physics, Faculty of Science and Technology, Keio University, Hiyoshi, Yokohama Sciences and Physiology/Biophysics, Mount Sinai School of Medicine, New York, 223, Japan; Physics Department, Graduate School, Tohoku University, Sendai NY 10029 and *Department of Biochemistry, University of Virginia School of 980-77, Japan; and CREST(Core Res. for Evolutional Sci. Tech), Nogawa, Medicine, Charlottesville, VA 22908 Kawasaki 216, Japan. In this paper we describe a simple two-state model of a two-component phos- Some cell organelles have membrane-linking pores (wormhole-like structure con- pholipid bilayer system. Application of Monte Carlo methods to this model per- sisting of lipid membranes). A nucleus, for example, has inner and outer membranes mits simulation of the observed excess heat capacity versus temperature curves which are interconnected with many such pores(nuclear pores). In this study, we of dimyristoylphophatidylcholine (DMPC)/ distearoylphosphatidylcholine (DSPC) found that lipid membranes, without assistance of proteins, can form cell-size en- mixtures as well as the lateral distributions of the components and properties re- velopes (giant liposomes) having membrane-linking pores. The envelopes together lated to these distributions. A comparison of the thermodynamic properties of with normal giant liposomes were prepared by simple hydration of phospholipids DMPC/DSPC mixtures with the configurational properties shows that the temper- and observed under a phase-contrast microscope or under a confocal microscope atures characteristic of the configurational properties correlate well with the maxima with fluorescent staining of the membranes. The diameters of the pores were sev- in the excess heat capacity curves rather than with the onset and completion tem- eral microns. The shapes of these envelopes are categorized as follows: a sliced peratures of the solid/fluid phase transition. In the two-phase coexistence region we lotus-root type which is a disk-like(flat) liposome having many pores(often more also found excellent agreement between the percolation threshold temperatures at than 10) connecting the opposite sides of the disk; a pretzel type where several different system compositions detected in Fluorescence Recovery after Photobleach- tube-like envelopes are interconnected with one another; a nucleus type which had ing (FRAP) experiments and the midpoint temperatures of the percolation of gel inner and outer membranes interconnected with multiple pores. The structures de- clusters calculated at the same compositions. (Supported by grants from the NIH scribed above were stable unless flow or evaporation in the surrounding medium and NSF.) occurred. rs-PA374I IV lvaivIVKJLPA%rluMEMBRANE14 iv, 17STRUCTUREJL Z% ti %., a lu A%JVI

Th-Posl63 Th-Posl64 ARE GIANTS UNILAMELLAR PHOSPHOLIPID VESICLES LYOTROPIC AND THERMOTROPIC PHASE BEHAVIOR OF REALLY UNILAMELLAR? A TWO PHOTON MICROSCOPY HYDRATED MONOACYLGLYCEROLS: STRUCTURE STUDY. ((E. Gratton, T. Plarasassi, W. Yui, and L. A. Bagatolli.)) LFD/Physics, CHARACTERIZATION OF MONOVACCENIN. ((H. Qiu and M. UIUC, 1110 WV. Green, Urbana, IL 61801-3080. Caffrey.)) Department of Chemistry, The Ohio State University, Columbus, OH 43210. Unilamellar liposomes are used for several purposes such as transport studies and drug deliserv. We want to utilize such liposomes composed of a single bilayer for the This work is part of a long-term research effort to establish the relationship between study of domain coexistence and for the estimation of the domain size. A variety lipid molecular structure and lyotropic and thermotropic mesophase propensity by of methods for their preparation are described in the literature. We used several comparing the equilibrium temperature-composition phase diagrams of a homolo- of those methods and we tused fluorescence microscopy and electron microscopy to gous series of monoacylglycerols. Here we report the temperature-composition phase assess the number of lamellae present in the vesicles following each preparation. diagram of monovaccenin (a C18:1cll monoacylglycerol) in water, constrlucted us- Using somiie of the methods reported in the literature, we were able to obtain only ing small-angle x-ray scattering in the temperature range of ca. 0 °C to 110 °C and a snmall percent of unilamellar vesicles in a population mainly composed of multil- the composition range of ca. 0% to 60%(w/w) water in the heating direction. The amellar vesicles. By using two-photon microscopy to image the vesicles labeled with interpreted equilibrium phase diagram is based on several hundred discrete x-ray 2-dimethylamnino-6-lauroylnaphthalene (LAURDAN), we observed that the vesicle diffraction measurements in temperature-composition space recorded as a function lamellar strtucture also depends upon the phase state of the lipids. In some cases, we of temperature in 5 °C increments and of composition in 4%(w/w) water increments were able to assess the ntumber of lamellae present in each vesicle in a preparation on average. The phases identified and characterized structurally in this system in- based oni the quantization of the intensity. By a comparison with red blood cell clude the lamellar crystaBline (Lc) phase, the lamellar liquid crystalline (Lo phase, membrane intensity, labeled with LAURDAN together with the vesicles, we could two inverted cubic phases (Q230, Ia3d; Q224, Pn3m), the inverted hexagonal (HII) estimate which vesicle, or which portion of them, may be composed of a single bi- phase, and the fluid isotropic (FI) phase. The monovaccenin/water phase diagram laver. WVe must point out that using the sectioning capability of the two-photon is very similar to that of the monoolein (a C18:1c9 monoacylglycerol)/water sys- excitationi nicroscopy to image LAURDAN labeled vesicles, we obtain an impres- tem. However, there are important differences in transition temperatures and phase sively clear representationi of the vesicle complex internal structure. Supported by boundary positions between the two that we attribute to differences in the effective the National Institutes of Health (RR03155). length and shape of the corresponding amphiphiles. The sensitive response to tem- perature and lipid identity of the average water channel radius in the fully hydrated, bicontinuous cubic Pn3m phase makes monoacylglycerol systems important poten- tial candidates for controlled drug release and merribrane protein crystallization.

Th-Posl65 Th-Posl66 GIXD FROM PURPLE MEMBRANE AT THE AIR/WATER THE TRIOLEIN IN DEPOCYTTM IS COMPLETELY OR ALMOST INTERFACE ((S.A.W. V,erclas,1'5 P.B. Howes,2 K. Kjaer.2 A. Frenzeni NI. COMPLETELY EXCLUDED FROM BILAYERS. ((J.F. Ellenat, Weygand,3 G. Biildt.4 NA.. Dencher5 and NM. Losche3.)) iBENSC R.Solis2, D.S. Cafisot. and MI.B. Sankaram2.)) 'Chemistry Dept. and Biophysics (NE/Biophvsics), HMNI Berlin, D: 2Physics, Riso National Laboratory. DK: Program, University of Virginia, Charlottesville, VA 22903: 2DepoTech Corp.. 3Physics, UIniversitv of Leipzig, D: 4IBI-2, Research Center Jiilich, D: 10450 Science Center Dr., San Diego, CA 92121. s1Biochemistry, TU Darmstadt. D DepoCytTM is a sustained-release formulation of DepoFoamTMl-encapsulated cy- X-ray diffraction from a single layer of purple membranes at the air/water inter- tarabine used for treatment of neoplastic meningitis. DepoFoam is a liposomal face in a Langmuir trough has been measured. Grazing incidence X-ray diffraction drug delivery system typically containing DOPC. DPPG, triolein. and cholesterol (GIXD) is demonstrated to be a powerful method to obtain structural information on in 0.9% (w/v) saline. DepoFoam consists of large (ca. 10-20 pm) spherical vesicles membrane proteins under physiological conditions. The method is so sensitive that with many internal. nonconcentric chambers. Small. Hamilton and collaborators diffraction can be measured from samples with only 1013 protein molecules in the have shown that i3C NMR can be used to assess quantitatively the amount of beam. The projected structure of the transmembrane protein bacteriorhodopsin in triglyceride residing in bilayer regions of lipid dispersions. W'e have prepared a De- purple membrane patches spread on the air/water interface has been reconstructed poCyt sample containing "C-carbonvl labeled triolein and performed quantitative using phases obtained from electron microscopy. Diffraction was observed up to i3C NMR experiments. No spectral component attributable to triolein incorporated the order h.k = 4.3 of a hexagonal lattice with a lattice constant2E(a)a= (b)a= in bilayers was observed. Spectral components attributable to triolein in a liquid- 60.6 A, corresponding to a resolution of 9 A. Compared with conven- like phase were observed and the intensity of these components accounted for all of tional X-ray diffraction on multilayer samples, the purple membrane at the triolein present in the sample within experimental error. Implications of these the air/water interface shows a reduction in the unit cell size from 62.5 A results for the mechanism of DepoCyt action will be discussed as will details of our to 60.6 A, independent of the surface pressure. A lattice constant of 57.5 quantitative analysis and uncertainties involved. A was observed for partially delipidated purple membrane, which is close to values observed with conventional methods. The work reported here is a first step toward 2D protein crystallography using grazing incidence X-ray diffraction at the air/water interface.

Th-Posl67 Th-Posl68 NEWS FROM THE EDGE OF MAMMALIAN BIOLOGY: THE BREAKDOWN OF CELL MEMBRANES BY ELECTRICAL AND PROGRESS IN UNDERSTANDING THE SKIN BARRIER MECHANICAL STRESS. ((J. Akinlaja* and F. Sachs)) Physics* and MEMBRANE ((J.L. Thewalt. D. Langlais. X. Chen. N. Kitson.)) Dept. of Physics. SFU. Burnabv. B.C. VX5A 1S6. Dept. of Phvsics., UBC. Xancouver. B.C. Biophysical Sciences, SUNY, Buffalo, N.Y. (Spon. by G. Willsky) V6T lZl. Div. of Dermatology. UBC. 835 XVr 10th Ave.. Xancouver. B.C. V4Z 4E8. Using cell-attached patches from HEK293 cells, we studied whether The interior milieu exists in large part because of the skiti's permeabilitv barrier. mechanical tension and electrical stress couple to cause membrane rupture. an intercellular msembrane in the upper epidermis. Mlounting evidence points to Tension was estimated from the applied pressure and geometry of the patch. primarily crystalline lipid packing in these lamellae. which are composed of roughly Voltage pulses of increasing amplitude were applied until we observed a sud- equal mole fractions of ceramide. saturated fatty acid and cholesterol. The molecular den increase in conductance. For pulses of 50.s duration, breakdown required packing and composition within these regions of complex crystalline membrane is not > 0.5V and was dependent on the tension. For pulses of lOOms duration, yet knosn. Our solid state deuterium studies of model previous N-\IR membranes breakdown 0.2-0.4V and wasindependent of tension. Apparently, two designed to duplicate selected essential features of the permeability barrier. namely required small headgroup size and low pH. showed that the majority of each of the three physically different processes can lead to membrane breakdown. major lipid species is immobile at skin temperature. XVe here explore the effects of We could best explain the data by assuming that rupture in the high chain length and chain heterogeneitv of the fatty acid and ceramide lipid fractions voltage range was caused by electrical breakdown ofthe lipid bilayer, while in on the amount of crystalline membrane formed at body temperature as well as the low voltage range, it was caused by a different, tension independent, proc- the phase behaviour of the membranes as they are heated. Two features of this ess. For the short pulses, the critical electromechanical energy per unit area for system are of particular interest: (1) a large amount of cholesterol appears to be breakdown (a 4 dyne/cm) was in agreement with earlier results on bilayers. incorporated in these mernbranes in a solid state: (2) these membranes are good One ofthis model is that (at least in a patch) the bilayer holds a sig- candidates for formation of long-lived compositionallv distinct microdomains. prediction nificant amount (a 40 %) of the membrane tension. Breakdown voltages for long pulses were in agreement with earlier work on algae, but the mecha- nism(s) of breakdown remain unclear. Supported by MDA and NIH MEMBRANE STRUCTURE A375 Th-Posl69 Th-Posl70 CORRELATED SCANNING FORCE AND ELECTRON MICROSCOPY SURFACE PROPERTIES OF PULMONARY SURFACTANT SYSTEMS USING A OF BILAYER NANO-TUBULAR STRUCTURES OF PULMONARY CAPTIVE BUBBLE TENSIOMETER((F.Possmayer, J. Hutzal, K. Inchley, S. SURFACTANT. SchOrch and N. 0. Petersen)) University of Western Ontario, London, ON, ((K. Nag, G. Munro, S. H. Heam, Y. Rasmussen, N. 0 Petersen and F. CANADA, N6A 5A5 Possmayer)) Obstet. & Gyn.& Biochem. (MRC Group) and Chemistry, University ofWestern Ontario, London, Ontario, Canada N6A 5A5. The biophysical properties of dipalmitoylphosphatidylcholine:phosphatidylglycerol (DPPC:PG), (70:30); DPPC:PG:surfactant protein-B (DPPC:PG:SP-B) (70:30:01) and bovine lipid extract surfactant (BLES) were studied at 0.5, 1.0 and 1.5 mg/ml with a Tubular myelin (TM) is composed ofatypical bilayer lipid-protein tubes which captive bubble tensiometer. SP-B enhanced adsorption of DPPC:PG mixtures to are presumed to be the precursor ofsurface active films in the alveoli responsible equilibrium surface tension (approx. 22 mN/m). With DPPC:PG mixtures initial for pulmonary stability. Scanning force microscopy (SFM) and electron compressions of 60 to 80% were required to achieve surface tensions near zero and microscopy (EM) were performed on Lowacryl embedded sections ofTM. As subsequent compressions showed no improvement. With DPPC:PG:SP-B initial compressions of 30% (0.5 mg/ml) and 20-15% (1.0 and 1.5 mg/ml) sufficed to achieve previously observed, EM ofthe tubular myelin indicated that lipid bilayer square low surface tensions. This was followed by decreases to approx. 15% for all three tubes of40-45 nm diameter were organized in square lattices, and the center of samples by the 5th cycle. At 0.5mg/ml BLES samples (which contain 45% DPPC) such tubes probably contained surfactant proteins. The correlated SFM (in required 70% compression during the first cycle but only approx. 30% in subsequent tapping mode) ofsimilar unstained sections indicated that the surface topology cycles to achieve low tensions. At higher concentrations initial compressions of 40- ofTM consisted ofhemispherical bumps of5 nm height and separated by 50-60 30% were sufficient to achieve low tensions and final surface tensions of about 25 mN/m were achieved by the 5th compression. These results indicate SP-B (and nm spacing. These bumps were determined to be the central core region ofthe possibly SP-C) not only accelerate surfactant film formation but promote either the tubes by using phase and friction imaging modes of the SFM, although no selective DPPC adsorption or the formation of surface-stabilizing multilayers. protein structures in the core could be observed. EM of sections of TM with Furthermore selective adsorption and/or multilayer film formation is highly lipids removed indicated cross-hatching ofproteins spaced in rectangular tubes susceptible to alterations in bulk phase surfactant concentrations. and the spacing between the tubes was increased from the native form. These (Supported by a Group Grant from the MRC of Canada). studies suggest that correlated scanning force and electron microscopy can provide information on lipid-protein arrangements in atypical membranes. (Supported by MRC, Canada).

Th-Posl71 Th-Posl72 A MODEL SYSTEM FOR CELL ADHESION: INTERACTION OF FORCE SPECTROSCOPY ON ADHESIVE CELLS AND VESICLES. SUPPORTED MEMBRANES AND GIANT VESICLES FUNCTIO- ((A. Albersdorfer, R. Bruinsma*, and E. Sackmann)) TU-Munchen, NALIZED WITH THE RECEPTOR PROTEIN CONTACT SITE A (csA), Physik Department (E22), James-Franck-StraB3e, D-85747 Garching, EXPERIMENTS UNDER LATERAL SHEAR FLOW. *Department of Physics, University of California, Los Angeles, ((A. Behrisch, A. Kloboucek, R. Simson, G. Gerisch* and E. Califomia, (Spon. by E. Sackmann) Sackmann)) TU-Munchen, Physik Department (E22), James-Franck- Stral3e, D-85747 Garching, *Max-Planck-lnstitut fOr Biochemie, Am Abstract. - Cell adhesion is tuned by a complex interplay of specific Klopferspitz 18a, D-82152 Martinsried (Spon. by E. Sackmann) (lock and key) and universal forces leading to the formation of domains of tight adhesion (pinning centers). The contact site A glycoprotein is a developmentally regulated We study the local clustering of the receptors in model membrane homophilic cell adhesion molecule from Dictyostelium discoideum. A systems and propose a method to measure weak point forces exerted model system for cell adhesion based on specific receptor interaction is on cells or giant vesicles adhering to solid surfaces by sub micron established. The biological membrane is mimicked with phospholipids pinning centers under conditions of thermal equilibrium. The method and the glycocalyx with lipopolymers. Applying various techniques is based on the analysis of the shape of the contact line between (Film Balance, Micro Fluorescence, FRAP), the purified GPI-anchored vesicle and substrate using reflection interference contrast protein was reconstituted in solid supported bilayers and giant vesicles microscopy. Force levels below one pN could be measured, consisting of uncharged phospholipids and PEG-lipopolymers. considerably below that accessible to available force probes. We Interaction of the functionalized giant vesicles with the supported illustrate the procedure with a study of the adhesion of giant vesicles bilayer was observed with a Reflection Interference Contrast adhering to a supported membrane by biotin-streptavidin-biotin Microscope (RICM). Depending on the protein concentration, lateral linkages. phase separation leading to the formation of domains of tight adhesion (pinning centers) separated by areas of weak adhesion can be observed. The RICM-technique allows quantitative studies of surface profiles near the contact area. We estimated adhesion energies and by applying an extemal shear flow rupture forces of pinning centers.

CALCIUM SIGNALLING Th-Posl73 Th-Posl74 FOCAL AGONIST APPLICATION RESULTS IN SPATIALLY RESTRICTED CHARACTERIZATION OF CALCIUM RELEASE PATHWAYS AND THEIR Ca RELEASE AND CAPACITATIVE Ca ENTRY IN CULTURED RELEVANCE FOR CAPACITATIVE CALCIUM ENTRY IN VASCULAR VASCULAR ENDOTHELIAL CELLS ENDOTHELIAL CELLS ((J. Htser, J.R. Holda, J. KockskAmper# & L.A. Blatter)) ((Jaclyn R. Holda, Gregory A. Mignery, and Lothar A. Blatter)) Depl of Physbogy, Sritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA; #A.G. Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Muskelphysiologle, Ruhr-UniversiMt Bochum, 0-44780 Bochcwn, Germany (sponsored by K.L. Byron) Maywood, IL 60153, U.S.A. ATP stimulation of cultured vascular endothelial (CPAE) cells leads to Ca release from internal IP3-sensitive stores and subsequent Ca entry from the extracellular environment. The latter has Depletion of intracellular Ca2+ stores activates a capacitative Ca2+ entry channel to been shown to be triggered by Ca depletion of intracellular storage sites displaying the characteristics of capacitative Ca entry (CCE). The molecular identity and the activation promote refilling of these stores in endothelial as well as many other cell types. It has mechanism of CCE in endothelial cells has yet to be determined. We have studied the subcellular been suggested that the putative capacitative Ca2+ entry channel is the type-3 organization of agonist-induced Ca release and capactative Ca entry using confocal imaging of inositol tnsphosphate (IP3) receptor, based on reports of its localization to the plasma Fluo-3 loaded endothelial cells. Focal activation of purinergic receptors in Ca-free solution by membrane. To investigate this premise, the various isoforms of IP3 receptors were pressure application of ATP resulted in localized Ca release in the stimulated cell region. Ca identified and characterized in vascular endothelial cells derived from bovine release frequently propagated some ten microns into unstimulated regions but failed to extend pulmonary arteries (CPAE cells). Total RNA was extracted from these cells and was throughout the cell. Subsequent readdHion of Ca to the bathing solution caused typical Ca entry reverse transcribed producing single stranded cDNA. This product was used as template for polymerase chain reactions (PCR) with 'consensus' oligonucleotide - ATP - ATP (CCE) signals in those regions where Ca release had been observed. Previously non- primers which hybridize and amplify all IP3 receptor isoforms (i.e. type-1, 2, and 3). The excited regions either displayed no rise in PCR products were Southern blotted with type specific oligonucleotides to establish FIo [Ca], upon Ca readdition or a [Ca], rise of slow which isoforms of the IP3 receptor were present in CPAE cells. The predominant IP3 kinetics and lower amplitude. The latter were isoform was type-2, however, there was no type-3 IP3 receptor found in this cell type. 2 readily explained by intracellular Ca diffusion In addition, Westem blot analysis using a mouse monoclonal anti-ryanodine receptor from excied regions. Activation of CCE in the (RyR) antibody confirmed the presence of RyR type-1 and/or RyR type-2 in this stimulated region of the cell occurred without endothelial cell line, although their functional significance has yet to be determined. _ 2 _ _ complete depletion of the Ca stores. In In CPAE cells store depletion through the IP3 pathway, but not the RyR pathway, 1CS1 _ contrast, global superfusion with ATP leads to the activation of capacitative Ca2+ entry. Furthermore, the results show that 120 initiated release throughout the cell, with the type-3 IP3 receptor cannot be responsible for this influx as Southern blot analysis Foal of ATP readdition of Ca eliciting fast Ca entry (CCE) suggests its absence in this cell line. Taken the data do not applicati, (1 pM) reesi,n spatiallyrestritedm these together, support the CareleainthestimAatedP ce reglo (), b attaut signals in all regions monitored. From hypothesis that the IP3 receptor is the putative capacitative Ca2+ entry channel in region (# 2) aboiut50 dsant frotm theeofsitesmulaion. data it is concluded that activation of CCE by CPAE The CCE transbt upon readisitiofdeotacelutr Ca was aso Ca loss from intemal stores does not involve cells. reridkted to te previously excited cel region. a 'long-range' diffusible messenger. A376 A3-6 CALCIUMIACIM SIGNALLINGIGAHN Th-Posl75 Th-Posl76 THE ROLE OF Na+/Ca2+ EXCHANGE, PLASMA MEMBRANE Ca2+-ATPase ANION-DEPENDENCE OF CAPACITATIVE Ca2+ ENTRY IN VASCULAR AND CALMODULIN FOR [Ca2+]i REGULATION DURING CAPACITATIVE ENDOTHELIAL CELLS Ca2+ ENTRY IN VASCULAR ENDOTHELIAL CELLS ((Andrey Klishin, Marina Sedova and Lothar A. Blatter)) ((Marina Sedova, Andrey Klishin and Lothar A. Blatter)) Department of Physiology, Loyola University Chicago, Maywood, IL 60153 Department of Physiology, Loyola University Chicago, Maywood, IL 60153, USA Depletion of intracellular calcium stores activates calcium entry across the plasma We have investigated the contribution of two Ca2+ extrusion systems, the Na+/Ca2+ membrane (capacitative Ca2+ entry, CCE). Using the Ca2+-sensitive dye indo-1 and the exchanger (NCX) and the plasma membrane Ca2+-ATPase (PMCA), to the regulation of perforated patch-clamp technique we investigated modulation of the CCE by intracellular calcium concentration ([Ca2+]i) during capacitative calcium entry (CCE). In membrane permeable and impermeable anions in single vascular endothelial cells single vascular endothelial cells (CPAE cell line) CCE was activated after store depletion with (CPAE cell line). Intracellular calcium stores were depleted with thapsigargin (1 gM) in thapsigargin (1 pM) in Ca-free solution and monitored as changes of [Ca2+]J after 2 Ca2+-free solution (1 mM EGTA). Readdition of extracellular Ca2+ led to a CCE- mM extracellular Ca2+ CCE-induced of were a (2 mM) was restored. changes [Ca2+]1 biphasic with induced biphasic change of [Ca2+l with a rapid elevation followed by a gradual decrease rapid increase in peak [Ca2+]i that was followed by a decline of [Ca2+]1 to a sustained level. removal of extracellular Ca2+ in a of to a sustained higher plateau level. CCE transients were recorded under current- and plateau Subsequent resulted decrease [Ca2+]i voltage-clamp conditions, using the perforated-patch technique. Gramicidin (0.1 to basal levels. The time constant T of a monoexponential fit of this decline was used to mg/ml) characterze the rate of Ca2+ efflux. Inhibition of NCX in Na+-free solution enhanced the was used as the anion-impermeable pore-forming agent, in order to preserve the peak of CCE transient but had no effect on the plateau phase or the rate of decline of physiological regulation of intracellular anion composition. A reduction of extracellular [Ca2+]i to basal levels. In contrast, PMCA inhibitors affected the typical time course of the Cl- from 144 to 10 mM (equimolar substitution with impermeable gluconate) completely CCE transient by abolishing the decrease of [Ca2+]i to the plateau and by significantly blocked CCE in unclamped cells. Current-clamp recordings under the same conditions slowing the rate of decline of [Ca2+]i after removal of extracellular Ca2+. Although the rate revealed a membrane depolanzation of 35 mV and therefore a reduction of the driving of decay of [Ca2+]i was slowed in the presence of the PMCA inhibitor lanthanum (1 mM), force for Ca2+ influx. CCE, however, was also blocked in voltage-clamped cells when the [Ca2+]j eventually decreased to control levels. Additional inhibition of NCX, however membrane potential was kept constant. In contrast, substitution of extracellular Cl- by completely prevented this decay of [Ca2+]i. The typical biphasic time course of the CCE the permeable anions Br- or 1- facilitated CCE, with 1- having the more pronounced transient could be attributed to the delayed activation of the PMCA. The delayed effect. The chloride channel blocker NPPB (50 tiM) greatly reduced CCE with all activation of the PMCA was mediated by calmodulin and was abolished in the presence substitutes for extracellular Cl-. of the calmodulin antagonist W-7. These results demonstrate that various monovalent anions have profound effects on We concluded that the plasma membrane Ca2+-ATPase is the main Ca2+ extrusion CCE in endothelial cells. With chloride, this effect may by mediated by Cl--dependent mechanism during CCE in CPAE cells. Contribution by Na+/Ca2+ exchange is minimal changes of the membrane potential or through a direct interaction with CCE channels. under control conditions, but becomes most important during inhibition of PMCA.

Th-Posl77 Th-Posl78 DOES NITRIC OXIDE NHIBIT CAPACITATIVE CATION INFLUX IN DIPYRIDYLTRIAZOLES: A NOVEL SERIES OF STORE-OPERATED HUMAN PLATELETS? ((E.S. Trepakova, R.A. Cohen and V.M. Bolotina)) CALCIUM CHANNEL BLOCKERS ((W.C. Zinkand, E.J. Warawa, R.C. Boston Medical Center, Boston, MA 02118 Falcone, J. Patel and E.P. Christian)) Respiratory, Inflammation and Neuroscience Department, Zeneca Pharmaceuticals, Wilmington, DE 19850. Nitric oxide (NO) inhibits the rise in intracellular free Ca2+ concentration and in but the of inhibition Store-operated calcium (SOC) channels provide a critical pathway for maintained (Ca2+in) aggregation platelets, mechanism this is calcium signalling in T cells and other cell types. However potent and selective unclear. Fura-2 we NO Using tested the hypothesis that inhibits the store- pharmacological blockers of SOC channels are still lacking. We have discovered and operated cation influx in platelets via acceleration of sarcoplasmic/endoplasmic optimized a series of compounds with a dipyridyltriazole backbone which show a clean reticulum Ca2+-ATPase (SERCA) and refilling of intracellular Ca2+ stores. pharmacological profile and a specific structure-activity relationship for SOC blockade. Thrombin caused Ca2+ or Mn2+ influx preceded by a transient rise in Ca2+i, due The most active analog, N-l-n-octyl-3,5-bis-(4-pyridyl)triazole (compoundl), to rapid Ca2+ release from the stores. Cation influx reached a maximum during reversibly inhibits thapsigargan (TG)-induced SOC influx as measured in Jurkat cells the first minute of Ca2+ release and decreased with time. When Ca2+ was loaded with FLUO-3 or FURA-2 dyes in both cell suspension and individual cell slowly released from the stores by inhibition of SERCA with t-butyl- calcium imaging paradigms. Fluorescence measures also revealed that compoundl did hydroquinone (BHQ) or thapsigargin (TG), Ca2+ influx gradually increased and not affect calcium release from endoplasmic reticulum stores or the sarco-endoplasmic was maximal only after 10-15 minutes. NO suppressed thrombin-stimulated reticulum calcium ATPase. Concentration-dependent SOC blockade (IC50= 7 AM) by Ca2+ influx of the time after In contrast, compoundl was confirmed in the patch clamp experiments in Jurkat cells. Patch clamp independently agonist application. studies also revealed a significant selectivity for SOC over voltage-dependent BHQ/TG-induced Ca2+ influx was affected by NO only during the first 1-2 calcium of of SERCA with of currents in dorsal root ganglion neurons. In a model of murine T-cell proliferation, minutes application inhibitors complete disappearance compoundl inhibited calcium-dependent (T-cell receptor activation) proliferation the effect after 10-15 minutes. These data are consistent with our (IC, hypothesis = 2 sM) selectively over IL-2-induced (i.e., calcium-independent; IC,0 = 8 JIM ) that the inhibitory effect of NO on the store-operated cation influx is indirect proliferation. Examination of other dipyridyl triazole analogs has demonstrated that the and mediated by acceleration of SERCA and faster refilling ofthe store. n-octyl side chain is necessary for activity and is of optimal length. Reduction in length to < 5 C ablates activity. In addition, we have found that one pyridyl ring, but not both, can be exchanged for a phenyl ring without loss of activity.

Th-Posl79 Th-Posl8O EFFECTS OF MELATONIN ON THE Ca2+ RESPONSE OF VASCULAR CAFFEINE-INDUCED [Cal] OSCLLATIONS IN NEURONS OF FROG SYMPATHETIC ENDOTHELIAL CELLS IN PRIMARY CULTURE: ROLE OF OXYGEN GANGLUL REACTIVE SPECIES. R. ((Z. Cseresny6s, Al. Bustarte and M.F. Schneider)) Department of Biochemistry and ((L. Pogan, Sauv6)) D6partement de physiologie, Molecuar Biology, Univ. of Maryland, Baltimore, MD 21201 Groupe de recherche en transport membranaire, Universit6 de MontreaI Canada H3C 3J7 (spon. J.Y. Lapointe) Cae release from calium stores of the endoplanic retculum (ER) i sympathetic gangon neumne was ahown to potenlat Ca2' uptake hfb the ER via r vd calcium ansport, An overproduction of reactive oxygen species (ROS) has been associated to an or RACT (Ceeny6s et al., 1997). We now ahow that RACT may be iwolved hI makahing number of [Cal ocat°n kidueed by adivaion of Ca2 reeas dn prolnd applicafion of 10 mM increasing endothelial dysfunctions. Melatonin, an hormone secreted caffeine. Two ditinct osclafon pattrns were deteced. in pattern 1', the Ist [Cal ansiet by pineal gland, is suspected to be a potent antioxidant and an hypertension wa 4-7 tmes hiher than the absequet peals, whereas hi pattern 2" the ffst and absequet normalizer. A study was therefore undertaken to compare the effect of [Cal peaks wwre of appro e qual ampitude. Transitions between the two patte of melatonin to known antioxydant agents on rat aortic endothelial cells (RAE) in osecbion were also obsenved wilhin one cel. The oetion were blocked by ryanode and primary culture. of RAE cells to the hypoxantine-xantine oxidase were also detected via motoing [Ca2l hi the ER uswin te AM form of 1uo-3FF loaded rlo te Exposure ER. Emplyn a compuler model that descrbes Ca2' flues in frog sympathetc neurons (Friel, (HX-XO) free radical generating system (90-120min), caused a significant 1995; Cwere et al., 1997), we were able to aiiuate pattem 1 when RACT was hiciuded hi increase of the basal [Ca + I level while impairing in a time-dependent manner the model as a non-ieer feedback mechanim. Sbity analysis of te model system (StcWdand the release of Ca2+ from internal stores in response to ATP stimulation. Somogyi, 1994) howed that in he presence of RACT, an hireased ER Ca2' leak due to caffeine Coincubation with deferoxamine, vitamin E and vitamin C protected partly coLid reat* in mai ed pattem 1 oscatios without calcim-induced calium release (CICR) the of and wihott elvated Ca2' infdu The latter firxwas also confirmed by expenments performed against effects ROS, while melatonin and catalase provided almost in zero eaacelar Ca2+ in which pattem 1 oscdlations were also observed. Pattern 2 oscilation complete protection. Furthermore, the InsP3 sensitive (IPS) and thapsigargine can be expained and aimulated by hciudng CICR, whout RACT, as a non-linear feedback sensitive (TAS) internal Ca2+ stores appeared differently affected by ROS, with mechanism in the model (Friel, 1995). Consequeny, RACT and CICR may coribute, separately IPS stores being affected to greater extent than the TAS stores. These or together, to the complex [Cal oscillation pattems in frog sympathetic neurons. observations suggest that melatonin restores the RAE responsiveness to ATP, by (Suported by NIH R01-NS33578). a scavaging action on ROS, including a decrease production of H202. These Csereany6s Z., Bustamante A.l., Klein M.G., and Schneider M.F. (1997) Neuron, 19, 403-419. phenomena could explain the protective effect of melatonin in cardiovascular Friel D.D. (1995) Biqphya.J., 68, 1752-1766. dysfunctions such as hypertension. Supported by a grant from the MRC Canada StukiJ.W, and Somogryi R. (1994) Biochim. Biophys. Ata, 1183,453-472. (MT 7767) t-AL't-IUIVIIAl NI If M alkyINIPLULAINksClNNANlAl lINC. A.3AA77/ /

Th-Posl81 Th-Posl82 THE EFFECT OF CALCIUM RELEASE AND CALCIUM INFLUX ON FUNCTIONAL RECONSTITUTION OF A PUTATIVE CA" CHANNEL FROM MITOCHONDRIAL NAD(P)H FORMATION. ((T.Rohics, A.Spat)) SALIVARY GLAND PLASMA MEMBRANE VESICLES. ((Timothy Lockwich, Semmelweis Univ. Med., Dept. of Physiology, Budapest, Hungary H-1444 Nelson Arispe'. Jvoti Chauthaiwale, Suresh V. Ambudkar2, and Indu S. Ambudkar)) NIDR, NCI2, NIH: and USUHS', Bethesda, Maryland 20892. Elevation of cytoplasmic calcium ion concentration ([Ca2'],) leads to the rise of mitochondrial matrix calcium concentration ([Ca2'],,). The main physiological role We hase previously reconstituted a passive Ca-+ influx pathway from rat parotid of [Ca2'] signaling is the stimulation of the Krebs cycle. The resulting NAD(P)H gland basolateral plasma membrane vesicles (BLMV) into proteoliposomes (PrL) formation can be detected by fluorimetric techniques. In the present study we (Lockwich et al. J. Membr. Biol., 148, 277-285, 1995). Here, we have purified this measured mitochondrial NAD(P)H formation and cytoplasmic Ca2' signaling activity fronm both rat parotid and submandibular gland BLMV by fractionation of simultaneously in single adrenal glomerulosa cells. [Ca2' J, was detected with the the octylglucoside solubilized extract of BLMV (OG-extract). Gel filtration of the Ca2' sensitive dye fura-PE3, and mitochondrial NAD(P)H production was OG-extract and reconstitution of fractions into PrL showed a single peak of 45Ca"+ followed based on intrinsic fluorescence. We compared the effect of inositol 1,4,5 influx activitv, 20-30-fold higher than in PrL prepared from the OG-extract. The trisphosphate (IP) induced Ca2' release with that of Ca2' influx from the apparent molecular weights for the parotid and submandibular gland preparations extracellular space. Intracellular Ca2' release was evoked by the Ca2' mobilizing were similar ( 167+55.9 kD and 135 +30 kD, respectively). The OG-extracts were hormones angiotensin II (AII) and vasopressin (VP). Both AII M) and VP (10-9 also fractionated using wheat germ followed either (10-6 M) induced a higher NAD(P)H formation than the Ca2' influx induced by the agglutinin (WGA) sepharose by elevation of K' from 3.6 to 5.6-7.6 mM. In each experiment NAD(P)H formation Concavalin A-sepharose (ConA) or Lens culinaris-sepharose (LcH). In both cases, was expressed as a percentage of the response induced by the K' concentration 45Ca2' influx into PrL prepared from the fraction binding to ConA, or LcH, was that evoked the same amplitude Ca2' signal that AII or VP. All induced 263±34 % 60-90-fold higher than PrL from the OG-extract (about 200-fold higher than (n=6, p<0.01), VP 210±41 % (n=9, p<0.05) NAD(P)H response. When Ca2' actisitv from BLMV). PrL with enhanced Ca2+ influx actisity generated similar influx was evoked by Ca2' readdition, the effect of Ca2' release (237±41 %, cation channel activities when fused to planar lipid bilayers. Zn2+- and La"- n=9) was again superior to Ca2+ influx (117±19 %), in respect of NAD(P)H sensitive Cs' and Ca" currents were measured. Consistent with the gel filtration formation. These data indicate that Ca2+ released from IP, sensitive stores data, a protein of molecular weight 109-118 kD was detected in all PrL with (presumably in the vicinity of mitochondria), is a more effective stimulator of enhanced activity. The data demonstrate the presence of a Ca +channel in rat NAD(P)H formation than Ca2' influx. Our data confirm the biological significance parotid and submandibular plasmna membrane which appears to be a glycoprotein of previous observations, showing that IP, induced Ca"2 release increases [Ca' ], with an apparent functional molecular weight betweenl 137- 160 kD. to a greater extent than extracellular Ca2'.

Th-Posl83 Th-Posl84 SLOW INTERCELLULAR Ca22 WAVES IN CARDIAC MYOCYTES AND CNS CARBACHOL-ACTIVATED Kc.. CURRENTS ARE INITIATED BY IP3- ASTROCYTES: COMPARISON IN CULTURES FROM WILDTYPE (WT) AND Cx43 INDUCED CA" RELEASE AND SUSTAINED BY STORE-OPERATED NULL (Cx43(4-)) MICE. ((S.O. Suadicanl1,2,3; E. Scemesl,3 and D.C. Sprayl,3)) 1Dept. CA2 INFLUX IN HSG CELLS. ((Xibao Liu', Eduardo Rojas and Indu S. Neurosci., AECOM, NY, USA; 2Dept. Biol. USJT, Brazil; 3Dept.Physiol.,USP, Brazil. Ambudkar )) NIDR' and NIDDK-, NIH, Bethesda, MD 20892 In order to further evaluate the role of gap junction channels In propagation of 2 of these waves in cardiac intercellular Ca waves, we have examined properties Carbachol K' current in HSG cells was measured using the myocytes and astrocytes from neonatal Cx43 (4-) mice. Although Cx43 is the major (CCh)-stimulated gap junction protein expressed in these cell types In WT mice, Cx43 (4-) cardiocytes whole-cell patch clamp technique. At 0 mV, I to 10 jaM CCh induced and Cx43(-/-) astrocytes are coupled: junctional conductance values are about 15-20 oscillatorv currents. while 100 lM or higher induced a biphaste current with a nS In both WT cell types and about 5 nS In Cx43 (-/-) cardiocytes and <1 nS in Cx43 reversal potential corresponding to that of K'. Replacenment of K' with Cs', or (4-) astrocytes. Rate of spontaneous beating in cardiocytes and the spread of Ca2' intemal of Ba eliminated CCh-induced the currents. waves Induced by mechanical stimulation In both cell types were measured application ratiometrically in Indol-AM loaded cultures using the Nikon RCM 8000 real time Charybdotoxin (50 nM), but not apamin (50 nM), reversibly blocked CCh- confocal microscope. Cx43 null cardlocytes beat less frequently and less induced responses. Thapsigargin ( lOiM), BHQ (1OpM) and IP5 (I10 to 100 pM), rhythmically than did WT sibling cultures; variance of beat rate was 20 times higher. but not ryanodine or caffeine. caused increases in the Kca current, and blocked Conduction velocity and number of cells recruited into spread of mechanically CCh-induced Loading HSG cells with BAPTA (10 elicited Ca2' waves were slightly but significantly lower in Cx43 (-/-) cardlocytes than subsequent responses. tm1M) In WT. Conduction velocity, Ca2' response amplitude and number of participating completely abolished the CCh-induced K' current. La" and Gd '. but not Zn". cells were all slightly but significantly reduced In Cx43 (4-) astrocytes. In both cell mimicked the effect of remosvitg extracellular Cai>+ by preventing Ca> stores types, the purinergic P2 receptor antagonist suramin minimally affected parameters refilling aild diminishing CCh-induced oscillatory and sustained K(, cLirrents. wave whereas of the junction Inhibitor heptanol of Ca2' spread, application gap Direct measurements of intracellular in HSG cells. reduced all parameters. We conclude that under the conditions of these experiments Ca'2concentration single and In these cell types, the primary route of Intercellular Ca2' wave spread is showed that CCh (100 pM) induced a biphasic ilicrease in [Ca>+], in a manner through junctional channels. Because Ca2' wave spread is maintained with only similar to CCh-induced KC;, currents. The data are consistent with the hypothesis slight deficits in tissues from mice lacking the major gap junction protein coupling that CCh-induced Kc- current in HSG cells is triggered by intracellular Ca> other connexins in these WT cells, the residual coupling provided by expressed release from Ca` stores and is sustained Ca' tissues appears to contribute a high safety factor for slow Ca2' wave propagation. IP;-sensitive by store-operated (Research supported by FAPESP to SOS, NIH 5POI-NS07512 to DCS and ES). influx via a paithway sensitive to La3+ and Gd . but not to Zn

Th-Posl85 Th-Posl86 ANALYSIS OF THE TOPOLOGY AND SUBUNIT FUNCTIONAL EXPRESSION OF INOSITOL OLIGOMERIZATION OF THE TYPE-1 INOSITOL (1,4,5)-TRISPHOSPHATE RECEPTOR TYPE I (INSP3R-I) IN 1,4,5-TRISPHOSPHATE RECEPTOR ((Lnmma Borrepo-I)iaz. l'ablo Perez. HEK293 CELLS ((E.V. Kaznacheyeva. V. 1). L,upu and 1. B4ezpro,zvanny.) Dariel (;alvan and (;regory \lignerv .) Departnient of Phbssiolo-_y. 'Stritch School Dept of Physiology. I.T Southwestern Mled Ctr. Dallas. TX 752:35 of M\edicine. Lovola Vniversits Chicagit. \Mavwsood. II 6015.3 Rat InsP'31-1IDlNA wvas transiently expressed in 1E11K293 cell line. flxpressed lhe topological orgaiiization and seqiuetices involved in the oligonmerizatiott of Insl'31 prote'in -vsas detected by Western blotting, and confocal fluorescence nlo- the itiositol 1.4.3 -trisphospthate reteptor (Insl R ) subunits were exansiied by traii- croscopy tisitng anti-InsP;3I?-I polvclonal antibody. In biniding assay con(fidictedi usinig sient transfectioti of ('OS tells vsith receptor expression plastitids containing varying, 10 INI [3lljltsP3. density of specific InsP:3 binding sites in microsoniies isolated fronm nitmlers atid combitiatiotis of putative inienilt)ratie spanning -seiuetices. Selective ittld InsP:RI- tranisfected cells was 10- 15 pmol/mng of protein compare to 0.1 pnmol ,Ing of complete pernieabilizatioii of traitsfecte( ('OS tells follosed by itiittiiitiocsntochertiica.l proteini it unttranisfected cells. X't'hen microsomes isolated from Insi'3R-I transfected anal sis with amino-tertinial. carboxv]-tertitinal. intertial and etrerologous antitbod- cells were ftuse(i to planar lipid bilaver membrane. InsP3-gated calciullm hianntels were ies confirni thtat the reteptor sparis tt'e etidoplasntic reticulttit six timzes. Sequentes observed in niost of the experinients. In control experitiients with niicrosotine's iso- involved iti the n,iiltiitterization of the Insl'ttjl itito tetrainiers wsere defiined b% the lated frorii itiock (vector alone) transfected HE};29:3 cells or front untratisfected cells sediiiientation properties of recombinant proteiti on 5-207% sucrose gradients and the InsP3-gated channels were not observed. indicatitig that contaniinatiol with endoge- ability to co-assenible scith ati amino-tertifinallk truncated receptor contaittitig all nous ItnsI'3R is insignificant itn our planar lipid bilaver assay. Detailed comnparison iiiernbrane spanning sequences. These stuidies reveal that the first two niembrane of iitain ftiiictional properties of recombinant rat InsP3R-I with the properties of spaniiing sequences (residties 227-5-23261 ,tre sufficient for suib- iitit assembly. Thf native rat cerebellar InsP3R will be presented. Futictional expression of InsP3R-I stability atid extent to swhich the menibrane spanning deletioni niiutants form nmu1l- cl)D X in IlEl29E3 cells opens an avenue for a future structure-functional analysis timers is augatiented hi! the cvtoplasmic carboxyl-ternini of the receptor. Ntitanits of InsP;31R properties. Stipportcd by .4H11 and The Robert A. ltl lch J'ourtdation. coritaitting oiil% the thtird atid fourth (resi(lues 2332-2407) or fifthi i.nt sixtIi (residues 21-1 1-25'-s9) meriibrarie sparining domaiiis litiked to the carboxyl-termttitnal 160 anilito acids nitiltitlnerized. Iogether these results shos that the reteptor transverses the endoplasmiic reticultini six times and suiggest that there are at least tsvo obligatory deterittitiants for niultinierization. These iticulde the presence of at least twso nierto- brane spantitiig settiiences atId the presetiie of the carboxvl-terltiini .Slipportcd by .\1H .11113i?67 and .\H111 IIL 51. A378 CALCIUM SIGNALLING

Th-Posl87 Th-Posl88 T CELL ACTIVATION STUDIED BY OPTICAL TRAP ((X. Wei. T. THE ROLE OF RYANODINE-RECEPTOR CHANNELS IN B. Erasieva. P. A. Negulescu. C. H. Sun. M. WV. Berns. G. J. Sonek. B. J. MEDIATING CYCLIC ADP-RIBOSE INDUCED Ca2+ RELEASE IN Tromberg. M. D. Cahalan.)) Dept. of Physiology M Biophysics. Univ. of SEA URCHIN EGGS ((Xiaoqing Guo and Peter L. Becker.)) Dept Physiology, California at Irvine. Irvine. CA 92697 Emory Univ, Atlanta, GA

T helper lymphocytes are responsible for the adaptive immune response and acti- Cyclic ADP ribose (cADPr) mobilizes intracellular Ca2+ in sea urchin eggs and vation other cells of the immune system including macrophages and B lymphocvtes. in a variety of mammalian cell types. Although not yet conclusively documented, In the bodv. individual T helpers need to be activated first by phvsical contact such Ca2+ release is believed to result primarily from gating of ryanodine-receptor wvith antigen-presenting B cells to develop into effector cells. T-cell contact wcith release channels (RyRCs), which have been found to be restricted to the cortical antigen-presenting B cells initiates an activation cascade which includes an increase region in sea urchin eggs. We examined the spatial features of the Ca2+ transients in T-cell intracellular calcium and leads to T-cell proliferation and differentiation. in intact sea urchin eggs induced by flash photolysis of caged cADPr using a con- Although T-cell/B-cell physical contact is required for an immune response. little is focal microscope operating in line-scan mode. After photolysis, the onset of rapid knos n about the patterns of cellular interaction and their relation to activation. W\e cADPr-induced Ca2+ release occurred after a delay of several hundred msec, but have combined fluorescence spectroscopy and imaging with optical manipulation to was spatially uniform, occurring in the center of the egg as soon as it was evident investigate the physical properties of T-cell activation. W*e study cell-cell contact in the cortical region. In eggs coinjected with caged cADPr and caged calcium requirements for T-cell activation using optical tweezers to control the orientation (to elevate cytosolic [Ca2+] and cADPr simultaneously), the onset of Ca2+ release of T-cell/B-cell pairs and fluorescence microscopy to measure the subsequent T-cell was immediately detected in both the cortical and interior regions. In an egg ho- intracellular calcium level ([Ca2+]i) response. B cells or beads coated with antibod- mogenate assay where Ca2+ release was monitored by the rise in free Ca2+ with ies to the T-cell receptor (TCR) are trapped with a near-infrared titanium-sapphire fluo-3, 50 uM ruthenium red (RR) blocked release induced by 5 pM cADPr. How- laser and placed at different locations along the T-cell, which has a polarized ap- ever, when homogenate Ca2+ release was detected by 4"Ca efflux in the presence of pearance defined by the shape and direction of crawling. T cells which are presented 5 mM BAPTA, 200 pM RR failed to block cADPr-induced Ca2+ release when the antigen at the leading edge have a higher probability of responding and a shorter ambient [Ca2+] was elevated to 1.6 pM coincident with addition of cADPr. Further, latency of response than those contacting B-cells or beads swith their trailing end. in caged-cADPr loaded eggs, coinjection of 100 pM RR did not significantly alter Alterations in antibody density and bead size are used to determine the spatial re- the kinetics and magnitude of the flash-induced [Ca2+] rise, although reuptake of quirements for T cell activation and the minimum number of receptors *vhich must Ca2+ was dramatically slowed. These result suggest that, although RyRCs may be engaged in order to transmit a positive signal. participate in cADPr-induced Ca2+ release, an additional unidentified channel may also be gated by this second messenger.

Th-Posl89 Th-Posl90 INHIBITION OF THE MUSCARINIC RECEPTOR-INDUCED PHOSPHATE ENHANCES ACTIVE 45Ca UPTAKE IN RAT BRAIN UPREGULATION OF R-TYPE CA2+-CHANNELS BY HALOTHANE MICROSOMES ((Xue-Jun Meng, R.T. Timmer, R.F.Abercombie, and R.B. AND ISOFLURANE ((G. L. Kamatchi, C. K. Chan, T. P. Snutch, C. Lynch Gunn.)) Department of Physiology, Emory University School of Medicine, Atlanta, III, and M. F. Durieux .)) Department of Anesthesiology, University of Virginia GA 30322 Health System, Charlottesville, VA 22906 and Biotechnology Laboratory, University of British Columbia, Vancouver, B.C., Canada V6T 1Z3 (T.P.S.) Fulcheri et al. (Biochem. J., 1993, 289: 299-306) have shown that the 45Ca ca- pacity of brain ER microsomes is strongly dependent on the presence of inorganic Voltage-gated calcium channels (VGCC) may present sites of anesthetic action, be- phosphate. We used similar measurements of 45Ca accumulation at 37 °C from a cause of their involvement in the regulation of cell excitability and neurotransmitter medium containing 5 pM CaC12 to verify their results at lower, more physiologic release. T'hese channels have been classified as T, L, N, P/Q and R -type based on [Ca2+] and to examine possible mechanisms of phosphate transport. Measurements their pharmacological and electrophysiological properties. These channels have been of ATP-dependent 45Ca uptake revealed two phases: an early rapid (1 min) phase shown to be modulated by all subtypes of muscarinic acetylcholine receptors. In our that was phosphate-independent and a second slower, steadily increasing phase (up investigation, we coexpressed ml muscarinic receptors along with the R-type of cal- to 30 min) that depended on the phosphate concentration (2-10 mM). In the ab- cium channels in Xenopus oocytes. Depolarization of these oocytes to 0 mV, using sence of phosphate, 45Ca accumulated to an apparent intra-luminal concentration two electrode voltage-clamping technique induced inward calcium current typical to of 400 pM [Ca]i /5 pM [Ca]o in the early rapid phase and then Ca was released R-type of channel. Administration of 10pM acetyl-) -methylcholine (MCh) upregu- from microsomes within five minutes. The slower, phosphate-dependent, phase was lated the calcium current significantly. Though time to peak did not change, both independent of the sodium concentration up to 40 mM, was not inhibited by 1 mM peak and late currents increased significantly. In addition, both angiotensin II (AT arsenate (a Na-PO4 co-transport inhibitor) was not enhanced by 2 mM glucose- II; 100nM) and Iysophosphatidic acid (LPA; 100nM) also upregulated the Ca chan- 6-phosphate (a possible source for ER intraluminal phosphate) but was partially nel. This increase in Ca-current is not related to intracellular Ca, as BAPTA did blocked by the anion exchange and anion channel inhibitors DNDS (250 pM) and not influence this upregulation. However, administration of halothane (0.59 mM) niflumic acid (50 pM). These results suggest that at low (jiM) concentrations of and isoflurane (0.70 mMI) blocked the upregulation of the R-type channel induced by calcium, phosphate is required for the ER to maintain luminal Ca and that the MCh, AT II and LPA. All these receptors have a similar G,-protein mediated path- mechanism for phosphate transport into brain microsomes is unlikely to require way, which may mediate upregulation of Ca channel. In addition volatile anesthetic sodium ions or glucose-6-phosphate metabolism. blockade of upregulation rnay involve various steps in the pathway.

Th-Posl9l Th-Posl92 Quantitative two-photon excited calcium uncaging ((E.B. Brown, G.C.R. INHIBITION OF RECOMBINANT HUMAN L-TYPE Ca2+ Ellis-Davies#. and W.Wr. Webb.)) Dept. of Applied and Engineering Physics, CHANNEL BY THE ACTIVATION OF ANGIOTENSIN ATSA Cornell University. Ithaca. NY and # Dept.of Physiology, Allegheny University, RECEPTORS CO-EXPRESSED IN XENOPUS OOCYTES ((Murat Oz, Philadelphia. PA Nikolai M. Soldatov, Michael T. Melia, Kathryn Sandberg, Darrell R. Abernethy, and Martin Morad.)) Georgetown University Medical Center, Department of A techniquie for the measurement of the two-photon uncaging action cross section Pharmacology, Washington, DC 20007 of calciuim cages has been developed in this laboratory ". Using this technique, we report on the prognosis for effective two-photon excitation of a novel calcium Human L-type Ca2+ channel and angiotensin II receptors have already been cage. DMNPE-4 3. Be also describe calculations which predict the total amount of cloned and functionally expressed in Xenopus oocytes. In this study, type IA calcium ions tincaged and the temporal behavior of the liberated calcium distribution rat angiotensin (ATSA) receptor and a splice variant aIC,77 (EMBL Data Bank under given multiphoton photolysis conditions with a given cage. These calculations, #Z34815) of the human recombinant L-type Ca2+ channel were co-expressed in combined with the two-photon uncaging action cross sections that we have measured Xenopus oocytes, and the effect of human synthetic angiotensin IIon the function for a variety of calcium cages enable the quantitative study of calcium dependent of the channel was investigated. Ba2+ currents (IBa) were elicited by 1-s depolariz- processes with high spatial resolution within cells or thick tissue samples. The ing pulses from -90 mV to +20 mV using the two-electrode voltage clamp technique. ultimate intent of these studies is the application of these tools to the quantitative In oocytes expressing the human L-type Ca2+ channel alone, application of I pM study of the spontaneous transient calcium coiicentration elevations (osparkso) in angiotensin did not affect the amplitudes or kinetics of IBa- In sharp contrast, in developing skeletal muscle cells, probing the potentiation/inhibition relationships oocytes co-expressing the recombinant channels and ATIA receptors, application of between consecuitive sparks in one localized region as well as between sparks in angiotensin in concentrations ranging from 1 nM to 1 pM gradually (1-5 min) and spatially separate regions. 1. Shear, J. et al. Biophys. J.70(2) A211 (1996)2. Brown reversibly inhibited IgB. In oocytes pre-injected with Cs4-BAPTA, the inhibitory et al. Mlanuiscript in Preparation (1997) 3. Ellis-Davies, G.Biophys. J.72(2) A246 effect of angiotensin on IBa was completely abolished, suggesting that intracellular (1997) This work was carried out in the Developmental Resource for Biophysical Ca2+ may mediate in part this effect. In line with these findings, IBa through a Imaging and Optoelectronics (DRBIO) with funding provided by the NSF (grant splice variant of alc (alC,86; EMBL Data Bank #Z74996) missing Ca2+-dependent DIR 88002787) and NIH (grant RR07719 and RR04224). EB was supported as a inactivation, was not suppressed by up to 1 pM angiotensin when co-expressed with predoctoral trainee under NIH grant T32GN108267. G.E-D. wa supported under ATIA receptors. Our results show that human L-type Ca2+ channel co-expressed grant GM153395 with ATIA receptor in Xenopus oocytes may be regulated by angiotensin. Supported by AHA (Nation's capital Affiliate) and NIH grant HL16152. ~ ~ CALCIUMCLIMSINLIGA7SIGNALLING A379 Th-Posl93 Th-Pos194 EFFICACY OF AGENTS WHICH INHIBIT MITOCHONDRIAL Ca CALMODULIN TRAPPING AND AUTOPHOSPHORYLATION IN UPTAKE IN INTACT VENTRICULAR MYOCYTES ((Z. Zhou. D.M. CALCIUM/CALMODULIN DEPENDENT PROTEIN KINASE II. Bers.)) Department of Physiology, Loyola University Chicago, MIaywood. IL, 60153 ((A.V. Rotberg, P.B. O'Hara, A. Dosemeci, R.W. Albers.)) Amherst College Chemistry Department, Amherst, MA 01002. Laboratories of Neurobiology and We address how rapidly extracellularly applied inhibitors of mitochondrial (NIito) Neurochemistry, NINDS, NIH, Bethesda, MD, 20892. Ca uptake (e.g. ruthenium red or RuR and the more potent Ru360) gain access to the Mito in intact ferret ventricular myocytes in which Mito are strongly loaded Calcium/calmodulin dependent protein kinase II (CaM K II). a major component with indo-1. This is especially important for hydrophilic agents which do not readily of the post-synaptic densities (PSD's) in hippocampal neurons. is believed to act as cross the sarcolemma. In control cells. intentional sarcolemmal damage with a patch a signal transducing enzyme, converting calcium frequencv pulses into a chemical pipette results in rapid rise in cytosolic [Ca] and a large irreversible cell contracture, message. This role of CaM K II is closely linked to two main functional features without significant loss of cellular indo-1. This contracture is followed closely in of the enzyme: its initial dependence on calcium/calmodulin binding for activation. time (<10 s) by a rapid rise in Mito [Ca] toward the maximal fluorescence ratio and its subsequent ability to autophsophorylate at position Thr-286 (the 6Ao site) (Re,a,). This short lag reflects the rapid uptake of Ca by Mito when [Ca] around and become constitutively active. The phenomenon known as acalmodulin trap- them is very high (e.g. mM). When the cells are preincubated with 30 pM RuR for ping' refers to the fact that in the presence of calcium and ATP, the affinity of 20-60 min before pipette damage, there is a large time lag (up to 200 s) between CaM K II for calmodulin increases dramaticallv, thus slowing the dissociation of the the time when 90% of maximal cell contracture is reached and 90% of Rna, occurs. complex when intracellular calcium levels are lowered. The question remains as to This latency time (tk) reflects the block of the Mito Ca uniporter by RuR. This whether ATP binding or Thr-286 autophosphorylation is responsible for calmodulin tk value increases monotonically with incubation time in RuR and at only 5 min trapping. In addition, a second autophosphorylation site (Thr 306. the oB6 site.) RuR exposure there is no tk difference from control. Thus, >60 min exposure to 50 which is phosphorylated only in the absence of calcium and interferes with subse- pM extracellular RuR is required for maximal block of the MIito Ca uniporter. In quent calmodulin binding, thus has further effects on the aetrapping- phenomenon. contrast, Ru360, the selective and more potent Ca uniport blocker (Ki = 0.2 nM) By studying anisotropy changes in the binding of CaM K II to dansvlated calmod- reaches a comparable maximal tk after only 10 min with 10 pNI, making it practical ulin in adecalcified calmodulin/CaM Kinase/ATP system, we hope to further ex- to use in intact cells. Lipophilic agents which dissipate the Mito E, (FCCP & plore this question. As calcium is added, A-site phsophorylation will occur, and CCCP) produce smaller maximal effects on tk since they don't directly block Ca by subsequently chelating calcium with either EGTA or EDTA (shich deactivates uptake, but this smaller efect is immediate (e.g. <30 sec exposure to 1 pM FCCP). ATP,) the effects of B-site phosphorylation and subsequent calmodulin binding can This method allows us to conclude that 10 MM Ru360 allows sufficient Ru360 entry be explored. In addition, experiments on CaM Kinase II activation by a novel cross to block Mito Ca uptake in intact cells after a 10 min incubation period. linked form of calmodulin will also be described.

Th-Posl95 Th-Pos196 SINGLE CHANNEL CHARACTERISTICS OF INOSITOL ALDOSTERONE MEDIATES INTRACELLULAR CALCIUM (1,4,5)-TRISPHOSPHATE RECEPTORS FROM CARP OLFACTORY SIGNALS IN SKELETAL MUSCLE CELL CULTURES ((M. Estrada. M. CILIA. ((H. Cadioul. E. Thrower2, S. Bendahhou', G. Mollel and H. Miranda, J.L. Liberona. and E. Jaimovich.)) Instituto de Ciencias Biomedicas. Duclohier'.)) 1UMR 6522 CNRS-Universite de Rouen. Bd. M. de Broglie. 76821 Fac. Medicina, Universidad de Chile and C.E.C.S., casilla 70005, Santiago 7. Chile Mont-Saint-Aignan, France. 2School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K. Fast steroid effects are not compatible with the mechanism of action of these hor- mones characterized by a prolonged latency period before effects on gene expres- Olfactory transduction takes place primarily via a cAMP-dependent pathway al- sion. This "'non genomic 77 route in some cells appears to be mediated by intracellular though additional mechanisms are currently being discovered. One such route in- calcium ([Ca2+]i). Such a mechanism has not been described in skeletal muscle cells. volves the generation of inositol (1,4,5)-trisphosphate (InsP3 and activation of an Aldosterone(10-100 nM) effects were measured as fluo-3 calcium fluorescence using InsP3- sensitive ion channel (Honda, E., Teeter. J.H. and Restrepo, D. 1995. Brain either confocal microscopy or fluorescence microscopy using out of focus fluorescence Research 703, 79-85; Fadool, D.A. and Ache, B.W., 1992. Neuron 9, 907-918). To elimination. IP3 mass was determined by a radioreceptor displacement assay. We further study this, we have investigated InsP3-stimulated channel activity in fish saw two types of intracellular calcium changes after aldosterone addition: a relatively olfactory cilia fused to planar lipid bilayers. Carp olfactory cilia were isolated by fast (less than 2 min) calcium transient, frequently accompanied by oscilations and established procedure (calcium shock and ultracentrifugation) and membrane frac- a slow rise in [Ca2+]i that reaches its maximum after 30 min of aldosterone expo- tions incorporated into planar lipid bilayers (neutral mixture made of POPC:DOPE. sure. Both calcium signals were located mainly to the nuclear region. Intracellular 7:3) formed by either the "Montal-Mueller" or the "tip-dip" techniques. The elec- calcium responses seem rather specific for aldosterone since other steroid hormones trolyte solution, consisting of 140 mM NaCl, 50 mM BaCl2, 10 mM HEPES. pH 7.4. do not induce detectable changes in fluorescence even at 100 fold higher concentra- bathed both compartments of the bilayer apparatus. Addition of 5-20 micromolar tion. The mass of IP3 increases transiently from a basal level of 23.6 ± 8.9 to 33.8 InsP3 to the trans compartment at an applied potential induced channel activitv ± 9.7 upon addition of aldosterone; the IP3 transient is faster than the fast calcium and two conductance states were seen with values of 90 pS and 220 pS. Further signal. These results suggest the existence of a non genomic pathwav for aldosterone addition of 100 micromolar ATP to the same compartment caused the channel to in skeletal muscle cells involving nucleoplasmic (and possibly cytoplasmic) calcium switch to one conductance level wvith a value of 265 pS, indicating a regulatory role signals. probably mediated by 1P3. Supported bv FONDECYT 1950434, 1970760. for ATP. SDS-gel electrophoresis of the membrane fraction exhibits a single band at MDA, and The European Economic Communitv. 240 KDa comparable to that seen for InsP3 receptor purified from rat cerebellum. Preliminary Western analysis using antibodies raised against types 1. 2 and 3 InsP3 receptor suggests that this cilia-membrane associated InsP3 receptor is tvpe 3.

Th-Posl97 Th-Pos198 MECHANISM OF Ca2+ REGULATION IN OSTEOBLAST-LIKE THE IMPORTANCE OF PROTEIN PHOSPHATASE 1 IN CARDIAC CA2+ HOMEOSTASIS CELLS ((M.J. Park. S.C. Kwon. E.J. Park, J.H. Shin. S.R. Park, C.K. Suh.)) ((Palfreyman T., Hussain M., Drago G.A., Berrebi-Bertrand I*, Bril A.*, Orchard C.H. Department of Physiology & Biophysics, Inha University College of Medicine. 253 and Colyer J.)) Depts of Biochemistry, Molecular Biology & Physiology, University of Yonghyun-dong Nam-gu, Inchon 402-751, Korea Leeds, Leeds, & *SmithKline Beecham Lab Pharm, Saint Grigoire, France. Physiological activitv of osteoblast including bone formation is known to be closely Protein phosphorylation by protein kinases and subsequent dephosphorylation by protein related to the increase in intracellular CaS+ activity of osteoblast. CaS+ is an im- phosphatases play an important role in cellular signal transduction in cardiac muscle. The portant intracellular messenger in diverse cellular functions, and the regulation of role of 2rotein phosphatases in the regulation of phospholamban (PLB) phosphorylation its level is mediated bv the transmembrane Ca2+ movement via Ca2+ channels and and CaL+ homeostasis was investigated in isolated adult ferret ventricular myocytes. Na+-CaS+ exchange, and intracellular Ca2+ movement through the intracellular Phosphatase inhibition by either 10pM calyculin A, 10 pM okadaic acid or 10 pM Ca2+ store. In this study, we investigated the mechanisms of intracellular Ca2+ reg- tautomycin produced an increase in the level of PLB phosphorylation on both Ser 16 and ulation in osteoblast cells. Osteoblast-like cells (OLC) were isolated from femoral Thr 17 in response to 5 minutes of I-adrenergic stimulation by 200 nM isoprenaline. and tibia of neonatal rats, and cultured for 7 days. Cultured OLCs were loaded with Okadaic acid and Tautomycin at lower concentrations (0.1-1 PM) were without effect CaW+-fluorescent dye Fura-2, and fluorescence images were monitored with a cooled whereas calyculin A remained a potent phosphatase inhibitor. This identifies protein CCD camera. High K+ (25mM) solution did not induce any significant [Ca2+], phosphatase-l as a major phosphatase involved in PLB dephosphorylation. These increase. [Ca2+]i decreased when 0-Ca2+ solution was superfused, and returned to also identified phosphatase-l as an important enzyme in the basal level the normal experiments the control of the when Tyrode solution (NT) was reperfused. However. phosphorylation state of PLB. Inhibition of protein phosphatase-l by 1 pM calyculin when was applied with superfusion solution. the reperfu- A lpM thapsigargin O-CaS+ caused maximal phosphorylation of PLB without 5 sion of NT elicited a transient increase of [Ca2+]i above the basal level, implving the need for adrenergic stimulation. the existence of the store-dependent Ca+ influx. WVhen the extracellular [Na+] was Consistent with this, the Ca2+ transient kinetics were accelerated in the presence of replaced with N-methyl-D-glucosamine. an elevation of [Ca2+], was observed. Occa- Calyculin A. In ferret myocytes loaded with fura-2, calyculin A (0.2 PM) caused a sionally, a spike-like transient [Ca2+], increase was also observed, which was blocked significant acceleration of the rate of decline of the Ca2+ transient (T1/2 442.6 ± 20 ms, by 1pM thapsigargin. The results from this study suggest that the intracellular n=8 vs 215.0 ± 3.0 ms, n=5; mean and S.E.M. control vs 0.2 pM calyculin A; p<0.001) Ca2+ activity in OLCs is regulated by transmembrane Ca2+ entry via Na+-Ca2+ without affecting the time to peak. These results support the possibility that PLB exchange and intracellular Ca2+ stores. phosphorylation is mediated by phosphatase-1 inhibition which is associated with positive lusitropic effect. ra.70VA380 CALCIUtM4-22JLA-RIULTZ L7&%Yl'%ZSAJJUJLL'1%7SIGNALLING Th-Posl99 Th-Pos2OO BLOCK BY MONOVALENTS OF Ca2+-ENTRY THROUGH P2X-PURINOCEPTORS IN LABELING OF CALMODULIN WITH A NEW BIFUNCTIONAL BODIPY HUMAN B-LYMPHOCYTES ((Lohn, M., Klapperstuck, M., Markwardt, F.)) FOR IMPROVED FLUOROPHORE STABILITY IN ANISOTROPY J.-B.-Inst. for Physiology, M.-L.-University Halle, Germany MEASUREMENTS. ((Anka G. Ehrhardt 1. Hee Chol Kang 2. Richard A. Tuft 1. Fredric S. Fay 1, Mitsuo Ikebe 1.)) 1 UMMC, Dept. of Physiol., WVorcester, MA 01655: 2 Although the Ca2+-permeability of P2X-purinoceptors in human B- Mlolecular Probes, Eugene, OR 97402 lymphocytes is high (Bretschneider et al.,PflOgers Arch. 429), the increase in [Ca2+Ji after 10 min application of ATP4- is negli- The bifunctional probe BODIPY FL bisiodoacetamide (BOBI) was synthesized and used gible as measured by flow cytometry using Ca2+-sensitive dyes. To for intramolecular crosslinking of calmodulin (CaM) to produce a probe for fluorescence investigate this phenomenon in detail, ATP-induced membrane cur- anisotropy measurements of CaM/ CaM target complexes. In order to include the BOBI at- rents and intracellular calcium ion concentration (FCa2+Ji) were tachment sites, the two cysteine residues (approximately 0.7 and 1 nm apart from each other) measured simultaneously in Epstein-Barr-Virus-transformed and ton- were inserted into CaM by means of site directed mutagenesis. Two mutant CaMs were con- sillar human single B-lymphocytes by means of the voltage-clamp- structed: (R74D80CC) and CaM(E82R9OCC). These mutants were expressed in the E.coli and FLUO-3/FURA-red-fluorescence-technique. In cells clamped at strain BL21 (DEL) by IPTG induction, then extracted from the cells and purified by a series -80 mV, a 5 s application of ATP4- increased Ca2+ i by about of liquid chromatography steps. The purified mutants were then conjugated with BOBI and 50 the intracellular K+ Na+ reduced separated from free dye by gel filtration. The CaM activity of the BOBI conjugated mutants nmol/l. Replacing by this was examined with a myosin light chain kinase (MLCK) activity assay. The CaNt-BOBI [Ca2+2i-increase to about 25 nmol/l, substitution by Cs+ increased probes activate MLCK with the same calcium dependency and potency as wild type CaM. In it to 80 nmol/l, respectively. Substitution of the extracellular in vitro experiments the binding of CaM to different targets (MLCK, caldesmon, calcineuin Na+ by choline+ increased the ATP4--induced [Ca2+'i-enhancement to and whole cell extract) has been tested. The CaM probes yielded anisotropy values which 150 nmol/l. Under current-clamp-conditions, the lymphocytes de- are consistent with a rigid globular protein with a molecular mass of about 17 kDa, sug- polarized during application of ATP4-. The amount of depolariza- gesting that the fluorophore is tightly constricted to the CaM molecule and intramolecular tion was dependent on [Ca2+J1. The [Ca2+Ii-increase was negligible rotations are almost totally suppressed. CaM(E82R9OCC)-BOBI showed significant changes with Na+ but substantial with choline+ as extracellular monovalent in the anisotropy value with the binding of all targets tested. CaM(R74D80CC)-BOBI did cation. These data suggest that after ATP4--application to human not increase the anisotropy value with caldesmon, while it showed anisotropy increases with B-lymphocytes, an increase of the global [Ca2iJ- is inhibited by all other tested targets. These data support the suggestion, that caldesmon is binding CaM Na+ ions due to a direct inhibition of the Ca2+-permeation and a in a conformation which is different from the CaM conformation upon binding with other reduction of the driving force for Ca2+ entry by a Na+-carried target proteins. depolarizing current. (Supported by DFG, Ma 1581/2-2)

Th-Pos2Ol Th-Pos202 Parvalbumin Enhances Activity of Ca2+ Puffs/Sparks While Restricting TEMPORAL AND SPATIAL RESOLUTION OF MrTOCcONDRIAL MATRIX FREE ca2+ Ca2+ Wave Propagation In Xenopus Oocytes. ((L.Nl. John. MI. Zhang. P. DYNAMICS IN RAT AORTIC MYOCYISS. ((Monteith G.RI and Blaustein M.P)) Dept. Camacho and J.D. Lechleiter.)) Depts of Biomedical Engineering and ofPhysiol, Unmv ofMD, Balimore, MD, USA. ¶Presentaddress - Dept. Pharm, Univ Neuroscience. UVA Health Sciences Center. Charlottesville. VA. Dept of of QLD, QLD, Australia. Physiology and Dept of NMolecular Medicine/IBT, U;niversity of Texas HSC. San Antonio. TX 78245 Mitochondrial matrix free Ca2- ([Ca2-]m) was assessed using the Ca2e-indicator, Rhod-2 (1,2), in cultured aortic myocytes. Rhod-2 preferentially accumulated into Inositol 1.4.5-trisphosphate (IP3)-induced Ca2+ release is modulated by Ca2+ se- mitochondria; small amounts of dye remained in the cytosol, and some sequestered 260: 226-229. Nature 377: into the sarcoplasmic reticulum (SR), where it was saturated with Ca2e. Using questration processes (Science 438-441). Consequently. fluorescence digital imaging, dynamic changes in [Ca2+]m was resolved from the other endogenous Ca2+ binding proteins (CaBPs) are also likely to modulate Ca2+ sig- compartments; cytosolic free Ca2+ ([Ca2+Jcyr) was determined from the diffuse signal nalling dvnamics. In this study we focused on the effects of reversible Ca2+ binding and distinct Ca2l properties reported by the residual Rhod-2 in the cytosol. Results (buffering) by parvalbumin. Xenopus oocytes were injected with parvalbumin ( 50 show that photo-dynamic damage with high levels of dye excitation were associated pM final) and Ca2+ Green I indicator dye ( 12.5 pMI), 30-60 minutes prior to imag- with sustained increases in [Ca2+]m and loss of mitochondrial integrity. Such effects ing. Confocal imaging (BioRad 600 UV, 700 pm x 700 pm x 30 pm) at 1 sec were not observed at lower excitation intensity; hence, low light levels were used for intervals was performed following injection of IP3 ( 300 nNI final). Parvalbumin physiological studies. Physiological agonists such as ATP, and in some cases 50-200 pMII) inhibited IP3-mediated Ca2+ oscillations and only produced a slowly serotonin, induced increases in [Ca2l],. These effects were reversible. The degree developing Ca2+ tide. However, discreet Ca2+ puffs/sparks occurred when parval- of mitochondrial Ca2' sequestration, correlated with the level of peak bulk [Ca2J]C, bumin was lowered to 25 pM (n=10). These focal Ca2+ events either failed to as measured by fura-2; The temporal changes in [Ca2`]m were distinct from changes propagate or propagated asvmetrically for short distances ( 50-100 pm). WVe can in bulk [Ca2+cyT; the time to reach peak [Ca2+]J lagged behind peak [Ca2+lcy by - account for these observations by the Ca2+ binding kinetics of parvalbumin since it 2-3 sec. Similar results were observed in contracting smooth muscle cells. These binds Ca2+ slowly due to the rate-limiting step of Mg2+ dissociation (k,,ff 1 sec5 ). studies suggest that, at least in arterial myocytes, mitochondria exhibit graded Consequently, parvalbumin is unlikely to affect IP3R activation (which takes place responses during Ca2+ signaling: [Ca2+Jm responses associated with modest increases in 1-10 msec) while inactivation (which is a slower process 100-200 msec) may in [Ca2i+cyT, responses associated with higher [Ca2+]cyT loads, and those associated be reduced. Ca2+ diffusion is also likely to be reduced by parvalbumin, thereby with loss of mitochondrial integrity. We speculate that this spectrum of [Ca2+]m restricting propagation to neighboring release sites. The ability of a given CaBP responses (small and large reversible rises, and large sustained increases) may be to prevent propagation by restricting Ca2+ signals to local puffs/sparks, suggests a related to, physiological, pathophysiological, and cytotoxic cellular activities. novel function for endogenous Ca2+ buffers in Ca2+ signalling. Supported by NIH [11 Hajn6czky et al. Cell 82:415-424, 1995. [2] Mix et al. Biophy. J. 66: A97, 1994. grants to JL (GM48451) and to PC (GM55372).

Th-Pos2O3 Th-Pos2O4 TUMOR NECROSIS FACTOR-a (TNF) ACTIVATES A NOVEL S-NITROSOGLUTATHIONE AND CALCIUM SENSITIVITY IN AIRWAY SMOOTH CALCIUM SENSITIZATION PATHWAY IN BRONCHIAL SMOOTH MUSCLE ((C.M. Pabelick, W.J. Perkins, D.O. Wamer, K.A. Jones)) Mayo Clinic MUSCLE ((J.R.M. Parris, D.J. MacEwan, G.F. Nixon.)) Dept. of Biomedical and Foundation, Rochester, MN 55905 Sciences, University of Aberdeen, Aberdeen, Scotland AB25 2ZD Nitric oxide and NO-donors such as S-nitrosothiols play an important role in the TNF, released by inflammatory cells, induces a hyperresponsiveness of the airways, regulation of smooth muscle tone. This study investfgated the mechanisms by however, the cellular mechanism is unknown. The aims of this study were to inves- which S-nitrosoglutathione (GSNO) relaxes airway smooth muscle. Canine tigate the contractile effects of TNF on bronchial smooth muscle. Staph. aureus o- tracheal smooth muscle strips were loaded with fura-2/AM and mounted in a toxin permeabilized bronchial strips were contracted in pCa 6.3 and agonist-induced photometric system for simuitaneous measurement of force and intracellular Ca2+ sensitization was activated with carbachol (CCh). Strips were relaxed in Ca2+ calcium concentration ([Ca211,). Regulatory myosin light chain phosphorylation free, 1 mM EGTA buffer (Gl), incubated for 45 minutes in Gl with 1 pg/ml hu- (rMLCP) was measured using 2-D gel electrophoresis, and cyclic GMP was man recombinant TNF and the stimulation protocol repeated. In other experiments measured by radloimmunoassay. When force-[Ca2']i relationships were measured ceramide, a mediator of TNF effects (and activator of protein kinase C (), was liber- by sequentially increasing extracellular [Ca2l' during KCI-induced depolarzation, ated by addition of sphingomyelinase (SGMase). In intact bronchial smooth muscle, 100 pM GSNO decreased the force produced by a given [Ca2)without changing contractions to CCh were significantly increased by 200% after TNF incubation. the reltionship between extracellular and intracellular [Ca 1. GSNO also In permeabilized strips the pCa 6.3 contraction following TNF incubation was in- decreased the rMLCP produced by a given [Ca2'],. without changing the creased fivefold (TNF treated 370 + 49% of initial pCa 6.3 vs control 74 + 10%, n=6, relationship between force and rMLCP. When added to sustained KCI-induced p

Th-Pos2O7 Th-Pos2O8 FUNCTIONAL DIFFERENCES IN INTRACELLULAR Ca STORES IN CALCIUM TRANSIENTS AND MEMBRANE CURRENTS INVOLVED IN VOLUME PULMONARY AND RENAL ARTERIAL SMOOTH MUSCLE CELLS. REGULATION IN HUMAN OSTEOBLASTS. ((M. Weskamp and S. Grissmer)) Dept. of ((IL Janiak and J.R. Hume)) University of Nevada School of Medicine, Dqetment of Applied Physiology, University of Ulm, 89081 Ulm, Germany. Physiology and Cell Biology, Reno, NV 89557-0046. Intracellular calcium concentrations, [Ca2l,, in human osteoblasts and in a human osteoblast-like cell line, Cl, were investigated using fura-2. The cells were incubated for Recent evidence suggests that pulmonary arterial smooth muscle celis (PASMC) and 20 min. in Dulbecco's modified Eagle's medium with 10 % FCS and 4pM fura-2 / AM at renal arterial smooth muscle cells (RASMC) have two types of iitracellular calcium 37'C. Then the cells were washed three times in mammalian Na+ solution. Cell swelling was induced by applying hypotonic solutions. A fura-2 imaging system was used to stores: a caffeine-ryanodine sensitive calcium store and an 1P3 sensitive calcium store. In determine [Ca2 ] of individual cells before and during swelling. tis study we investigated whether in PASMC and in RASMC, these calcium stores Applying hypotonic solution leads to function independently. Smooth muscle celis were isolated from the tiird branch of an increase in [Ca2+], (see figure). pulmonary and renal arteries by enzymatic dispersion and were loaded with the calcium- Several seconds after changing the A. sensitive fluorescence dye, Fura-2AM, to measure free intracellular calcium 100% Na+ solution to a 50% Na+ concentration. In both types of cells, brief application ofcaffeine (CAFF, 15mM) caused solution containing 5 mM Ca a II I in or [Ca25l increased within 100 0,, rapid calcium transient. Brief perfusion with angiotensin (ANG, zM) PASMC seconds. Most of the cells were able O's phenylephrine (PHE, 1AM) in RASMC also elicited rapid calcium transients (from IP3 to regulate [Ca2+] to the initial level . sensitive calcium stores). Perfusion of PASMC with thapsigargin (TG, 5pM) or within 15 minutes. 0,2 .- Na', cyclopiazonic acid (CPA, 10 pM) completely abolished subsequent responses to ANG, Membrane currents were measured oil while a normal calcium transient could be elicited by CAFF. In contrast in RASMC, TG using the whole-cell patch-clamp °,,010 or CPA also blocked subsequent responses to the application of PHE, but also abolished technique. With high Ca + (RY) concentration in the patch pipeKte an outward current was seen at depolarized the CAFF transient. Block ofryanodine channels with 300 pM ofryanodine in both potentials. This current was not present, when the Ca2+ concentration in the patch PASMC and RASMC abolished the response to CAFF. In PASMC, ANG still elicited a pipette was kept low indicating that this current, possibly a potassium current, may be calcium transient in RYN, but PHE failed to elicit a calcium transient in RASMC treated Ca2+ dependent. In combination with a volume sensitive Cl conductance in the same with RYN. These results suggest that in the PASMC, caffeine-ryanodine dependent and cells (Steinert & Grissmer, J. Physiol. 500: 653.660, 1997) this current could contribute IP3 dependent calcium stores may function independently, whereas in RASMC these to cell shrinking by lowering the osmotic pressure inside the cell after exposure to stores may functionally overlap and share a common SR calcium ATPase. ( HL 49254). hypotonic solutions. Supported by a grant from the BMBF (iZKF Ulm, B4).

Th-Pos2O9 Th-Pos210 A LOW AFFINITY InsP3 BINDING SITE INCREASES InsP3-GATED EFFECTS OF TEMPERATURE ON CALCIUM SENSITIVE PROBES STUDIED IN CHANNEL ACTIVITY AT HIGH CYTOPLASMIC CALCIUM RELATION TO MEASUREMENTS OF INTRACELLULAR CALCIUM CONCENTRATIONS. CONCENTRATION IN HUMAN PLATELETS DURING CHILLING. ((EJ. Kaftan, B.E. Ehrlich# and J. Watras')) *Univ. of Washington, Seattle, ((Ann E. Oliver', Fern Tablin2, Naomi J. Walker2, and John H. Crowe')) WA, #Yale Univ., New Haven, CT and $Univ. of Connecticut, Farmington, 'Section of Molecular and Cellular Biology, and 2Department ofAnatomy and CT Physiology, School ofVeterinary Medicine, University ofCalifornia, Davis, CA 95616 Calcium-sensitive fluorescent dyes have been used effectively to monitor the Cytoplasmic calcium (Ca) is a potent inhibitor of inositol 1,4,5- intracellular calcium concentration in a variety ofcell types. Care must be exercised in trisphosphate (InsP3)-gated channel activity. In the presence of 2 ,uM InsP3, 5 choosing the appropriate dye, however, especially when sample temperature is used as an p.M cytoplasmic Ca completely inhibited channel activity. We found that independent variable. We have conducted a systematic study offive calcium-sensing when the cytoplasmic concentration was channel dyes in regard to the effects oftemperature. Chlorotetracycline, Quin-2, calcium green, InSP3 increased, activity Indo-l, and Fura-2 all show temperature-dependent effects on fluorescence in all or part persisted at Ca concentrations as high as 30 pM. InsP3 binding studies ofthe range tested (5-40 'C). The temperature-sensitivity varies from dye to dye in conducted over a broad range of InsP3 concentrations revealed a high affinity regard to the intensity ofthe effect, whether it is linear or non-linear, and whether the dye and a low affinity InsP3 binding site (KD= 50 nM and 10 lM). The affinity of would be likely to over- or under-estimate the actual calcium concentration. The dual- the 50 nM binding site decreased 3-fold as the Ca concentration was increased wavelength dye Indo-1, for example shows a sharp temperature-sensitivity during chilling to 10 pM. In contrast, InsP3 binding to the low affinity site appeared to be between 40 and 20 'C, but very little effect between 20 and 5 'C. Since the membrane unaffected by the addition of Ca. The observed Ca dependence of the InsP3- phase transition of human platelets has recently been shown to fall between 15 and 18 OC, gated Ca channel allows the cell to abbreviate the rise of intracellular Ca in the Indo-l is an appropriate calcium-sensing probe to use in measurement ofthe intracellular presence of low levels of InsP3, but also provides a means of maintaining high calcium concentration of human platelets during chilling through the liquid crystalline to intracellular Ca during periods of prolonged stimulation. gel phase transition. We have used Indo- l in this capacity, and the data indicate that the NIH GM51480 internal calcium concentration increases in human platelets during chilling through the Supported by grant phase transition. The mechanisms for this calcium increase probably include leakage from the dense tubule system, leakage from the external milieu, and ion flux through calcium ion channels. (This work was supported by NIH grant # NHLBI 57810-01). A382 A382MTOHNRACHNESADPYILGMITOCHONDRIAL CHANNELS AND PHYSIOLOGY Th-Pos2ll Th-Pos212 RU360 SPECIFICALLY INHIBITS Ca2+ UPTAKE INTO SIMULTANOUS MEASUREMENTS OF MITOCHONDRIAL [Ca2+] and [NADH] MITOCHONDRIA 1\V V'ITRO AND IA SIlL IN CARDIAC DURING INCREASED WORK IN RAT CARDIAC TRABECULAE. MYOCYTES. ((M.A. Matlib, Z. Zhou, S. Knight. S. Ahmed, K.M. Choi. J. ((Rolf Brandes, Robert Helm and Donald Bers)) Loyola University Chicago, IL Krause-Bauer, R. I'hillips, R. Altschuld, Y. Katstube, N. Sperelakis, and D.I. Blers.)) Pharniacol. antd Cell Biophys.. Univ. of Cinicinnati, Cincinnati. OH A ith increased work and cytosolic [Ca2+] ([Ca2+]i), increased mitochondrial 15267-0575, Lovola ITniv, ('hicago. IH. Ohio State Univ, Colombus. OH. V [Ca2+] ([Ca2+]m) has been suggested to cause increased [NADH]m and consequently increased rate of oxidative phosphorylation (ox.phos). As we have Itutlheninilll red (RR) is a ssell-knosn inhibitor of Ca2+ uptake into NMiTOchondria shown previously, increased work caused an initial drop of [NADH],, followed by a slow recovery and an overshoot after work was returned to control (see Fig). int ritro. Hicoseser. its ntilitv as an inhibitor of Ca2+ uptake into MIITO in rivo Here, we have correlated the rise in [NADH]m with [Ca2+]m by simultaneously or iri situ in inttact cells is limited becatise of its inhibitorv effect on sarcoplasnlic measuring fluorescence of NADH and the Ca2+-sensitive dye Rhod-2, which reticrilrim (SR) Ca2+ release process. Ru360 has beeti shosvn to be a potent inhibitor preferentially loads in the mitochondria. Work, average Force and average of Ca2+ uiptake iinto IITO in vitro. We studied its effects on Ca2+ uptake and [Ca2+] were increased by increased pacing w x Na+-induced (a2+ effltux fromii MIITO. Ca2+ uiptake and Ca2+ release from the SR of frequency. Immediately following a work jump, -l, Hi¢b44s5. perriieabilized cardiorinvocvtes, Na+/Ca2+ exchange from sarcolemmal (SL) vesicles, 0.25-42 Hz, [NADH]m fell while [Ca +]m was still low, I, arid actomnsosin-ATPase iii mvofibrils isolated from rat heart. Ru360 was more suggesting a Ca2o-independent rise of the ox.phos potelnt (IC50=0.162 sNI) than RR (ICso=6.6 nrI) in inhibiting Ca2+ uptake into rate. Following continued stimulation, [NADH]m Recovery 0IITO. 0i3RBu360 bouind to isolated MITO with high affinity (Kd=0.2 nM, B,,, = recovered while [Ca52m slowly increased, consistent . .. , 1 38 fiiiols/itig). 10pmn Rur360 produced no effect on any of the other processes studied. with Ca2+-dependent stimulation of NADH production. Also, it prodtuced no effect on voltage-dependenit CaZ+ channels, cytosolic Caz± or When work was lowered (2-+.25 Hz) [NADH]m, . conitractioin trarisierits of sinigle cardiac tnvocytes. i13Ru360 was takeni up by isolated initially rose while [Ca0m remained high, consistent nisocstes in a bi-pliasic marinier. Furtlher. 101urn Ru360 outside the intact soltage- with continued stimulation of NADH production while lamped senitricular msocstes for 30 ruiin presented MITO Ca2+ uptake induced the ox.phos rate was reduced. As [Ca2 ]m fell e I [Ca [NADH]m returned to control value. On average, the tc, work bI sers stronig stimtilatioti (+l10mV) of Ca2+ influx via Na+/(a2+ exchatnge. NVe A01f conclude that Ru:360 is quite specific in blocking of MITO Ca2+ uptake and can be [Ca2+]m signal increased by 29% (T = 25 sec), while nised iin intact cells. [NADH]m recovered by 12% (x = 39 sec). -; We conclude: Ca2+ acts as a signal in controlling the mitochondrial energetic state during increased work.

Th-Pos213 Th-Pos214 MATHEMATICAL MODEL OF INTRACELLULAR COMPARTMENTALIZED ENERGY RAPID DIFFUSION OF GREEN FLUORESCENT PROTEIN IN THE TRANSFER: ITS USE FOR ANALYSIS OF EXPERIMENTAL DATA ((Saks V.A., Aliev M.K., Dos Santos P., Diolez Ph., Canioni P., Bonoron-Adele S., Besse P.)) Joseph Fourier MITOCHONDRIAL MATRIX. ((Arthur Partikian, Bence P. 61veczky, R. Univcrsity of Grenoble and INSERM Unit 441, Bordeaux, France). Swaminathan and A.S. Verkman)) UCSF, CA 94143. The mathematical model of compartmentalized energy transfer was constructed on the basis of experimcntal data on comparunentation of the creatine kinase (CK) activity in muscle cells, It is thought that the high protein density in the mitochondrial matrix results in ATP producLion in matrix, ATP-ADP translocation, production of phosphocreatine (PCr) in the coupled mitochondrial creatine kinase reaction, limited permeability of the outer severely restricted solute diffusion and 'metabolite channeling' from one enzyme mitochondrial membrane porin channels for adenine nucleotides, diffusion of substrates in the to another without free aqueous-phase diffusion. To test this hypothesis, we myoplasmic space and regeneration of ATP by myoplasmic CK. 31PNMR was used to measured the diffusion of green fluorescent nt protein (GFP) expressed in the quantitate high energy phosphates, [Ca2+1-linked changes in mechanical output and myocardial mitochondrial matrix of fibroblast, liver, skeietal muscle and epithelial cell lines. oxygen consumption (MVO2). Spectra acquisitions were performed before and after blockage of of GFP with a 100x micron encrgy rcscrve achieved by a 98 % decrease in CK activity obtained by perfusion of Spot photobleaching objective (0.8 spot diameter) iodoacetamide (IA), in glucose (G) and pyruvate (P) langendorff perfused rat hearts. gave a half-time for fluorescence recovery of 15-19 ms with greater than 90 % of Before IA [ATPI [PCRI [CrI RPP MOV2 the GFP mobile. As predicted for aqueous-phase diffusion in a confined G+LICa2+1 11.8 16.7 11.7 31900 35.3 compartment, recovery was slowed or abolished by increased laser spot size or G+H[Ca2+1 11.3 13.6 14.8 50400 52.7 After IA bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach G+LlCa2+1 10.3 9.9+ 18.5+ 25600 34.2 data using a mathematical model of matrix diffusion gave diffusion coefficients of G+H[Ca2+1 8.4 9.6+ 18.8+ 21900+ 30.2 2-3 x 10-7 cm2/s, only 3-4 fold less than that for GFP diffusion in water. RPP=Ratc Prcssurc Product (mmHg/min);MVO2 (mmolO2/min/gdw);[ATPI [PCrI and [CrI of GFP rotation time-resolved a rotational are exprcssed in mM; lCa2+l:L=l.25mM ;H=3.5. Measurement by anisotropy gave Increase in mechanical performance was accompanied by more pronounced increase in ATP correlation time of 23.3 + 1 ns, similar to that of 20 ns for GFP rotation in water. hydrolysis products in G than in P perfused hearts. Energy reserve blockage results in complete The rapid and unrestricted diffusion of solutes in the mitochondrial matrix abolition of contractile (and oxygen consumption) reserve with both substrates.These data were suggests that metabolite channeling is not required to overcome diffusive interpreted according lo a mathematical model of compartmentalized energy transfer in the cell. Also, the modcl was used to analyze the published experimental data on the transgenic mouse barriers. Clustering of mitochondral enzymes may thus occur not for metabolite deficient in CK. The results conform to non-equilibrium well-coordinated functioning of channeling, but for establishing an uncrowded, relatively enzyme-free aqueous diffcrent CK isoenzymes in the muscle cells. space in which solutes can freely diffuse.

Th-Pos215 Th-Pos216 THE RAPID MODE [RaM] OF MITOCHONDRIAL CALCIUM Photoactivated calcein generates reactive oxygen species (ROS) that are TRANSPORT ((L. Buntinas. R. Elisees. K.K. Gunter. T.E. Gunter.)) capable of changing Ca2+ accumulation dynamics in isolated rat liver Department of Biophysics. Unisersity of Rochester. Rochester. N\' 14642 mitochondria. ((Renken, C., Eriksson, O., Ricchelli, F., Jori, G., and Bernardi, P.)) CNR Centers for the Study of Biomembranes and Metalloproteins and Departments of sing the svstem developed bs Sparagna. et al. for creating and measuring calcium Biomedical Sciences and Biology, University of Padova, Padova Italy pulses in a cuvette similar to those found in viso. wce discovered a nes mode of calcium uptake in mitochondria. The duration of this transport is <.2 sec and has Isolated rat liver mitochondria were incubated in a cuvette with 40 pM calcein and exposed been to visible light at a power of 0.3 W/cm' for a duration of 10 minutes. Ca2+ accumulation obsersed in heart. liser and brain mitochondria. Evidence for this Rapid Mode and retention was then followed with Arsenazo III in a dual wavelength spectrophotometer. (RaMI) is indicated bh making pulses of various duration and measuring net calcium Mitochondria exposed to light and calcein took up less Ca2+ than control mitochondria uptake by mitochondria suspended in solution and placed in the cuvette. A non-zero or mitochondria exposed to calcein or light alone. Only mitochondria treated with both value for calcium uptake is found wshen extrapolating to zero pulse wvidth. Mlultiple light and calcein rapidly lost accumulated Ca2+. Both Ca2+ accumulation and retention by pulses of calcium shossed considerably greater uptake than a single pulse of the same these mitochondria were reverted to the control pattern by the addition of Cyclosporin A, total duration and pulse magnitude. Control experiments ruled out calcium binding suggesting that treatment with light and calcein activated the mitochondrial permeability or exchange. The RaM behaves very differentls in heart and liver mitochondria. transition pore. To study the mechanism by which calcein may be affecting the pore, In heart mitochondria ANIP and spermine both had an inhibitory effect on RaMI. calcein at pM concentrations was incubated in a solution of PBS, and 02 consumption was is hile liver mitochondria swere not effected by AMIP and spermine wcas an activator of measured. Illumination of calcein with the same power density initiated a process of 02 uptake by the RaNI. Ruthenium red had little effect on uptake via the rapid mode in consumption, which was enhanced by the addition of photosensitive aminoacids such as His heart mitochondria. In liver mitochondria. ruthenium red inhibited both the RaM or Trp. The addition of 1 mM NaN3 inhibited 02 consumption by His, while even 100 mM and the but different NaN3 failed to inhibit 02 consumption by Trp. These data suggest the presence of mixed uniporter. oser concentration ranges. While resetting RaM photosensitization mechanisms, involving singlet oxygen and possibly other ROS which may in liver mitochondria can take as little as .75 sec. it takes the order of a minute explain the effects observed on the pore. We note that the total amount of energy incident to reset RaM in heart mitochondria. A rapid efflux mechanism has been observed on our samples was 6 pJ/pm2. A confocal microscope focusing 10 mW of energy into an folloswing rapid uptake. under non-physiological conditions. Sparagna. G.C.. K.K. area of less than 1pm2 delivers 10 fJ/pm2/msec of incident energy to the sample. Thus, Cunter and T.E. Gunter. A System for Producing and Monitoring in Vitro Calcium probe-dependent mitochondrial alterations are a potential source of artifacts in confocal Pulses Similar to Those Observed in Vivo". Anal. Biochem. 219 pp.96-103. 1994 microscopy of isolated cells. Sparagna. G .C.. K.K. Gunter and T.E. Gunter, "Mitochondrial Calcium Uptake from Physiological-type Pulses". J. Biol. Chem. 270 No. 46 pp.27510-27515. 1993 Gunter. K.K. and T.E. Gunter. Transport of Calcium by MIitochondria'. J. Bioen. and Biomem. Vol. 26. No. 3. 1994 MITOClFlONDRIALMvFCASAs CHANNELS(UANIN5 rANfIAND-PPHYSIOLOGYJ fJ AA A383i3R3y Th-Pos217 Th-Pos218 MitoKTp REGULATES STEADY-STATE MATRIX VOLUME DIFFERENTIAL ABILITIES OF THE 3 MOUSE VDAC ISOFORMS ((M. Jaburek, P. Paucek, and K D. Garlid) Dept. of Biochemistry and MoL Biology, TO CONFER PERMEABILITY TO THE MITOCHONDRIAL Oregon Graduate Institute of Science and Technology, P. 0. Box 91000, Pordand, OUTER MEMBRANE. ((Xiaofeng Xu. William K. Decker. William J. Craigen OR, 97291-1000). and Marco Colombini.)) Labs. of Cell Biology, Dept. of Zoology. Universitv of Maryland, College Park. MD 20742 and Dept. Molecular and Human Genetics. We have hypothesized that opening and closing mitoK-w wil cause the mitochontdial Baylor College of Medicine, Houston, TX 77030 matrix to swell and contract, and that matrx volume should oscilate between steady states of zero net K flux, maintained by the K+/H+ anniporter. We have now The mouse has 3 genes that express 3 isoforms of the mitochondrial channel confirmed this conjecture directly in isolated rat liver mitochondrna (Figur). called VDAC: VDAC1, VDAC2. and VDAC3. Each of these was introduced (via a Mitochondria were brought into steady state volume (1.7 p1 water/mg) during single-copy plasmid) singly into yeast cells whose major VDAC gene (POR 1) had + in K+ been knocked out. Each complements to variable degrees the temperature sensitive respiration (succinate rotenone) 150 mM isolated mitochondria salts. NlitoKATP is open, because no inhibitory growth phenotype caused by lack of VDAC. However, the show very different levels of outer membrane permeability. Mitochondria containing no additiorm VAL/ ligands are present. Addition of 0.2 mM ATP mouse VDAC1 are 2 to 3 times as permeable to NADH as those with mouse VDAC2 inhibits mitoKvrp, and volume contracts to 1.4 gl and 4 times as those with mouse VDAC3. This was the case despite the fact that water/mg). Volume is restored to 1.7 ±l/mg when the expression levels and the ability of these proteins to increase the permeability ATP tees mitoK.vn is opened by 20 pM cromakalim. This of liposomes to non-electrolytes were similar. Electrophysiological differences were effect of cromakalim is blocked by 10 RM seen upon reconstitution into planar phospholipid membranes. VDAC1 and VDAC2 glibenclamide, which inhibits mitoKtvnr. MitoKxn' insert readily and have almost the same overall properties except that VDAC2 chan- controls matrix volume over a range of about 0.3 nels are more heterogeneous with lower mean conductance and selectivity. VDAC3 ATP GLY CRiM VAL pl/mg protein, indicating a profound effect on the inserts very poorly and shows little voltage dependence. VDAC3 undergoes alter- volume of the intermembrane space. (Supported by native splicing that introduces a single additional amino acid into the protein. The AHA grant 9630004N to PP and NIH grant extra methionine at amino acid position 39 within the VDAC3 protein does not Ghi31086 to KDG). significantly alter the permeability of membranes containing VDAC3. The large differences in mitochondrial outer membrane permeability likely reflect inherent dif- ferences in the mouse VDAC proteins or differential regulation of the mouse VDAC channels by the yeast regulatory mechanisms.

Th-Pos219 Th-Pos220 ATP TRANSPORT THROUGH A SINGLE VDAC CHANNEL, STUDIED DIRECT EVIDENCE OF THE MITOCHONDRIAL PERMEABILITY BY NOISE ANALYSIS. ((T.K.Rostovtseva, and S.M.Bezrukov)) LPSB/NICHD, TRANSITION PORE IN SINGLE LIVING CELLS ((V. Petronillil 3. P. NIH, Bethesda, MD 20982 Bernardi"3. MI. Canton2, R. Colonnal 3. G. Miotto2 and F. Di Lisal.2.)) CNR Unit for Biomembranes' and Depts of Biochemistry2 and of Biomedical Sciences3. The "molecular Coulter counter" concept (Bezrukov et al., 1994. Nature 370, University of Padua, v.le G. Colombo 3. 35121 PadualItaly 279) has been used to study transport of ATP molecules through a nanometer- scale aqueous pore of the voltage-dependent mitochondrial ion channel, WVe developed a direct technique for monitoring the mitochondrial permeabilitv VDAC. We examine the ATP-induced current fluctuations and the change in transition pore (MTP) in intact cells. When calcein-AM loaded cells were incubated average current through a single fully open channel reconstituted into a planar in the presence of 1 mM CoCl2. calcein fluorescence was quenched in both cytoso- lipid bilayer. At high salt concentrations (IM NaCl), the addition of ATP lic and nuclear compartments, whereas mitochondria fluorescence was unaffected reduces both solution specific conductivity and the channel conductance, but resulting in their appearance as glowing bodies against a dark background. Treat- the effect on the channel is several times stronger and shows saturation ment with MTP inducers caused mitochondrial calcein fluorescence to decrease con- increase in both cytosol and nucleus. the behavior even at 50 mM ATP concentration. These results and simple steric comitant with the fluorescence Although addition of MTP inducers resulted in rates of calcein release svhich wvere consistent considerations indicate strong attraction of ATP molecules to VDAC's aqueous with MTP opening, the rapid decrease of mitochondrial calcein was not completely pore and permit us to evaluate the effect of a single ATP molecule on the prevented by Cyclosporin A (CsA) and in many cases mitochondria retained appre- channel conductance. ATP addition also generates an excess noise in the ionic ciable amounts of calcein. These two features made MTP opening in intact cells current through the channel. By relating low-frequency spectral density of the differ from what can be observed in isolated mitochondria and permeabilized cells. noise to the equilibrium diffusion of ATP molecules in the aqueous pore, we Besides a rapid efflux from mitochondria after treatment with MTP inducers. the calculate a diffusion coefficient D=(1.6-3.3)x10-" m2/s. This is one order of quantitative analysis of calcein fluorescence variations revealed a constant decrease magnitude smaller than the ATP diffusion coefficient in the bulk. This value is of mitochondrial calcein in untrated cells. On-the other hand. in CsA treated cells. in with recent results multi-channel membranes the fluorescence of mitochondrial calcein was quite stable. suggesting that MTP good agreement obtained with under using the luciferinAluciferase method for detection of ATP in small is likely to fluctuate between open and closed states in intact cells resting concentrations (Rostovtseva and Colombini, 1997. Biophys. J. 72, 1954). conditions.

Th-Pos221 Th-Pos222 THE ROLE OF LOW PHOSPHATE CONCENTRATIONS IN REGULATION REACTIVE OXYGEN SPECIES-INDUCED OPENINGS OF THE PERMEABILITY OF MITOCHONDRIAL PERMEABILITY: MODULATION OF MATRIX TRANSITION PORE IN SINGLE ISOLATED MITOCHONDRIA Ca2+ CONCENTRATION. ((Yulia E. Kushnareva, Lisa M. Haley, and Patricia ((J. HOser & L.A. Blalter)) Deptt o Physiology, Sitlch ScoAo oftMedicine, Loyola Uriversity Chicago, Maywood, IL 60153, USA M. Sokolove)) Dept. Pharmacol., Univ. MD Med. Schl., Baltimore, MD 21201. We have used confocal microscopy to image changes in the electrical potential gradient across the inner mitochondrial membrane (AT) in single organelles with the potentiometric indicator TMRE. A'l in single isolated mitochondria from rat heart We have reported that both mitochondrial signal peptides [Arch. Biochem. displayed rapid spontaneous drops to levels not further sensitive to depolarization by Biophys. 336: 69] and butylated hydroxytoluene (BHT) [J Bioenerg ADP or FCCP. The depolarizations were caused by openings of the mitochondrial Biomembr. 28: 199] induce a pore(s) in the inner mitochondrial membrane that permeability transition pore (MTP), a cyclosporin A-sensitive ion channel in the inner membrane. MTP openings depended on laser illumination and dye concentration. Pore is distinct from the classic Ca2+-dependent and cyclosporin A-sensitive gating in the single organelle displayed a variable latency from the onset of laser permeability transition pore (MPTP). One characteristic common to all of these illumination. This variability was likely to be caused by qualitative differences between processes is sensitivity to inorganic phosphate While the stimulatory effect individual organelles, e.g. differences in the content of pore modulators such as adenine (Pi). nucleotides. By averaging the fluorescence response over 30-40 individual of high (>1 mM) Pi levels on the MPTP is well-documented, the role of lower Pi mitochondria we obtained the 'ensemble response'. In the averaged trace MTP was concentrations in the regulation of mitochondrial permeability remains unclear. evident as a gradual decay of AI', with the slope indicating the rate of mitochondrial permeabilization under a given set of Here we investigated the effects of Pi (slmM) on mitochondrial swelling 15- conditions. The light-induced voltage decay induced by triggers of the classic MPTP, by BHT, and by signal peptides, with CAT was significantly slowed by the sulfhydryl the reducing agents dithiothreitol and reduced following results: (1) A hitherto unrecognized ability of low Pi to delay 30 HT glutathion indicating the involvement of classic MPTP induction different was identified. (2) All of the by triggers thiol oxidation in MTP opening. effects of low Pi, delay of the MPTP induction, blockade of BHT-induced -01t ~ ~ x t ~ ~Furthermore, scavenging reactive oxygen swelling, and stimulation of signal peptide-induced swelling, reflect the ability species (RO S) by eit er of to Ca2+ in the mitochondrial matrix. In contrast to MPTP or BHT- 0 100 200 300 butylhydroxytoluene (BHT) or even more Pi complex ts efficiently with catalase had a marked induced permeability, peptide-triggered swelling is inhibited by Ca2+. The data protective effect on AT. Interestingly, ROS- 'Ensemble respose' showing the protectie effect of induced pore opening in the isolated suggest the existence of multiple sites for involvement of Ca2+ in the regulation ROSseevegeersBHTl(5sIW)andcatalases(0.5mg/ml. organelles occurred in the absence of of mitochondrial permeability. [Support: Amer. Heart Assoc. #94007080] elevated [Ca2+]. A12A 4lrTn(Un%N IrA I -V^ANN.1.RZ ANM PUY.CrInlr.nV-

Th-Pos223 DOES THE Ca2" UNIPORTER FORM THE MITOCHONDRIAL PERMEABILITY TRANSI- TION PORE? ((Nickolay Brustovetaky and Janet M. Dubinsky)), Univ. ofMinnesota, Depart.of Physiology, Minneapolis, MN 55455 (Spon. by David Levitt) In search of the molecular identity of the mitochondrial permeability transition pore (PTP), we hypothesized that mitochondrial Ca2+ uniporter under some circumstances might be converted into the PTP. To investigate this hypothesis we used isolated rat brain mitochondria. The PTP induction was monitored by following mitochondrial transmembrane potential (Aytm) with tetraphenylphosphonium (TPP+) and a TPP+-sensitive electrode. In the presence of 3 mM P, addition of 25 PM Ca2" to mitochondria oxidizing 3 mM succinate induced long lasting depolarization. Ap,m was restored on addition of I AM cyclosporin A (CsA) identifying the PTP as a conductive pathway responsible for the depolarization. BSA added before Ca2+ did not prevent the depolarization but enhanced the effect of CsA. The restoration of Asl. with CsA was observed only in the presence of Mg2+. Similar repolarization of the mitochondria was obtained with 100 lsM ADP or ATP plus 1 pM oligomycin with or without Mg2'. GDP, GTP or UTP were ineffective. Ruthenium Red (RR, I iM), an inhibitor of the Ca2+ uniporter, prevented Ca2+-induced depolarization. Surprisingly, RR added after the depolarization very efficiently restored A\p,, CsA added after RR as well as RR added after CsA had no further effect. CGP- 37157 (10 jM), an inhibitor ofNa+/Ca2+ exchanger, did not restore Ai,m ruling out Ca2' cycling as a cause of the depolarization. At a higher Ca2+ concentration (50 pM) the effect of RR was weaker. In these conditions CsA could not recover AN/, by itself but enhanced the ability of RR to restore AI,,. Thus, we propose that following Ca2' overloading, the Ca2+ uniporter can be reversibly converted into a CsA-sensitive conductive pathway, depolarizing brain mitochondria. This pathway may represent the PTP in a low conductive state.

CYCLIC NUCLEOTIDE-GATED CHANNELS Th-Pos224 Th-Pos225 TWO OPPOSING EFFECTS OF UV LIGHT ON THE ACTIVATION OF RELATIVE IMPORTANCE OF THE FINAL BINDING STEP IN PROMOTING CYCLIC NUCLEOTIDE-GATED CHANNELS. ((T.R. Middendorf1, D.A. OPENING OF CYCLIC NUCLEOTIDE-GATED CHANNELS. ((Y. He and J. W. Baylor1 and R.W. Aldrich2)) 1Dept. of Neurobiology and 2Dept. of Molecular Karpen)) Department of Physiology & Biophysics, University of Colorado School of and Cellular Physiology and Howard Hughes Medical Institute, School of Medicine, Denver, CO 80262 Medicine, Stanford University, Stanford, CA 94305. The importance of cyclic channels in visual and olfactory transduction We have used in situ UV modification to investigate the activation nuclcotide-gated mechanism of cyclic nucleotide-gated (CNG) ion channels. UV irradiation is well established, although relatively little is knosvn about how binding of cyclic of excised membrane patches from Xenopus oocytes expressing bovine rod nucleotides to multiple sites causes opening. By using the photoaffinity analog APT- or rat olfactory CNG channels produced dose-dependent, irreversible cGMP to covalently tether cGMP moieties to binding sites, we are able to study channel changes in channel current. Two distinct effects of UV were observed: a activation at the level of individual binding events. Cloned bovine retinal cGMP-gated decrease in the current evoked by saturating cGMP, and a concommitant channel a subunits, or different ratios of a and [3 subunits wvere expressed in X Iaevis increase in the spontaneous (ligand-independent) currents. The UV effect at saturating concentrations of ligand was altered by oocytes and studied in excised membrane macropatches. Atop 80% covalent perturbations that affect the thermodynamics of channel activation. The activation, binomial statistics predicts that the majority of channels that remain to be sensitivity to tJV was lower when: i) 10 uM Ni++ was present on the fully activated will be missing only their last ligand. This allows us to study the last cytoplasmic face of the patch, ii) olfactory, rather than rod, CNG channels binding step in isolation. It has been showvn previously that cAMP is only a partial were irradiated, and iii) cGMP rather than cAMP was used to activate the agonist of the retinal cyclic nucleotide-gated channel. We found that cAMP activates currents. These findings suggest a mechanism for the effect at saturating about 1% of the maximum cGMP-induced current in expressed a subunit ligand concentration: irradiation decreases patch current by lowering the Hosvever, atop covalent efficacy of channel opening. On the other hand, spontaneous currents in homomultimers, and 8% in expressed heteromultimers. 80% rat olfactory channels increased after UV exposure. They were blocked by activation, cAMP becomes a much more potent agonist of the channel: it activates internal Mg++ with the same affinity as currents evoked by saturating about 40% of the remaining current in expressed homomultimers, and 80% in cGMP, thus identifying them as CNG currents. Taken together, these data heteomultimers. Our data indicate that cAMP potency depends on the level of covalent suggest that UV light has two opposing effects on CNG channels. The ability activation. This suggests that the last binding step is not the sole determinant of the of cyclic nucleotides to promote channel opening is decreased, but the channel opening equilibrium. Rather, the quality of each binding event helps to channel's intrinsic ability to open in the absence of ligand is increased. determine the efficacy of opening. (Supported by EY01543, EY06351, the McKnight Foundation, and the Howard Hughes Medical Institute.)

Th-Pos226 Th-Pos227 MOLECULAR MODELING OF THE CYCLIC NUCLEOTIDE BINDING SYMMETRY OF DIVALENT CATION BINDING TO THE PORE OF DOMAIN OF THE [ SUBUNIT OF THE RETINA CHANNEL ((S-P. Scott and A CYCLIC NUCLEOTIDE GATED-ION CHANNEL. ((S.H. Rho and J.C. Tanaka )) University of Pennsylvania, Philadelphia, PA 19104 (Sponsored by C.-S. Park.)) Dept. of Life Science, Kwangju Institute of Science and Technology G.B. Wells). (K-JIST), Kwangju, 506-712, Korea Cyclic nucleotide gated channels (CNGC) are formed from a and [ subunits. A conserved glutamate residue within the pore-forming region has been identified Both subunits have a cyclic nucleotide binding domain in the C-terminal region. as a critical determinant for several permeation characteristics of cyclic nucleotide- Heterologously-expressed subunits produce no current in response to cyclic gated (CNG) channels. In the case of bovine retinal CNG channel, the residue nucleotides while a subunits express nucleotide-activated currents. Previous (E363) determines both the affinity and the size selectivity of external divalent molecular modeling of the a subunit binding domain predicted that residues 61 on cation binding to the conduction pore constructed by a homo-multimer (likely a strand [5, 83 on strand [37, 119 and 127 on the Ca helix (CRP numbering) interact tetramer) of channel subunit. In this study, we investigated the symmetry of divalent with the purine. Recent mutagenesis work supports these predictions. cation binding using dimeric channels in which two retinal CNG channels were Molecular modeling of the [ subunit binding domain of human retina CNGC connected tandemly with a short linker. The dimeric channels were expressed in predicted residue interactions with the purine nrng. Of the residues predicted to Xenopus oocytes and their permeation characteristics were studied using tight-seal interact in the a subunit, Thr 83 and Asn 127 are retained. Lys 119 is not predicted to patch clamp method. When examined at the single channel level, a dimeric channel interact with the purine, but Met 123 does interact. The Met interaction is similar to (for example, D-E, a dimeric channel of high-affinity mutant, E363D, and the wild that which occurs between a methionine and a conjugated ring of Phe or Tyr in type) exhibited single channel conductance and gating characteristics unique to the channel protein cores. The a subunit has an Ile in this position, which is unable to interact those of homomeric wild type and E363D. Divalent cations blocked with the Significantly, Leu 61 no longer interacts with the purine via ring- currents through both E-D and D-E dimers with affinity in between that of wild purine ring. exact of various the residues of the a subunit models. type and E363D mutant channel. Since we can measure the affinities ring interactions as predicted between aromatic E-D or D-E, we could use them to reveal the We suggest that a ring-ring interaction with the purine is essential for inter-domain divalent cations for WT-WT, D-D, and [ symmetry of binding at the site composed of (four) carboxylates provided bv each communication mediated through the [5 strand. In conclusion, the subunit likely those channels fell halfway bind the but the lack channel subunit. The blocking curve of Sr2+ for hybrid uses three amino acids, Thr 83, Met 123, and Asn 127, to ligand; between the two parent channels but that of Mg2+ closer to the high affinity parent of an interaction with the [35 strand may account for the inability of the homomeric [3 curve. These results strongly suggest that the divalent cations of different size channels to be activated with cyclic nucleotides alone. may interact with the carboxylate groups in the external binding site with different geometrical coordination. -~ ~~ ~ ~~~~~YLCNCETD-AECYCLIC NUCLEOTIDE-GATED CHANNELSHNESA8 A385 Th-Pos228 Th-Pos229 DO PERFUSION-RELATED CONDITIONS CONTRIBUTE TO THE CYCLIC NUCLEOTIDE-GATED CHANNELS: PROPERTIES OF APPARENT DIFFERENCES BETWEEN CLONED AND NATIVE CYCLIC CYSTEINE-SUBSTITUTED MUTANTS IN THE PORE REGION. ((A. NUCLEOTIDE-GATED CHANNELS? ((S. E. Gordon, J. 1. Crary, A. L. Becchetti, P. Roncaglia, K. Gamel and V. Torre.)) Biophysics Sector, SISSA, rrieste, Italy. Zimmerman.)) University of Washington, Seattle, WA 98195-7290 and Brown University, Providence, RI 02912 We applied cysteine-scanning mutagenesis to the aminoacid residues (V348-S371) in the P region of the a subuiiit of the cGMP-gated channel, cloned from bovine retina. Mutant Several properties of expressed homomultimeric rod cyclic nucleotide-gated ion channels cRNAs were expressed in Xenopus oocytes. Single-channel or macroscopic currents were have been reported to differ from those of native rod channels. Hypotheses to explain these measured in inside-out patches, 1-4 days after injection. We have studied the following differences have included modulation present in one case but not the other contributions mutants: V348C, S350C, L351C, T359C, T360C, 1361C, T364C, P366C and S371C. In all of by the beta subunit to native channel properties, and species differences. Ve have found an them, the dose-response to cGMP was poorly affected. The ion selectivity of S350C, T364C artifact of a perfusion configuration that appears to exaggerate some observed differences and S371C was similar to that of w.t. channel (Na = K > Rb = Li > Cs). T359C and P366C' between homomultimeric channels and native rod channels. Using a system in which metal selectivity was similar to that previously measured in E363S, E363A and E363G, with needles were used to connect the solution reservoirs to the cell chamberswe have found increased permeability to the large alkali cations. In symmetric sodium solutions, the w.t. a sag in outward current at positive potentials and a small increase in inward current at channel shows linear macroscopic currents, the single channel conductance is about 28 pS. negative potentials during 100-msec pulses. These effects appeared to be caused primarilv The open probability (Po) in saturating cGMIP is 0.8. The macroscopic currents in mutants by an unidentified blocking agent released from the metal needles. The effects varied in T359C and T364C were linear and maximally activated by 1 mM cGMP. However,in T364C magnitude. but were increased by exposure of the needles to millimolar levels of EDTA or at negative membrane potentials the channel openings were briefer and more flickering EGTA, and were not eliminated by switching to a different brand of needle. Removal of than at positive ones, with the appearance of conductance substates. The rnaximal Po all metal from the perfusion pathway, including embedding the AgCl pellet in agar. yielded was not different in the two conditions (0.8). Such behavior resembles that observed in channels more similar in behavior to native channels: they had an increased apparent affinity E363D and T364M. T360C had lower Po and inward rectificatioii (0.1 at positive and 0.3 for cGMP, increased maximal activation by cAMIP, greater voltage-dependence of activation. at negative potentials). Curreiits from S350C were strongly outwardly rectifying (Po < and striking gating kinetics. Ve suggest that the voltage-dependent inhibition by the metal- 0.05 at negative and > 0.8 at positive potentials). 1361C had a very low Po (less than derived artifact masked or distorted the true properties of the expressed homomultimeric 0.05) at all potentials and small macroscopic currents with no rectification. Ctirrenits from channels. The absence of this artifact in studies of the native channel could reflect either a V348C, P366C and S371C did not rectify.Thus, cysteine mutations in the P region are not difference in types of perfusion systems used to study native vs. homomultimeric channels functionally neutral. Moreover, currents measured in the presence of sulphydryl-specific and/or a reduced sensitivity of beta-subunit containing channels to the unidentified blocker. reagents show dependence from gating state and membrane potential.

Th-Pos230 MOLECULAR MECHANISM FOR MODULATION OF OLFACTORY CYCLIC NUCLEOTIDE-GATED CHANNELS BY CALMODULIN ((M. D. Varnum and WV. N. Zagotta.)) Dept. of Physiology and Biophysics and Howard Hughes Mledical Institute, University of Washington, Seattle, WA 98195 Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons and retinal pho- toreceptors are composed of four homologous subunits, each of which contains a single cyclic nucleotide-biniding site. Cyclic nucleotide binding initiates conforma- tional changes that lead to channel opening. To investigate the role of interdomain and intersubunit interactions in CNG channel activation and regulation, we have expressed various CNG channel intracellular domains as fusion proteins in bacte- ria. We observed a specific interaction in vitro between the amino-terminal domain (RolfN) and the carboxyl-terminal domain (RolfC) of the rat olfactory CNG chan- nel. Calcium-calmodulin, which modulates CNG channel activity during odorant adaptation, blocked this interaction. Localization of regions necessary for the inter- action indicated that the cAMP binding domain in the carboxyl-terminal region of the channel was sufficient for the interaction. Furthermore, deletion of amino acids 62-91 of RolfN, previously shown to constitute part of the calnmodulin binding site (Liu et al., 1994), disrupted the interaction in vitro and altered the gating properties and calmodulin sensitivity of expressed channels. We propose that calmodulin regu- lates CNG channel activity by disrupting the interaction between an amino-terminal autoexcitatory domain and the carboxyl-terminal gating machinery.

SYNAPTIC CHANNELS AND MEMBRANE RECEPTORS Th-Pos231 Th-Pos232 STOCHASTIC MODELING OF FACILITATED NEUROSECRETION BUILDING A BILAYER MODEL OF THE NEUROMUSCULAR ((M.Bykhovskaia, M.K.Worden, J.T.Hackett.)) Dept. of Mol. Physiol. and Biol. SYNAPSE. ((D. J. Woodbury.)) Department of Physiology, WVayne State Phys., University of Virginia, Charlottesville, VA 22906-10011. University School of Medicine, Detroit, MI 48201 Presynaptic processes of quantal mobilization and release are simulated using the The design and construction of a simple model of synaptic transmission are pre- Monte-Carlo method. The simulation is based on the stimulus-dependent model of sented. The construction is divided into five phases: (1) vesicle-membrane fusion neurosecretion, which incorporates an exchange between the total store and the im- without proteins, (2) using proteins to enhance fusion, (3) detecting neurotrans- mediately releasable store of quanta. Each presynaptic action potential is suggested mitter release, (4) activating fusion with Ca2+, and (5) controlling Ca2+ entry with to enhance both quantal mobilization and release. The simulated synaptic output voltage-gated channels. The use of planar lipid bilayers for the successful completion successfully reproduces the distribution of quantal content obtained by extracel- of phases 1, 2, and 5 is reviewed and the feasibility and uncertainties of phases 3 and lular recordings of synaptic currents from lobster neuromuscular junctions during 4 are discussed. One solution for phase 3 is to reconstitute the acetylcholine receptor frequency facilitation evoked at different stimulation frequencies (f). The rates of (AChR) into a bilayer as an autoreceptor (as found in an autapse). With this config- exchange between the total store and the immediately releasable store of quanta uration, the planar membrane represents both pre- and post-synaptic membranes. as well as the number of extra vesicles mobilized with each stimulus are assumed The major difference is that ACh, released on one side of the bilayer, diffuses parallel to be independent of f. The release probabilities are assumed to depend on f due along the same membrane to an AChR rather than across the synaptic cleft to the to an increase in the residual calcium. According to this model, frequency facili- post-synaptic membrane. Mathematical modeling shows that with reasonable initial tation occurs mainly because fewer quanta are demobilized from the immediately conditions, there is a high probability of detecting ACh release following fusion of releasable store as the period between successive stimuli shortens. For comparison, synaptic vesicles with a AChR-containing bilaver. The inevitable construction of experimental synaptic output was fit by non-uniform binomial statistics. which as- a full working model of the synapse will mean that the minimal structures neces- sumes quantal release from a fixed number of active zones with non-uniform release sary for synaptic transinission are identified. This will open the door for defining probabilities depending on f The Monte-Carlo simulation fits the experimental regulatory and modulatory factors of transmitter release. Supported by NIH grant data with a fewer number of fitting parameters compared to non-uniform binomial MH50003. statistics, therefore supporting the stimulus-dependent mobilization model. A386 ZI.awOAlzs~V SVNAPTICA&%, ('ANIICHANNELS ANIDIIANT MEMBRANEMMUAWrivuLMIr DUVJCWFfAyLDRECEPTORS Th-Pos233 Th-Pos234 Lateral redistributionof postsynaptic receptors provides NERVE GROWTH FACTOR ACUTELY INHIBITS CHEMICAL TRANS- activity-dependent control of synaptic efficacy ((L.P.Savtchenkot, MISSION VIA POST-SYNAPTIC TYROSINE KINASE RECEPTORS IN THE D.A.Rusakov2, S.M.Korogodt.)) ILab biophysics, Dniepropetrovsk State SQUID GIANT SYNAPSE. ((H. Moreno, M.Sugimori, and R.Llinas)) Department of Univ., Physiology and Neuroscience, New York University Medical Center, New York, NY Ukraine 2Dep.of biology, the Open Univ., Milton Keynes. UK 10016 A novel mechanism of functional synaptic changes including the activity depen- Neurotrophins are regulators of CNS development, synaptic plasticity and neuronal dent alignment of postsynaptic receptors and presynaptic release sites has recently survival, and exert both short and long term effects on synaptic physiology. We been described ( Xie et al., Proc Natl Acad Sci. USA. 1997, 94, 6983), whereby explored the effects of peptide growth factors on the synaptic transmission at the squid high-frequency synaptic activity could increase synaptic efficacy. WN'e explore and giant synapse. Local application of 100 ng/ml ofNerve Growth Factor (NGF) produced a rapid reduction of post synaptic potentials (PSP) amplitude. NGF had no effects on test theoretically a physical basis of this mechanism. This involves individual recep- presynaptic calcium currents (ICa) Since neurotrophins can signal through the tor - channels (AMPAR), which are capable of lateral electrodiffusion in the post activation of tyrosine kinase receptors, (RPTK) genistein a protein tyrosine kinase synaptic membrane. We analyze theoretically and simulate lateral re-distribution (PT'K) inhibitor was used to determine whether NGF modulation of the PSP was PTK of AMPARs driven by: (i) electrostatic interaction with each other; (ii) transient dependent. Intracellular post synaptic microinjection of genistein prevented the potential generated by currents through individual open channels; (iii) stochastic inhibition of PSP upon NGF superfusion. More importantly, the same effect was Brownian diffusion; and (iv) friction in the membrane bilayer. In these condition. obtained upon post-synaptic microinjection of the new class of protein tyrosine kinase simulated synaptic release of neurotransmitter induces clustering ( time scale 5-10 inhibitor SU4984 ( Science 276, 955-960. 1997) while the related compound SU 5402 did not affect NGF effect on PSP at the same concentration (5 gm). These two ms) followed by de-clustering (time scale 40-50 ms) of AMPARs. Given the Gaussian compounds differ in their specificity for PTK's inhibition, for instance SU 5402 is lateral distribution of neurotransmitter in the cleft and given the four-states kinet- FGFRK specific, while SU 4984 exhibit broad specificity. aFGF, EGF, NT-3,and ics of AMPARs, the model predicts an increase in the total post-synaptic current BDNF were also tested. These peptides demonstrated no noticeable acute effect on response when repetitive synaptic activation reaches a frequency of 30-50 1/s. This synaptic transmission as evaluated by monitoring normal presynaptic ICa and the related mechanism is considered to be a plausible cadidate for the expression of longer-term PSP. These data indicate that a post synaptic Trk-A type RPTK modulates chemical that which are normally anchored transmission probably by inhibiting glutamate receptors. The results are also in synaptic efficacy changes, provided AMPARs, in agreement with experiments showing that changes of tyrosine phosphorylation levels in the post-synaptic membrane, are temporarily de-anchored in the course of inten- squid giant synapse influence synaptic transmission through pre and post synaptic sive synaptic activation, with a possible role of a transient increase in post-synaptic mechanisms (PNAS 94, 1990-1994. 1997). Acknowledgrment: NS13742. calcium.

Th-Pos235 Th-Pos236 ION CHANNELS IDENTIFIED BY PATCH CLAMPING WHOLE CLONING AND HETEROLOGOUS EXPRESSION OF SYNAPTIC VESICLES FROM TORPEDO CALIFORNICA ((M. L. GLUTAMATE-GATED CHLORIDE CHANNELS FROM FLEAS: Kelly, D. A. Przywara, and D. J. Woodbury.)) Departments of Physiology and HIGH AFFINITY ACTIVATION BY IVERMECTIN PHOSPHATE Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201 ((A. Etter, R. Brochu, D. Cully, J. Friedman, P. Paress, J. W. Warmke, and C. J. Cohen.)) Merck Res Labs, Rahway, NJ 07065. Synaptic vesicles are quite small (90 nm diameter), and are generally considered too small to patch clamp. However, using very fine pipettes, we patch clamped pure The avermectins target glutamate-gated Cl channels (GluCl) in nematodes and synaptic vesicles (SV) from Torpedo californica. This resulted in the observation insects. A single GluCI subunit has been cloned from the cat flea, Ctenocephalides of a multiple conducting anion channel and a second channel. The identity of this felis, by homology to the previously characterized Drosophila GluCl. The two en- second channel is unclear but may represent a subconductance state of a previously coded proteins are ;85% identical. Heterologous expression of this flea cDNA in identified SV channel (Kelly and Woodbury, 1996, Biophys. J. 70:2593-2599) or an Xenopus oocytes produces homomeric GluCl channels (CfGluCl1) that can be di- altered state of a SV transporter. To test the later hypothesis, the proton pump rectly activated by either glutamate or ivermectin. Most biophysical properties of inhibitor, DCCD, the acetylcholine transport blocker, vesimacol, and ATP were CfGluCll are similar to those exhibited by Drosophila GluCl. Both channels are ac- tested on membrane patches. DCCD and ATP (1 mM) activate the anion channel. tivated by glutamate with an EC50 t10 ILM. Neither channel is activated by GABA, It is concluded that the anion channel represents an altered state of the hydrogen glycine, histamine or acetylcholine, which are agonists for some insect ligand-gated pump. The second channel appears to regulate osmotic conditions within the vesicle. chloride channels. Prolonged exposure to glutamate causes channel desensitization; Supported by NIH grant MH50003 to DJW. the rate of desensitization increases as the membrane potential becomes more neg- ative. Ivermectin-phosphate (IVM-P04) directly activates the channel and slows channel desensitization. The apparent potency of IVM-PO4 is voltage-dependent; EC50 ;z3 nM at 0 mV and w40 nM at -80 mV. The kinetics of channel activation by IVM-PO4 indicate that there are at least 3 binding sites for drug per channel. IVM-PO4 also potentiates the effect of glutamate, but only in depolarized cells and only at concentrations comparable to those that cause direct activation.

Th-Pos237 THREE DIMENSIONAL MODELS OF GLUTAMATE RECEPTORS ((XI.J. Sutcliffe, A.H. Smeeton. Z.G. *'o, R.E. Oswald.)) Dept. Chemistry, Leicester Universitv. Leicester. LEt 7RH, UK and Dept. of Pharmacology, Cornell University, Ithaca, New York 14853. USA

Structural models of glutamate receptors have been produced as part of a multidisci- plinary study of neuronal function (both ligand/receptor interactions and ion transport) at the atomic level. Glutamate receptors are the primarvexcitatorvneurotransmitter receptors in vertebrate brain and play an important role in a weide variety of normal and pathological processes. The models have concentrated on the agonist binding and transmembrane do- mains of ionotropic glutamate receptors (iGluRs), and have aided our understanding of the molecular determinants of ligand binding and channel activity. Experimental determination of the transmembrane topology was an essential prerequisite for the modeling studies (Wo & Oswald (1994) Proc. Nati. Acad. Sci. 91:7154). The model building process involved a combination of homology modeling, distance geometry, molecular mechanics. protein-ligand and protein-protein docking. electrostatic calculations and manual adjustment, in conjunc- tion with restraints from site-directed mutagenesis, ligand binding and electrophvsiological studies. The initial models (Sutcliffe et al. (1996) Biophys. J.70:1575) were used to produce hypotheses which were tested experimentally; these models have beensubsequently refined as part of an extremely effective multidisciplinarystudy using an iterative molecular model- ing/experimental verification cycle in which restraints derived from experimental studies are used at all stages. and the findings from one round of modeling are used as restraints in the next. Bystudying a variety of agonists and antagonists. details have been built up of those residues involved in ligand binding and the role of agonist binding (i.e., agonist-induced conformational change) in channel gating. The models also aid our understanding of the conductance properties of the channels. PEPTIDE CHANNELS A387 Th-Pos238 Th-Pos239 USE OF 5-FLUORO TRYPTOPHAN AND SOLID STATE NMR TO CONFORMATIONAL PREFERENCE OF [VAL5]gLW and PROBE THE ROLE OF LONG-RANGE ELECTROSTATIC [VAL5ALA8]gLW CHANNELS IN PHOSPHOLIPID BILAYERS ((S. INTERACTIONS IN GRAMICIDIN A CHANNEL. ((NI. Cotten'. D. Shobana', G. Saberwal', O.S. Andersen', D.V. Greathouse2 and R.E.Koeppe Busath2. T. A. Cross'.)) 'Department of Chemistry. National High Mlagnetic Field 12.)) 'Cornell University NIedical College, New York, NY 10021 and 2Univ. Laboratorv. Florida State University. Tallahassee. FL 32306-4005 2Department of Arkansas, Fayetteville, AR. Biologa. Brighanm Young University. Provo. Utah 84602 The gLNVs are analogues of gramicidin A (gA) in which the Leu and Trp residues are Lowering the energy barrier of ion transport in conducting channels requires specific interchanged relative to their positions in gA. The gLWs themselves form channels functional niodules for swhich a structural pattern may be identified. Interestingly. that are double-stranded (DS), as well as right-handed (RH) and left-handed (LH), some amino acids have been assigned both structural and functional roles. Here, wce single-stranded (SS) dimers. An Ala' -Val5 substitution precludes the formation of have used solid state N.NIR to study possibly functionally-significant long-range elec- conducting DS channels. Using Ala5 Val5 substituted gLW's we have determined trostatic interactions betwveen indole dipole of tryptophans and a cation monopole the conformational preference between LH and RH, SS channels in bilayers formed in gramicidin A (gA), a channel-forming polypeptide of knoscn structure and well- by DOPC and DPhPC. For V5gLW, the average lifetimes of the RH,SS channels are characterized channel properties. The orientation and dynamics of its tryptophans 20 ms and 30 ms in DOPC and DPhPC bilayers, respectively: the corresponding side chains which project outside of the pore are believed to be critical for func- values for LH,SS channels are 1100 ms and 1500 ms. In the case of V5A8gLW, tion. In the present study. substituting 5-fluoro trvptophan for tryptophan in gA the average lifetimes of the RH,SS channels are 20 ms and 24 ms in DOPC and has offered a wcay to vary the indole dipole moment components while minimizing DPhPC. respectively; the corresponding values for LH.SS channels are 730 ms and the structural effects of the substitution. Gramicidins containing one d4-indole 5- 670 ms. The relative appearance rates for LH over RH channels are tin both DOPC fluoro tryptophan at position 9. 11. 13 or 15 plus an '5N-amide nitrogen label have and DPhPC. The free energy difference (LH/RH) for V5gLW SS channels is -3.6 been incorporated into oriented lipid bilavers. The 15.N backbone chemical shifts KCal/mol; in V5A5gLWV, it is about -3 KCal/mol. The results show that the lipids recorded have confirmed that the peptides adopt the channel state conformation. do not significantly affect the conformational preference of V5gLW and V5A8gLW The 2H splittings obtained have been used to constrain the indole and thereafter which justifies the use of molecular dynamics simulation methods to analyze the free their dipole moment orientations. These results wsill be compared to the ones pre- energy differences between RH and LH channels and how they vary as a consequence viously determined for the tryptophans of gA (Hu. WV. and T. A. Cross. 1995. of amino acid substitutions. Biochcmzstry. 34:14147-14153). Possible mechanisms for the indole dipole moment orientation in gramicidin will be discussed to bring a more detailed understanding of the function of channels in terms of their structure at the molecular level.

Th-Pos240 Th-Pos241 INI tLUENCE O5F AN AMIITh-It)-EsTlER Rl PLACEMENT IN TI.E GRAMICImIN CIANNEL POLYLYSINE DECELERATES CHANNEL KINETICS OF NEGATIVELY BACKBONE- ()N 1 AN) CHANNEL STABILITY CHARGED GRAMICIDIN AS SHOWN BY SENSITIZED PHOTOINACTIVATION. )N PEIRMEA31I1TY ((A.V. Krylov'2, Y.N. Antonenko', A.A. Yaroslavov2, T.I. Rokitskaya', E.A. ((Jude, AR*, Providence, LL', Andersen, OS", Greathouse, DV*, Koeppe, RE, Kotova', R.E. Koeppe, I13, D.V. Greathouse3, O.S. Andersen4 )) 'AN Belozersky 11*)) *Dept. Chem. Biochem., Univ. Arkansas, Fayetteville, AR 72701, "Dept. Phys. Chem. Biol. lnst. and 2High Mol. Compounds Dept., Chemistry Dept., Moscow Physiol. Biophys.. Cornell Univ. Medical College, New York, NY 10021. Univ., Moscow 119899 Russia; 3Dept. Chem. Biochem., Univ. Arkansas, Fayetteville, AR 72701; 4Dept. Physiol. Biophys., Comell Univ. Medical College, New York, NY 10021. Amino acid side chains and peptide backbone groups are important for the folding An important factor regulating membrane protein function is the supramolecular and function of soluble and membrane proteins. The methods of site-directed organization of the proteins. We have addressed this issue by studying how mutagenesis and chemical synthesis are used extensively to substitute or modify charged polymers affect the channel formation kinetics of gramicidin A (gA) in side chains. Chemical modifications of the backbone are more difficult, but planar bilayers. gA channels are transmembrane dimers stabilized by hydrogen backbone carbonyl groups may participate directly in the transport of ions through bonds between monomers; the kinetics oftheir formation and dissociation could be cation channels. To assess the of the affected if the channels were organized into larger structures, which could be voltage-dependent importance peptide induced by the binding of extemal polymers. To study the channel kinetics, we backbone for channel properties, we have made use ofgramicidin A (gA) channels, used sensitized photoinactivation, in which current transients are induced by a flash in which the pore wall is formed by the backbone, and have synthesized a modified in the presence ofphotosensitizer. It has been shown previously (Rokitskaya et al. gA with a single backbone amide replaced by an ester bond. The ester analogue of Biochim. Biophys. Acta 1275:221, 1996) that the time course of the flash-induced formyl-Val-Gly, designated f-Val-O-Gly, was synthesized, characterized, and current decrease in most cases is fit by a single exponential decay with a time incorporated at the N-terminal of gA by solid-phase synthesis. The resulting [f- constant (tau) that correlates well with the single-channel lifetime. Addition of polylysine does not affect tau for gA channels, but causes a substantial increase in Val-O-GlylgA has the correct mass of 1882 ± I and forms both homodimeric tau for channels formed by O-pyromellityl-gA (with three negative charges). This channels and hybrid channels with gA in planar bilayers. The single-channel effect is reversed by addition of the polyanion polyacrylate. Calcium ions do not conductance (average duration) in I M CsCI at 200 mV is 4 pS (1 ms), compared affect the tau. The deceleration ofthe photoinactivation kinetics is attributed to the to 45 pS (500 ms) for gA, and 12 pS (4 ms) for gA/[f-Val-O-Gly]gA hybrid interaction of positively charged polymer molecules with negatively charged channels. The results demonstrate the critical importance of the backbone amide pyromellityl-gA, which would stabilize the channel state by reducing the rotational and lateral mobility of monomers and dimers, and thus increase the single channel carbonyl groups for channel folding and function. lifetime.

Th-Pos242 Th-Pos243 POLARIZABILITY EFFECTS ON IONIC BEHAVIOR IN A GRAMICIDIN- INFLUENCE OF EXPLICIT BULK WATER ON IONIC SELECTIVITY IN LIKE CHANNEL. ((K.A. Duca' and P.C. Jordan')) Program in Biophysics' GRAMICIDIN CONFORMERS. ((V.L. Dorman, M.B. Partenskii, and P.C. and Dept. of Chemistry'", Brandeis Univ., Waltham, MA 02254-9110, USA Jordan)) Dept. ofChemistry, Brandeis Univ., Waltham, MA 02254, USA Previous work showed that explicit inclusion of polarizability significandy in- Using a semi-microscopic Monte Carlo approach, we have described ionic se- creases correlational effects in a gramicidinlike channel (Duca & Jordan, Biophys. lectivity in the head-to-head (HH) [6.3-helical and anti-parallel double stranded Chem., 65, 123 [1997]). Here we separately consider how water and helix polariza- (APDS) forms ofgramicidin (Dorman, Partenskii and Jordan, Biophys. J., 70, tion modify structure in the ion-water single file of the head-to-head `6 helix. 121 [1996]; ibid 72, A396 [1997]). That analysis placed special importance on We contrast mid-monomer channel properties for two cations (Na+ and Cs') and electrostatic interaction between channel moieties and bulk solvent. Since ion- four polarizability scenarios: all polar groups polarizable; water polarizable; helix dipole and dipole-dipole forces are poorly described using cut-offs, our earlier polarizable; no polar groups polarizable. To ensure comparability, group dipole moments in non-polarizable cases are adjusted to mimic either mean interaction work, treating bulk solvent as continuum dielectric, provided a rigorous por- energetics or mean group dipoles of the polarizable water-filled channel. With a trayal ofthe long range problem (at the cost ofsuppressing molecular detail at non-polarizable helix, ion-carbonyl correlations weaken and ion-water correlations the channel mouth). The present study (a precursor to extending our method to strengthen; with non-polarizable water, the opposite holds. The consequences of other channel systems) is more realistic and, without loss ofrigor, incorporates a helix dipole fluctuations are quantitatively more important. few explicit bulk water molecules near the channel mouth, the remainder ofthe In studying coordination near minima and maxima in mid-monomer, it appears solvent again being a continuum dielectric; only 25-30 such waters are needed to that the increased translocational energy barrier for Na+ (with respect to Cs') cor- adequately treat long range effects. Here the explicit solvent waters are both relates with differences in translocation induced changes in ion-water and ion- translationally and orientationally mobile; the ion is not tethered to a specific carbonyl correlation. In traversing the local maximum, the shortest Na+-water site. We confirm the cation selectivity ofboth channel conformers and note that distance increases while the corresponding Cs'-water distance decreases; increased the cationic permeation free energy profile is less structured than the anionic ionic interaction with the innermost carbonyl can not compensate for the energy one; differences between the profiles depend significantly on the effect that po- penalty. larity has on ionic interaction with bulk water. A388 PPIECANLPEPTIDE CHANNELS Th-Pos244 Th-Pos245 THE INFLUENCE OF ELECTROLYTE CONCENTRATION AND CHARGE SIZING TWO POPULATIONS OF CHANNELS FORMED BY OF PHOSPHOLIPID MOLECULES ON VOLTAGE SENSITIVITY OF SYRINGOMYCIN E USING WATER-SOLUBLE POLYMERS ((Yui A. Kaulint, SYRINGOMYCIN CHANNELS ((Ludmila V. Schagina', Yuri A. Kaulin' , Alexander Ludmila V. Schagina2 and Joseph G. Brandt)) 'Monell Chem. Senses Ctr, Philadelphia, USA; 2II;L M. FeiginW, Jon Y.Takemoto3, John H.Teeter2 and Joseph G. Brand2)) 'Institute of Cytology, Cytol., St Petersburg, Russia. RAS, St. Petersburg, Russia; 'Monell Chemical Senses Center, Philadelphia, USA; 'Utah State University, Logan, USA. We demonstrated earlier that syringomycin E (SRE) fonms in planar lipid bilayera two major popuations ofchannels - "small" and "large" ones that are six-fold different in their conductance. To discriminate between two possibilities: i) the large channels are clusters ofsmall ones and ii) the large We demonstrated earlier (J.Membr.Biol., 1996, 149, 41-47) that syringomycin E (SRE) and small channels are two entirely different stuctur we studied the influence of neutral polymers added to one (cis) side of a lipid bilayer forms potential-dependent ion channels. A shift of such as poly(ethylene glycol)s (PEGs) of different molecular sizes on channel conductance. Our voltage (at cis-side) from positive to negative results in closing of these channels and in a results show that the addition of solutes to _meban bathing solution changes conductance of the decrease in macroscopic conductance over time. In the present study we investigated how this large and small channels in the same way and the aqueotus pore size ofsmall and large channels does response of SRE voltage-induced conductance change depends on the presence of negatively not diflr by more t}ian 2%. The dependence ofthe PEG-induced conductance reduction on polyme charged phospholipids in the bilayer and the concentration of electrolyte in bathing solution. molecular weight allows to conclude that both types of channels have the sieving radius of about I We observed that in bilayers formed from equimolar mixture of DOPS and DOPE bathed in tm. These data, together with the fact that the large and snall channels have the same anion-cation diluted electrolyte solutions (0.01-0.1 M NaCI) the conductance of bilayers modified with SRE selectivity, suggest that large SRE channels are clusters of small ones. Comparison ofour results on is greatly increased over time with positive voltage, and decreased with negative voltage. the influence of PEG on channel conductance to the corresponding alpha-toxin channel data (Macromolecules, 1996, 29, 8517-8522) shows that partitioning of PEG into SRE channel pores However, when we increased the NaCI concentration up to 0.6 M, the conductance of these exhibits much smoother transition between penetrating and excluded polymers. It may reflect membranes remained stable after the switch of potential sign from positive to negative. deviations ofSRE channel pore geometry from a eight cylinder suggesting a conical shape. Moreover, at I M NaCI an inversion of the response was observed (increase in membrane conductance at negative voltage and a decrease at positive). The conductance of bilayers made from neutral phospholipid (1,2-dioleoyl-sn-glycero-3-phosphocholine) and doped with SRE in 0.1 M NaCI, showed the response to voltage changes qualitatively similar to the one that we observed in DOPS/DOPE bilayers at IM NaCl. The results suggest that the charged groups of the lipid molecules facing the water phase participate together with the charged groups of SRE molecules as the voltage sensor.

Th-Pos246 Th-Pos247 CHARACTERIZATION OF A FUNCTIONAL HOMOPENTAMERIC MODEL STRUCTURAL CHARACTERIZATION OF THE M2 NICOTINIC ACETYLCHOLINE RECEPTOR CHANNEL. ((*C. M. Sciortino, AC. S. TRANSMEMBRANE PEPTIDE BACKBONE USING SOLID STATE Walker, ^W. R. Gray, ^1. M. Olivera, *J. Ma, and *K. -J. Shon)) *Dept. ofPhysiology and NMR. ((F. A. Kovacs, S. M. McNiel, J. K. Denny, J. R. Quine,T. A. Cross.)) Biophysics, Case Westers Reserve Univ., Cleveland, OH 44106; 'Dept. of Biology, Univ. National High Magnetic Field Laboratory, Institute of Molecular Biophysics, of Utah, S.L.C., UT 84112. (Spon. by J. Ma). Department of Chemistry and Department of Mathematics, Florida State University, Tallahassee, FL 32306-4005 The nicotinic acetylcholine receptor (nAChR) is a 290 KDa heteropentameric transmembrane protein consisting of acxpy8 subunits. The second transmembrane M2 The ion channel formed by the M2 protein from Influenza A plays an important domains from each subunit associate to fonn a cation selective heteropentameric ion role in viral infection. This tetrameric channel functions as a proton channel and channel. We have chemically synthesized a functional 21 KDa homopentameric model allows the uncoating of the viral RNA. A peptide corresponding to the single pu- channel consisting of five M25 transmembrane domains linked to a peptide template which tative transmembrane segment (M2-TMP) of M2 possesses the basic properties of retains the ability to transport ions. In building the model channel, we used a modular the protein: it adopts a primarily o-helical structure in DMPC and DOPC vesicles, strategy in which M28 and a linear template peptide were built separately, and then linked has ion channel activity, is pH gated, and reversibly blocked by amantadine. Orien- via thiol-maleimido couplings. The functional characterization of the homo-pentamer, tational constraints were obtained from SS NMR by observing "5N chemical shift, presented here, is studied by reconstitution into a planar lipid bilayer separating two wells 15N -'H dipolar and quadrupolar interactions for labeled sites of M2-TMP, which containing asymmetric solutions of KCI; and using voltage clamp techniques to measure were incorporated in both DOPC and DMPC hydrated bilayers, and mechanically channel conductance. The nAChR open channel blockers (e.g. lidocaine, chlorpromazine) aligned between glass slides. Our initial model was based on an ideal helix where are used to test whether the model channel can effectively be blocked. Results show that the the helix orientation in the bilayer was characterized by 2 Euler rotations: p, a homopentameric channel conducts in a cation selective, voltage sensitive manner and has a rotation about the helix axis, and r, the angle between helix axis and the bilayer linear current-voltage relationship. Micromolar concentrations ofchlorpromazine normal. These two parameters provided the basic information required to pack a effectively block the ability ofthe model channel to conduct, similar to concentrations symmetrical array of these transmembrane peptides. This was expanded to include needed to block the native nAChR channel. In addition, the model may be blocked by a mathematically modeled non-ideal helices which were rotated over all possible val- novel y-conotoxin, which has been reported to inhibit the nAChR at a site other than the ues of p and 7, computing the value of the SS NMR interactions for the measured acetylcholine binding site (Shon et al, Biochem. 36:9581-9587, 1997). The y-conotoxin, sites, related to each other by standard helical parameters, and then finding the with five positive charges, has been hypothesized to interact with a negatively charged ring (p, r) pair which best fit the experimental data for that model. A x2 analysis was at the vestibule of the nAChR pore. performed on each model to determine its statistical acceptability.

Th-Pos248 Th-Pos249 BIOPHYSICAL CHARACTERIZATION OF M2GLYR PEPTIDES IN SYNTHETIC PEPTIDES DERIVED FROM THE BRAIN GLYCINE LIPID AND AQUEOUS ENVIRONMENTS. ((Z. Tavakkol, K.E. RECEPTOR FORM CHANNELS IN PLANAR LIPID BILAYERS Mlitchell, C.A. Ambler, J.B. Stucky, T. Iwamoto, J. Tomich.)) Dept. of AND INCREASE CHLORIDE CONDUCTANCE IN MDCK CELLS Biochemnistry, Kansas State Univ. Manhattan, KS 66506 ((K.E. Mitchell, J. Tomich, T. Iwamoto and L. Freeman.)) Dept. of Biochemistry and Dept. of Anatomy & Physiology, Kansas State Univ. WHe bave synthesized a family of peptides based on the transmembrane sequence Manhattan, KS 66506 of the brain glvcine receptor (M2ClyR). We increased the aqueous solubility of these peptides by adding various numbers of lysine residues (1-6) to either the C- Recently, we have shown that a family of peptides derived from the pore-forming or N-terminus. The M2GIyR peptides have been analyzed for their ability to form sequence of the a-subunit of the brain glycine receptor (M2GlyR) increases short- chloride-conducting channels in lipid bilayers. Circular dichroism analysis of the circuit currents and water transport in monolayers of Manin-Darby canine kidney lysine-modified peptides shows that they form random coils in aqueous solution and (MDCK) cells. To enhance solubility, the native M2GlyR sequence was modified o-helical structures similar to the unmodified peptide in SDS micelles or 40% TFE. by addition of lysine residues to either terminus. The mean open times and single- These results indicate that the lysine modifications at either terminus do not dis- channel conductances for C-K4 and N-K4, the M2GlyR peptides modified with four rupt the helical character of the native peptide. Partitioning experiments reveal lysine residues at the C- and N-termini, are similar (5-6 ms and 50 pS). Reversal that the addition of Ivsine residtues to the sequence enhances its ability to partition potentials measured under asymmetric conditions are consistent with the M2GlyR into large unilamellar vesicles (LUV). The effects of ionic strength and lipid compo- channels being anion selective. Also, no channel activity was observed when glu- sition on partitioning are consistent with electrostatic interactions contributing to conate was used as a substitute for Cl-. Antibody to the C-terminus of the C-K4 enhanced partitioning of the lysine-modified peptides. We have also used digestive peptide blocks C-K4 channel activity while having minimal effect on N-K4 activity. enzymes (particularly lys-c) to investigate the orientation of C-K4-M2GIyR peptide In contrast to the unmodified M2GlyR channel, the C-K4 M2GlyR channel shows in LUV. In preliminary studies, the C-terminal lysine residues were susceptible to inward rectification which can be eliminated by direct application of endopeptidase digestion when the peptide was in solution or partitioned into LUV. This indicates lys-c to the bilayer chamber. In MDCK cells, an increase in whole-cell current con- that the lysine residues do not cross the lipid bilayer but remain free in solution. The sistent with an increase in Cl- conductance is observed shortly after introduction knowledge gained in the structure-function analysis of the M2GlyR peptide family is of C-K4 M2GlyR into the extracellular bath solution. Furthermore, the Cl- current significant in the development of a therapeutic agent for treatment of cystic fibrosis. induced in whole-cells by C-K4 M2GlyR shows inward rectification similar to that observed with this peptide in planar lipid bilayers. PEPTIDEapJgUyrA AJLIFl WI CHANNELS4- ALAXILLCHANNU14 I 11 LT.g A707A3899 Th-Pos25O Th-Pos251 ION CHANNEL FORMATION AND MEMBRANE PERTURBATION BY THE ION CHANNELS FORMED BY ALZHEIMER'S DISEASE PEPTIDE AP1-42 ((Y. NEUROTOXIC ALZHEIMER AMYLOID FRAGMENT p25-35 IN ITS Hiraktr*, Y. Kirino, and B. L. Kagan5)) *School of Pharmaceutical Sciences, The AGGREGATED FORM ((Y. Hiralwra, Y. Satoh, T. Suzuki, and Y. Kirino)) School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, University of Tokyo, Hongo, Tokyo 113, Japan. 'Departnent of Psychiatry, UCLA Japan Neuropsychiatric Institute, L.A. CA 90024-1759. The P-amyloid peptide (PAP) is a major proteinaceous component ofsenile plaques and cerebrovascular amyloid deposits found in the brain ofpatients with Alzheimer's disease. Substantial biochemical and genetic evidence implicates amyloid AP peptides in the It consists of 39-43 amino-acids and is reported to be neurotoxic only when it is etiology ofAlzheinees Disease. Various reports have indicated that AP 1-40 can form aggregated. The molecular basis of such aggregation-specific neurotoxicity remains cation sdeetive ion channels in planar lipid bilayers. However, recent evidence indicates unclear. In the present study, we focus on the ion channel hypothesis of the PAP- that AP1-42 is the predominat species in the hallmark senile plaque of Alzheimer's neurotodcity which argues that formation ofion channels by PAP itselfimpairs neuronal cells. We report here that the neurotoxic core of PAP, PAP-25-35 (P25-35), perturbs disease. Furthenrmore, APl-42 is more resistant to deVadation and forms agegates membranes composed ofphospholipids exclusive ofsphingomyelin, and only forms ion inside yssomes ofculured neurons leading to lysosomal disruption and cell death. We chaels in a buffer condition where the peptide spontaneously aggregates and facilitates report here that AP1-42 can form weakly cation selective, voltage-independent ion fiomation ofP-shoet stucture. In the saen caditions, the peptide also exhibits significant hemolytic activity on mammalian erytuhcytes which are poor in sphingomyelin. In channels at neurotoxic concentrations in planar lipid bilayers. The channel exhibits contrast, P25-35 in its nmeric rndom coil structure does not perturb lipid membranes multiple conductance levels. It shows substantial irregularity of activity perhaps significantly, and exhibits no hemolytic activity. Examination of time course of the indicatin a sto dependence on the state ofaggregation ofthe peptides. The properties suggests that large aggegates which precipitate by ultracentrifugation are not ofthis chanml differ substantially from that of 25-35 which is also neurotoxic. The active on membranes. Hence, soluble, probably small aggregates are responsible for the AP membrane-perturbing activity. The ability of P25-35 to interact with membranes highly properties of this channel would likely render it neurotoxic to relevant neurons in correlates with its neurotoxicity reported previously. The present study thus suggests a vivo. novel and intriguing mechanism of ion channel fonnation by the aggregated peptide molecules, which may constitute a molecular basis ofthe peptide's neurotoxicity.

Th-Pos252 ALZHEIMER'S DISEASE AMYLOID P-PEPTIDE [1-401 INDUCES AN INCREASE IN Na' AND Ca2+ PERMEABILITY IN A HUMAN ASTROCYTOMA CELL LINE (J.M. Alarc6n' and E. Rojasl,2) 'Dept. of Physiol. Biophys., Fac. Of Med., U. of Chile and 2LCBB, NIDDK, NIH, Bethesda, MD. Incorporation of Alzheimer's amyloid (3-protein (APP) molecule into arfificial and neuronal bilayer membranes leads to the formation of cation-selecffve channels (Arispe et al. PNAS. 90:10573, 1993; Kawahara et al. Biophys. J. 73:67, 1997). To determine the characteristics of A3P[1-401 channels spontaneously formed across natural membranes after exposure to the peptide, we loaded astrocytoma cells (132 1 NI cell line) with different cation reporter dyes (SBFI for Na and indo-1 for Ca2) by incubating the cells during 1 h in a physiological saline containing 1 FtM of the cell-permeant acetoxymethyl ester form of each dye. Loaded cells were resuspended in physiological saline. For fluorescence measurements we took aliquots (1 cm3) and placed this aliquot in a cuvette equipped with a magnetic stirrer. In astrocytoma cells loaded with SBFI the fluorescence rapidly rose to a saturating level after the application of AOP[140] (5 pM) demonstrating a rapid influx of Na+. Exposure of cells loaded with indo-1 to APP[1-401 (5 pM) also evoked a substantial but smaller fluorescence (405 nm) increase, most likely to be due to Ca2+ entry across APP[1-40] channels. Similar results were obtained with cells plated on glass cover slips. Since these cells express cholinergic muscarinic receptors (¶3protein coupled to phospholipase C), we also compared agonist-induced [Ca J] -rise in the absence and in the presence of muscarine. We noted a substantial inhibition of the signal in the presence of by AOP[1-40] (5 pM). Application of the peptide with the reverse sequence APP[40-1] (5 pM) had no noticeable effects. Taken together these results lend strong support to our channel model to explain the citotoxicity of amyloid (- peptides. (Supported by FONDECYT 1950774 and the Chilean Presidential Cathedra to E.R.)

SIGNAL TRANSDUCTION Th-Pos253 Th-Pos254 CHEMOTACTIC EXCITATION AND ADAPTATION KINETICS IN THE ACTIVITY OF PHOSPHATIDYLINOSITOL TRANSFER RESPONSE TO SMALL STEP STIMULI. ((Ravi Jasuja'. Jinsoo PROTEIN IS ENHANCED BY ETHANOL ((H. Komatsu, T.F. Taraschi Keyoung', David Trentham2 and Shahid Khan'.)) 'Dept. of Physiology and and N. Janes.)) Department of Pathology, Anatomy, and Cell Biology, Jefferson Biophysics, Albert Einstein College of Medicine. Bronx. NHY 10461. 2National Medical College, Thomas Jefferson University, Philadelphia, PA 19107 Institute of Medical Research, Mill Hill, London, NW7 1AA, UK. Phosphatidylinositol transfer protein (PITP) is known for its ability to induce phos- Signal transduction processes are central to several intra- and intercellular activ- phatidylinositol (PI) transfer among a variety of natural and artificial membranes. ities including gene expression. cell adhesion and motility. These systems may be The PITP is also essential for phospholipase C signaling and for constitutive and triggered by extra-cellular chemo-effectors and manifest physiologically observable regulated vesicular traffic among intracellular organelles and for secretion at the responses. Bacterial chemotaxis is one of the most well characterized signal trans- plasma membranes. In order to elucidate effects of ethanol on the function of PITP, duction svstems. In bacteria. the biochemical protein reactions involved in signal the activity of recombinant PITP-a in the presence of various concentrations of transduction have been characterized in-vitro and in-rivo utilizing mutant strains. ethanol was studied by monitoring the transfer of [3H]-PI from rat-liver microsomes Although, bacterial chemotactic responses to small stimuli have been under intense to sonicated liposomes composed of palmitoyl-oleoyl-phosphatidylcholine/bovine- investigation in several research laboratories. there is a disagreement in the available liver PI (98/2 in mol%) at 37 'C. The ability of PITP to mediate PI transfer from experimental results and theoretical predictions. Experimental advancements have microsomes to liposomes was enhanced by the presence of ethanol. Ethanol is a been hampered by the absence of well defined small stimuli that mimic physiological membrane perturbant that increases the fluidity of membranes. Previous workers conditions. WVe have utilized photorelease assavs for caged L-aspartate to study ex- demonstrated a positive correlation between membrane fluidity and PITP activity, citation and adaptation kinetics to small step attractant stimuli. Precise calibration an observation consistent with our findings. [We thank Dr. K. W. A. Wirtz for the of the photo-release is accomplished using caged fluorophore (8-hydroxy pyrinel.3.7 PITP.] tri-sulphonic acid). The response kinetics are further examined by varying basal methylation levels of Tar receptors using non-metabolizable substrate (D-aspartate) in the background. The results are analyzed as a function of fractional change in receptor occupancy and a model for excitation and adaptation kinetics will be presented. A390 A390SINLTASSIGNAL TRANSDUCTIONCIN Th-Pos255 Th-Pos256 MECHANISM OF ACTIVATION OF PROTEIN KINASE C BY CIS-UNSATURATED MEASUREMENT OF KINASE ACTIVITY IN SPATIAL REGIONS FATTY ACIDS ((Simon J. Slater, Shawn K. Milano, and Christopher D. Stubbs)) OF XENOPUS OOCYTES. ((C.-L. Lee, C.E. Sims, N.L. Allbritton.)) Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA Department of Physiology & Biophysics, University of California Irvine, CA 19107. 92697-4560 The mechanism of activation of protein kinase C (PKC) by cis-unsaturated fatty acids was investigated, which, despite numerous previous studies, remains unresolved. It was found that Kitiases are known to be integral to all cellular signal transduction, such as relaying while oleic (OA) and arachidonic acids (AA) alone were relatively poor activators of the Ca2'- receptor activation from the plasma membrane and modulating gene transcription. dependent isoform, PKCct, diacylglycerol- and phorbol ester-induced activity was Despite the central role of kinases in cellular physiology, it has so far been very synergistically enhanced by these fatty acids. It was hypothesized that this synergistic effect difficult to measure their activity in single, living cells. We are developing a method may proceed by a similar mechanism to that proposed previously for the enhancement of to measure the activation of these essential enzymes in subcellular regions of a single phorbol ester induced activity by diacylglycerol [Slater et al., (1996) J. Biol. Chem. 271, cell, the oocyte of the frog Xenopus laevis. This methodology draws upon principles 4627-46311. In that study, it was shown that PKCa contains two phorbol ester binding sites of from several different disciplines to develop a completely new paradigm in single low and high affinity, respectively, and that interaction of diacylglycerol with the low affinity cell measurement. The strategy is to use fluorescently-labeled peptides that are site resulted in an enhanced level of high affinity phorbol ester binding and activity. Here, it specific kinase substrates as detectors of kinase activity. An oocyte containing the was found that while high affinity phorbol ester binding to PKCex was indeed enhanced by peptide is sampled in a defined region with a capillary. The phosphorylated and both OA and AA, consistent with the amplifying effect on activity, neither fatty acid affected nonphosphorylated peptide are separated by electrophoresis through the capillary, low affinity phorbol ester binding, suggesting that the observed increase in the level of and quantitated by their fluorescence. The ratio of the two peptide species is a phorbol ester-induced activity does not involve interaction with this site. The OA- and AA- measure of the activity of the kinase. Preliminary work demonstrates the ability induced increase in high affinity phorbol ester binding and activity did not result from to subcellular enhanced perform rapid, sampling followed by electrophoresis and detection of membrane-association, and neither fatty acid affected the PS and Ca2"-requirements a protein kinase C substrate peptide in that sample. Peptide phosphorylated in for this the effects of OA and AA to be on the of process. Rather, appeared activity fully rico can be measured in a obtained from a small membrane-associated The effects of OA and AA were since saturated cytoplasmic sample region of an PKCa. highly specific With further acids, metabolites of AA and also a branched chain of all intact, living oocyte. refinement, we expect the technique to have fatty analog OA, 11-anthroyl-OA, the and resolution failed to affect activity. These results and the finding that the low concentrations of OA and sensitivity spatial required to measure subcellular activation of AA used had negligible effects on membrane structural properties known to influence PKC kinases in an oocyte responding to physiologic stimuli. This work was supported activity (e.g. head group spacing, etc.), are consistent with a specific fatty acid binding site by the Arnold and Mabel Beckman Foundation, the Searle Scholars Program and a within the enzyme molecule. fellowship from the National Defense Medical Center, Taiwan R.O.C..

Th-Pos257 Th-Pos258 SIGNALING PATHWAY LEADING TO INHIBITION OF MYELOBLASTIC ML-1 CELL RECONSTITUTION OF N-ADRENERGIC SIGNALLING IN SF9 CELLS. PROLIFERATION BY SUPPRESSION OF VOLTAGE-GATED K' CHANNELS. ((D. Xu and (M. Lachance, N. Ethier. G. Wolbring' and T.E. Hebert) ) L. Lu)) Department of Physiology and Biophysics, Wright State University School of Medicine, Centre de Recherche, Institut de Cardiologie de Montrhal, Departement de Dayton, OH 45435. biochimie, Groupe de le recherche sur le systeme nerveux autonome, de PQ, Canada A K' channel was found in ML-1 cells. Serum Universite Montrdal, Montreal, 4-aminopyridine-sensitive highly expressed 1 of Medical of starvation suppresses K' channel activity whereas stimulation of serum-starved ML-1 cell with Department Biochemistry, University Calgary fetal bovine serum (FBS) and epidermal growth factor (EGF) restores the channel activity. 02-adrenergic receptors were co-expressed with various G protein Suppression of K' channel activity by K' channel blockers arrests ML-1 cells at G0 phase of the subunits in Sf9 cells using recombinant baculoviruses. When co-expressed cell cycle. Thus, K' channel activity is required for ML-I cell proliferation. In the present study, with the Gsa subunit, maximal receptor-mediated adenylyl cyclase we found the possible linkage between K' channel activity and growth factor-mediated mitogenic stimulation was greatly enhanced (155.1±4.0 versus 225±11.4 pmol signaling pathway. Increased MAP kinase Erk-2 activity in response to both FBS and EGF were cAMP/min/mg protein) and high affinity agonist binding was detected. When strongly inhibited by suppression of K' channel activity by 2 mM 4-AP. Other K' channel Gjy subunits were co-expressed as well, receptor-stimulated GTPase inhibitors, TEA (10 mM), Quanine (30 pM) and Ba2' (5 mM) all had, though in less extent, the activity were also demonstrated in contrast to when the receptor was similar effect on Erk-2 activity as did 4AP. This is consistent with the previous patch clamp expressed alone and higher than co-expression with just Gsa. Other that 4-AP showed the most on K' in ML-1 study potent effect the channel cells. The robust properties of the receptor were unaltered including receptor desensitization increase in Erk-2 ester stimulation was not affected 4-AP. activity following phorbol (TPA) by and to inverse antisera an These results indicate that suppression of K' channel activity by channel blockers inhibits ML-1 response agonists. Using against epitope-tagged cell proliferation through blocking growth factor-mediated mitogenic MAP kinase activation and P2AR, both Gsa and Py subunits could be co-immunoprecipitated with the is independent of protein kinase C (PKC). These results also suggest a mechanism involving S2AR under conditions where subunit dissociation would be expected given upstream elements of growth factor-mediated signal transduction pathways. We further looked current models of G protein function. A desensitization-defective 02AR at the effects of K' channel activity on activation of other MAP kinases i.e. JNK-1 and P38. (S261, 262, 345, 346A) and a mutant which is constitutively desensitized Activation of JNK-1 or P38 was induced by osmotic stress (600 mM sorbitol). No significant (C341G) could also co-immunprecipitate G protein subunits. These results change in JNK-1 and P38 activities was found with suppression of K' channel activity by 4-AP, will be discussed in terms of a revised view of G protein-mediated which suggest that K' channel activity is specifically associated with the signaling pathway that signalling. This work was supported by the MRCC, HSFC and the FRSQ. activates Erks. In conclusion, our results provide new information that K' channel activity is an essential component in the initial event of growth factor-mediated mitogenic signaling pathways.

Th-Pos259 Th-Pos260 M2 RECEPTORS ACTIVATE A NOVEL CHLORIDE CURRENT IN LOPERAMIDE ACTIVATES CALCIUM RELEASE-ACTIVATED XENOPUS OOCYTES. ((Y.-X. Wang, P.D. Dhulipala. J. Benovic+. & MI. CURRENT IN PANCREATIC B-CELLS ((D. Mears'. C. Zimliki Jri.. J. Kotlikoff.)) Dept. of Animal Biology, Univ.of Penn. and +Depart. of Microbiology Harper2, J.W\. Dal 2. E. Rojast and I. Atwater'.)) 'LCBB and 2LBC. NIDDK. and Immunology. Thomas Jefferson Univ.. Philadelphia. PA 19104 NIH. Bethesda. MID 20892. Pancreatic B-cells respond to stimulatorv glucose levels svith bursts of electrical We examined 12 muscarinic receptor signaling pathways in Xenopus oocytes activity and oscillations of intracellular calcium and insulin secretion. Recently, using the two-electrode soltage clamp technique. Application of the muscarinic we have shown that the biphasic glucose response and muscarinic augmentation of agonist methacholine (mACH. 50 pMI) in nominally Ca2+-free solution activated the glucose response are driven by a calcium release-activated current. To further the role of this current in the we a sustained chloride current (VH=- 60 m\') in oocytes injected with MI2 receptor investigate B-cell, used loperamide, an opioid recep- in tor agonist that increases the levels of intracellular calcium maintained by calcium cRNA. Conversely. oocytes expressing MI3 receptors, mACH induced only the release-activated transient, calcium-activated chloride current The calcium channels in non-excitable cells. Loperamide had a tripha- IC,(C.). M2 receptor-activated sic effect on electrical in chloride current seas not effected by inhibition of C activity llmM glucose, consisting of a prolonged spiking phospholipase (U-73122). IP3 a transient silent and finally a burst pattern. These changes were block or chelation of intracellular phase. phase, rapid receptor (heparin), Ca2+ (BAPTA-AM). swhereas identical to those elicited by the muscarinic agonist but were not blocked all abolished the The chloride current carbachol, M13 receptor response. NI2 receptor-activated by an antagonist atropine or bh the opioid receptor antagonist naloxone. When has an anion conductivity distinct from that of sequence the Ca2+_activated Cl- loperamide was present during a step increase in glucose concentration, the initial current. Anti-Gi, antibodies blocked the MI2 (not M3) response. swhereas anti- prolonged burst reached more depolarized potentials than during the control situa- iq, antibodies inhibited the M3 (not M12) current. Injection of a 3ARK peptide tion. Intracellular calcium measurements showed that loperamide released calcium fragment that binds G3, or coexpression of the 3.ARK fragment cRN\A. had no effect from an intracellular store in insulin secreting cell lines. In whole-cell patch clamp on M12 receptor-actisated currents. although 5I3 receptor activation of ICl(Cl) seas experiments. inclusion of BAPTA and IP3 in the pipette resulted in the activation blocked. The 5'12-activated chloride current sEas not actisated by the diacylglycerol of a non-selective cation current that was blocked bv lanthanum and enhanced bv analog OAG. although the PKC inhibitors chelersthrine and calphostin C abolished increasing the extracellular calcium level. The current was not increased by lop M12 current coupling. These findings indicate that heterologously expressed 512 eramide. suggesting that the drug activates the current by calcium release in these receptors activate a novel Ca +-independent Cl- current in Xenopus oocytes. that cells.or that direct augmentation of the current byloperamide requires an intracel- coupling is mediated bv G,, -subunits, and requires the stimulation of protein kinase lular messenger that is lost in the whole-cell configuration. The data indicate that C. Supported by VIH HL45239. a calcium release-activated current,indirectly or directly activated byloperamide, plays a role in B-cell electrical activity. SIGNAL TRANSDUCTION A391 Th-Pos261 Th-Pos262 HORMONAL STIMULATION OF EXOCYTOSIS IN HUMAN EFFECTS OF EXTRACELLULAR CALCIUM ON ELECTRICAL PANCREATIC B-CELLS INVOLVES BOTH PROXIMAL AND BURSTING AND INTRACELLULAR AND LUMINAL CALCIUM DISTAL REGULATORY STEPS IN THE STIMULUS-SECRETION OSCILLATIONS IN INSULIN SECRETING PANCREATIC ,3-CELLS COUPLING. ((Krister Bokvist and Jesper Gromada.)) Islet Cell Physiology, ACCORDING TO CHAY'S STORE-OPERATED MODEL ((T.R. Novo Nordisk A/S, Fruebjergvej 3, DK-2100 Copenhagen, Denmark. Chay.)) Department of Biological Sciences, University of Pittsburgh. Pittsburgh, PA 15260 USA In this study we have used a combination of patch-clamp, capacitance and intracel- lular Ca2+ ([Ca2+]j) measurements to study the mechanisms underlying stimulation The extracellular calcium concentration, [Ca2+]5, has interesting effects on burst- of insulin release from human /-cells by the incretin hormones glucagon-like-peptide- ing pancreatic /-cells. With a medium containing a moderate amount of glucose., an 1(7-36)amide [GLP-1] and glucose-dependent insulinotropic polypeptide [GIP]. Both increase in [Ca2+]n lowers the repolarization potential. raises the plateau potential, hormones caused a >4-fold* increase in the capacitance step (=exocytosis) follow- and shortens the spike amplitude and plateau lengths. When extracellular Ca2+t is ing a voltage-clamp depolarization. This was paralleled by 40-50%' increase in the depleted in a perifusion medium, the cell depolarizes while the intracellular Ca2 corresponding integrated Ca2+ -currents and [Ca2+]i-transients. The stimulatory ac- concentration, [Ca2+],, decreases to the lowest level. As to why [Ca2+]o exerts such tions of GLP-1 and GIP on both Ca2+ currents and exocytosis were mimicked by an interesting effect on the bursting has been a mystery. By incorporating a low- forskolin and reversed by 20 MM Rp-8-Br-cAMPS, suggesting that GLP-1 and GIP threshold transient inward current to the store-operated bursting model (TR Chay, act by increasing the cAMP level and activating protein kinase A (PKA). In the Biol. Cybern. 75:419-431, 1996), we elucidate the role of [Ca2+]o in influencing presence of BAY K8644, which stimulate L-type Ca2+-channels, GLP-1 caused no electrical activity, [Ca2+],, and the luminal Ca2+ concentration, [Ca2+11m, in the further Ca2+_current increase but was still able to elevate the exocytotic response endoplasmic reticulum (ER). In addition, we explain how these three variables re- by 142±28%*. Flash release of caged cAMP in the presence of a constant (buffered) spond when various pharmacological agents are applied to the store-operated model. [Ca2+], caused a 3-4-fold' stimulation of the rate of exocytosis. In conclusion, our The existence of the transient low-threshold inward current (which may be different findings suggest that GLP-1 and GIP activate PKA which enhance exocytosis by from a transient Ca2+ current) should be elucidated experimentally. This current increasing the Ca2+-influx through L-type Ca2+-channels and by a more direct in- is essential to explain the extracellular CaS+ effect. The model predicts that i) teraction with late steps in the exocytotic process. Finally, in the presence of 5 [Ca2+]lsm oscillates slowly and ii) intra-granular Ca2+ in SGs is involved in exocy- mM glucose, GIP or GLP-1 caused a 30-40%' inhibition of the current carried by tosis. Experiments using fluorescent dyes such as mag-fura-2-AM or aequorin could the ATP-regulated K+-channels (K-ATP) thus sensitizing the human ,3-cell to glu- provide relevant information. The model also predicts that secondary messengers cose, the exact mechanism(s) underlying this K-ATP channel closure remain to be such as cAMP can greatly influence the frequency of bursting by activating the elucidated. ('p<0.05; n=3-5) calcium-releasing channel in the ER.

Th-Pos263 EVIDENCE FOR A CONCOMITANT TRANSPORT OF Mg2+ AND GLUCOSE IN RAT HEART FOLLOWING INSULIN ADMINISTRATION. [A. Romani, V. Matthews and A. Scarpa] Dept. Physiology and Biophysics - Case Western Reserve University, Cleveland, OH, 44106. Insulin physiologically counteracts several of the cellular effects mediated by - adrenergic receptor activation and cAMP formation. A broad number of reports indicates that ,- adrenoceptor stimulation induces a marked extrusion of Mg2+ from several mammalian cell types. To investigate whether insulin administration could counteract the observed mobilization of Mg2+, rat hearts were perfused in a retrograde Langerdoff system with a Ringer medium devoid of Mg2+, and the changes in extracellular Mg2+ content were measured by atomic absorbance spectrophotometry (AAS). The pre-treatment of the hearts with 6 nM insulin completely abolished the Mg2+ extrusion induced by the P-adrenergic agonist isoproterenol or by the cell permeant 8-Cl-cAMP. Also, insulin per se induced a marked accumulation of Mg2+ into the cells. This accumulation was small but detectable in the presence of 25 iM [Mg2+]out and became more evident at 50 or 100 iM [Mg2+]out. Insulin-mediated Mg2+ accumulation was abolished when the hearts were perfused with a medium devoid of glucose and when cytochalasin-B or phloretin were used to inhibit glucose transport, suggesting a link between Mg2+ accumulation and glucose transport. A similar Mg2+ accumulation was also observed when vanadate (0.8 mg/ml) was used to stimulate Glut4 operation. Insulin-stimulated 14C- glucose accumulation into the cells was also measured in the presence of varying [Mg2+]out. No 14C-glucose accumulation occurred when 15 jiM [Mg2+]out was present, a minimal concentration of 25-35 iM Mg2+ being required to observe a detectable transport. Glucose transport considerably increased when 50 aM Mg2+ was used, and it was almost maximal at 100 iM [Mg2+]out. These results would indicate that the operation of Glut4 transporters stimulated by insulin or vanadate is associated with an uptake of Mg2+ into cardiac cells, and suggest that extracellular Mg2+ may modulate Glut4 operation. [Supported by NIH HL 18708 and by Diabetes Association of Greater Cleveland 397-A-97].

TRANSPORT ACROSS BILAYERS Th-Pos264 Th-Pos265 POTENTIAL DISTRIBUTION OVER THE MEMBRANE/SOLUTION ELECTRIC POTENTIAL NEAR A SINGLE CONDUCTING ION INTERFACE DUE TO TETRACAINE ADSORPTION ((Cherny A.V.* and CHANNEL IN A CHARGED LIPID MEMBRANE ((V.G. Levadny+. V.S Sokolov#)) *Gorkii State University, Kharkov, Ukraine, tFrumkin Institute V.M. Aguilella and M.L. Belaya*.)) +The Scientific Council for Cybernetics, Russian Academy of Sciences; Vavilov str. 34, 333117 Moscow, Russia; Dept. de of Electrochemistry, Russian Academy of Sciences, Moscow, Russia (sponsor: Ciencias Exp., Univ. Jaume I, 12080 Castellon, Spain, *Institute of Plant A. Volkov). Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia Potential drops at the boundary ofthe bilayer lipid membrane due to tetracaine adsorption were investigated. To monitor the changes of boundary potential The movement of ions in an aqueous solution converging from bulk towards the we used the method based on the compensation of inner membrane field entrance of a single conducting channel placed in a charged lipid membrane is consid- ered. The aim of the study is to determine the electric potential distribution and the revealed by the second harmonic of capacitive current. These potentials were ion concentration profiles in the vicinity of the channel. Moreover, specific attention compared with potentials measured by the electrophoretic mobility of has been paid to the effect of the membrane surface charge on these profiles. The 3D liposomes with the help ofthe correlation spectroscopy method. The boundary Poisson equation has been used for the description of the electric potential profile in potential drops depended on electrolyte concentration for neutral membranes. the solution. The dependence of the ion concentration profiles on the electric poten- Potentials measured by electrophoretic method in the same conditions were tial has been obtained on the basis of the Nernst-Planck equation. An approximate smaller than the drops measured by inner field solution has been obtained, which is valid in the solution outside some hemisphere considerably boundary potential centered in the mouth of the channel. Our solution transforms to Gouy-Chapman's compensation method. theory at a large distance from the channel entrance. In the nearest vicinity of the To explain the obtained results it was suggested that a potential drop channel the above mentioned profiles are close to radially svmmetrical. For a neutral due tetracaine adsorption was located not only in diffuse double layer, but in a membrane they transform into earlier obtained expressions (Peskoff and Bers, 1988). layer inside the membrane. The latter was not screened by electrolyte solution We have analyzed the electric potential and ion concentration profiles for the case ions and could not be detected by electrophoretic method. when the contribution of the lipid charges and ion fluxes to the electric potential profile are of the same order. These profiles strongly depend on the direction of the A model of adsorption where charged groups of tetracaine molecules ion fluxes, and hence the potential and concentration distributions on both sides of are located in the membrane surface layer is in a good agreement with the the membrane are significantly different. As a result of the complicated interplay experimental data. between all electrical sources in the considered system there is a potential minimum in the nearest vicinity (ca. two Debye lengths) and a maximum relatively far away. A.37ZAAGI) TDAfI KAAA.TQPnVTalrux I IFLq-AVOD.eQQKvoa RDFDMIPL.AVI.'DeI irlpLa Th-Pos266 Th-Pos267 CHANGES OF THE TOTAL POTENTIAL ENERGY PROFILE FOR CATION COMPLEXES WITH THE RIGHT-HANDED DOUBLE-STRANDED THE INTERACTIONS OF CHARGED MOLECULES WITH LIPID DOUBLE-HELICAL (DSDHgf) DIMER OF GRAMICIDIN ((B. M. BILAYERS DUE TO THE ADSORPTION OF VERAPAMIL AND Burkhart, W. A. Pangborn, and W. L. Duax)) Hauptman-Woodward Medical PROPRANOLOL ((Elenca E. Pohl'. Auidrey V. Krvlov2, Michael Block', Peter Research Institute, Buffalo, NY 14203 Pohll'.)) 'Department of Mledical Physics and Biophysics. Mlartin-I.uither-Unisersitv, 06097 llalle/Saale. Gernianys 2AN. BPelozerskv Institute Gramicidin is known to conduct a number of univalent cations across lipid of Pihssico-(Cheunical Biology. Moscows State Universits, 119899 Mloscow. Russia membranes. The newly determined structure of the DSDHgt gramicidin dimer has been observed to crystallize in the presence of a number of metal salts: CsCl, TIhe effects of tthe organic calcitini chaicicel blocker verapamil acid thce beta-rezeptor RbCl, KI, KSCN, NaCl, and TlNO3. Initial characterization of these crystals blocker propranolol on dipole °d acid scirface 0, potentials of lipid bilayers were have revealed them all to be isomorphous to the CsCl structure, suggesting that stuidie(d ill details. The botindary potential Ob (Ob=Od+0,) of black lipid mernbranes the gramicidin in these structures also assumes the DSDHgp dimer structure. The olitainied bs the Inner Field ('ompensation (IFC) method and by conductance mea- crystal structures of these ion complexes will be presented demonstrating stirencients in the presence of nonactin was compared with g. calculated froni the differences in ion binding sites, ion occupancy, and anion significance. In iec trophoretical cmobilits of sniall uccilamiiellar vesicles. It was shown that, the in- addition, structural deformation of the gramicidin will be investigated as a crease of botiiiilarv poteictial was originated by an increase in surface potential clue function of cation radius (Cs+ > Rb+ > K+> Na+). Preliminary results on to the adsorptionc of the positisely charged propranolol. Althotigh, due to the ad- crystals of the Rb+ complex indicate ion binding that is significantly different sorptiocc of verapaciiil o, inicreases also, ob decreases. The dirccinishing of the total from that in the Cs+ complex. Each of these cations has been used as a probe in potential energy barrier is explained assuiiiicig that the adsorption plane of verapamil gramicidin electrophysiology, experiments and such structural differences may is localized deeper than tlhe oice of propatiolol. The resulting sharp decrease of inem- assist in the understanding of the vast gramicidin literature. brance dipole potential due to serapaiccil adsorption imay have important implications for both mencbrane translocatioic and partitioniing of small or hydrophobic ions alid 'harged groups in iiiembranie proteins. Firnancial support of the Kultusmincstesium Sachsecr Anhahlt (errnany (FA/: 221841 0085) is grateful acknotcledged.

Th-Pos268 Th-Pos269 THE AQUEOUS ER TRANSLOCON PORE IS SEALED AT ITS LUMENAL A MOLECULAR DYNAMICS STUDY OF THE PORES FORMED END BY BiP AND EXPANDS AFTER ASSOCIATION WITH THE RIBOSOME*NASCENT CHAIN COMPLEX. ((Brian D. Hamman and Arthur BY E. COLI OMPF PORIN IN A FULLY HYDRATED POPE E. Johnson)) Department of Medical Biochemistrv & Genetics, Texas A&M BILAYER ((D. P. Tieleman, H. J. C. Berendsen.)) Dept. of Biophysical University Health Science Center, College Station, Texas 77843-1114. Chemistry, UIniversity of Groningen, The Netherlands Secretory proteins are co-translationally translocated across the membrane of the endoplasmic reticulum (ER) through an aqueous pore that is sealed off from We have studied the properties of pores formed by OmpF porin from E. coli. the cytosol by a tight junction between the ribosome and the translocon. Short based on a molecular dynamics simulation of a system containing the OmpF trimer. nascent chains (<70 amino acids) are also sealed off from the ER lumen 318 palmitoyloleoylphosphatidylethanolamine lipids. 27 Na+ ions and 12992 water immediately after ribosomal targeting to the membrane. To determine what role, if molecules. In a production run of a nanosecond the OmpF trimer exhibits a C-o any, soluble proteins residing in the ER lumen play in the closing of the lumenal root mean square deviation of 1.8 A and a stable secondary structure. No evidence end of the translocon pore early in translocation, canine microsomes were is found for extracted with an alkali (pH 10.5) buffer to release the lumenal proteins, pelleted, large scale motions of the L3 loop. We have investigated the pore and then resealed upon neutralization. Translocation intermediates with dimensions, conductance and the properties of water inside the pore. This water fluorescent NBD dyes incorporated into nascent secretory proteins were forms a complicated pattern, even when averaged over one nanosecond of simulation prepared with microsomes that were either intact (purified and untreated), lumen- time, and has a net orientation along the pore-axis pointing from the extracellular extracted, or resealed (reconstituted) with one or more purified lumenal proteins. to intracellular side of the bilaver. Around the pore constriction site the water When NAD+ was added to these translocation intermediates, no collisional dipoles are highly structured in the plane of the membrane. oriented bv the strong quenching of NBD was observed in any sample. When perfringolysin 0 was added to introduce NAD+ into the lumen of these intermediates, collisional transversal electric field. The average water dipole moment of water molecules inside quenching of nascent chain dyes was observed in microsomes that lacked the pore can be over 50% of the dipole moment of a single water molecule. The lumenal proteins, but no quenching was observed in intact microsomes or diffusion coefficient of water inside the pore is greatly reduced compared to bulk. microsomes reconstituted either with total lumenal proteins or solely with purified BiP. Since addition of BiP-specific antibodies to total lumenal proteins prior to reconstitution prevented lumenal sealing of the aqueous pore, it is clear that BiP binds to the lumenal end of the translocon pore. BiP also seals the lumenal ends of translocons that are assembled in the membrane, but do not have a ribosome bound to the cytosolic end of the pore. Interestingly, experiments with collisional quenchers of different sizes reveal that translocon pores not bound to ribosomep have a much smaller diameter (10-20 A) than the exceptionally large 40-60 A diameter found in translocons functioning in translocation.

Th-Pos270 Th-Pos271 SQUALAMINE IS NOT A PROTON IONOPHORE ((11.5. Seliciskv. A. IMPLICATIONS OF OSMOTIC STRESS POTENTIATION OF IFrau,giosi. R.\I. Snmith. and ID.. Pedersen*.)) Chemistry Departmnent. Villanova AMPHOTERICIN B AND DERIVATIVES' CHANNEL-FORMING t'licversitcs. V'illaiosa, PA 19085. acid *('ollege Misericordia, Checnistry ACTIVITIES ((S.C. HARTSEL. P. MIKULECKY. T. RUCKWKARDT. A. Iuipartment, I)allas, PA 18612 SCOTT, J. SCOTT.)) Department of Chemistry, University of Wisconsin-Eau Claire, Eau Claire, WVI 34702 Sqtialamine is a potenit, broad spectruni aictimicrobial agent isolated from the clogfish sthark. Recently. a syncthetic analog of sqitalarciine synithesized frocii bistior- The effective. but nephrotoxic. antifungal agent Amphotericin B (AmB) forms 5 -cholenic acid wcas demonstrated to act as a proton ioccophore in anionic lipid ion selective channels in membranes and their stability/formation is enhanced by cmec'cmbtranes ((;. D)erg, T. I)ewa. S.L. Regen (1996) .1. Arner. Chem. Soc., 118, sterols. It has long been regarded as a model oligomeric ion channel and is commonly 8975). WVe have tested sqtialatuiine as a proton ionophore. Large unilamellar vesi- thought to require sterol for channel formation. We show conclusively using svn- cles ssere prepared front phosphatidylglycerol or pliosphatidvlcholine. contaictiirg up thetic phospholipid (POPC) membrane vesicles that AmB can form specific cation to 2 tiiole percenct squalaiciine, encapstilating the pIt sensitive fluorescent (lye 8- selective channels in the absence of sterols. Potency depends upon concentration. hlix iroxvpyreiie-1.3.6-tristilfociic acid. Sqcialacicine exhibits no iotiophoric activity at self-association state and other factors. Channel formation is particularly enhanced sctcalamine/plcospholipid rriolar ratios of tip to 2 iTIOle percent. When added ex- by transmembrane osmotic stress (e.g.. fluorescence-detected K+ ion currents are ternallv to phiospholipid vesicles. sqtialancicce lyses the vesicles, with release of the enhanced about 90-fold by increasing osmotic differential from 0 to 326 mOsm/kg). flicorescent dve. 'I'Iese restults indicate that sqicalanciine does not act as a proton The AmB methyl ester derivative (ANIE) shows much less nephrotoxicit' than AmB ioccophore. and natist be added to prefociiied phospholipid vesicles to induce lysis. despite being found in much higher concentrations in the kidneys in animal models. (Supported bs NIII Al 39757A) We believe that this seeming paradox can be reconciled by the greater osmotic stress potentiation of AmB as compared to AmE. The region of the kidney tubules most damaged by AmB is the ascending branch of the loop of Henle. the region also under the most inside to outside osmotic stress. Another AmB derivative. MIS-8209. is far more active than AmB against sterol-free unstressed vesicles yet its activity is not potentiated by osmotic stress. Consistent with our hypothesis. MIS-8209 shows a better therapeutic index in animal disease models. -~~~ ~ ~ ~ ~~~~RNPOTARSTRANSPORT ACROSS BILAYERSIAESA9 A393 Th-Pos272 Th-Pos273 ARACHIDONIC ACID INCREASES WATER PERMEABILITY OF EXCHANGE OF MONOOLEOYLPHOSPHATIDYLCHOLINE AS SARCOLEMMAL VESICLES FROM RABBIT SKELETAL MUSCLE MONOMER AND MICELLE WITH MEMBRANES CONTAINING ((K.C. Olbrich, O.F. Hutter', D. Needham)) Duke University, Dept. of Mech. Eng., POLY(ETHYLENE GLYCOL)-LIPID ((D. Needham, N. Stoicheva, D. V. Durham, NC 27708; 'Inst. of Physiology, University of Glasgow, G12 8QQ, UK. Zhelev.)) Department of Mechanical Engineering and Materials Science, Duke The area expansion modulus, lysis tension and water permeability of sarcolemmal vesicles University, Durham, NC 27708-0300 derived from rabbit psoas muscle were measured using the micromanipulation technique as previously described [1, 2]. Briefly, individual sarcolemmal vesicles were aspirated into a Surface-grafted polymers, such as poly(ethylene glycol) (PEG), provide an effec- holding pipet. Area expansion moduli and lysis tensions were determined by measuring the tive steric barrier against surface-surface and surface-macromolecule interactions. In in area of the vesicle in to change response increasing membrane tension. The water the present work, we have studied the exchange of permeability was measured by transferring individual vesicles into a hyperosmotic solution and mnonooleoylphosphatidylcholine tracking the volume change with time. All experiments were conducted at 21°C. (MOPC) with vesicle membranes containing 750 molecular weight surface-grafted The area expansion modulus of the sarcolemmal vesicles decreased from 376 ± 13 (mean + PEG (incorporated as PEG-lipid) from 0 to 20 mol % and have analyzed the ex- S.E.M.) mN/m to 241 ± 13 mN/m in the presence of 4-gM arachidonic acid (corresponding to perimental results in terms of thermodynamic and stationary equilibrium models. I8 mol% in the membrane [3]). The lysis tension decreased from 13.0 ± 0.3 mN/m to 7.6 ± Micropipet manipulation was used to expose a single lipid vesicle to a flow of MIOPC 0.3 mN/m under the same conditions. The water permeability of the sarcolemmal vesicles solution (0.025 uM to 500 uM). MOPC uptake was measured by a direct measure increased from 16.7 ± 1.2 un/sec to 29.2 ± 1.1 um/sec with the presence of arachidonic acid. of the vesicle area change. The presence of PEG(750) lipid in the vesicle rnembrane The of surfactants as arachidonic and bile decrease presence (such acid, lysolipids, acids) inhibited the partitioning of MOPC micelles (and to some extent area expansion modulus and lysis tension [4-6]. The increase in water permeability is microaggregates) consistent with increased free volume and disruption of lipid packing induced by the presence into the membrane, while, even up to 20 mol% PEG-lipid, it did not affect the ex- of arachidonic acid in the membrane [2]. change of MOPC monomers both into and out of the membrane. The experimental 1. Olbrich, K.C., et al., J. Physiol., 1997. 504: 40P. data and theoretical models show that grafted PEG acts as a very effective molecular 2. Olbrich, K.C., Ph.D. Thesis, 1997, Duke University. scale "filter" and prevents micelle-membrane contact, substantially decreasing the 3. Hutter, O.F., J. Physiol., 1997. 499: 133P. apparent rate and amount of MOPC taken up by the membrane, thereby stabilizing 4. Evans, E., et al., in Bile Acids in Gastroenterology Basic and Clinical Advances, 1994, Kluwer Academic Publishers: London. 59-68. the membrane in a solution of MOPC that would otherwise dissolve it. This work 5. Nichol, J.A. and O.F. Hutter, J. Physiol., 1993. 467: 333P. is supported by grant GM 40162 from NIH. 6. Nichol, J.A. and O.F. Hutter, J. Physiol., 1996. 495: 83P.

ERYTHROCYTE MEMBRANES Th-Pos274 Th-Pos275 NETWORK TOPOLOGY VERSUS INTRINSIC MOLECULAR ELASTICITY IN EXPLAINING DEFICIENCIES IN MEMBRANE PROTEINS OF THE RBC AFFECT THE SHEAR RIGIDITY OF ALTERED RIGIDITY OF BIOLOGICAL MEMBRANES ((Knowles D.W., Mohandas N., Evans CELLS BUT NOT THE DEFORMABILITY OF THE MEMBRANE-SKELETAL NETWORK. E.A+., Chasis J.A.)) Lawrence Berkeley National Laboratory, University of Califormia, Berkeley, CA. & ((Gimm J.A., Peters L.L., Lux S.E., Mohandas N.)) UCSF/UCB Bioengineering Graduate Group +Physics & Pathology Departments, University of British Columbia, Vancouver, BC. and Lawrence Berkeley National Lab., University of California, Berkeley, CA 94720. The Jackson Laboratory, Bar Harbor, ME 04609. Harvard Medical School, Boston, MA 02135. Recent work to understand the elasticity of large single biological polymers has indicated that molecules like DNA and titin behave as entropic springs. The mechanical character of these polymers is described by Protein 4.1 and band 3 are major red blood cell proteins which play roles in linking the lipid bilayer two intrinsic molecular features, contour length and inherent elasticity. In erythrocytes, spectrin is the large to the underlying membrane-skeleton and in regulating red cell membrane mechanical properties. structural polymer which determines the mechanical properties of the membrane skeleton such as In the membrane-skeleton, 4.1 binds to spectrin, the most abundant filamentous skeletal protein, membrane rigidity. Increased interactions of spectrin with transmembrane proteins has been shown to and actin oligomers at the junctional complexes. 4.1, in addition, binds to the cytoplasmic domain rigidify the erythrocyte membrane but the essential question of the underlying molecular mechanism is of band 3 and also to the glycophorin C linking that protein to the spectrin network. The unknown. Specifically, does erythrocyte membrane rigidification, induced by increasing network transmembrane protein band 3 functions as a structural protein linking to the spectrin network via interactions, involve shortening the contour length of spectrin by restricting it's ability to extend and there ankyrin. Protein 4.2 also binds to the cytoplasmic domain of band 3 but its binding site is different by altering the topology of the network, or alternatively, by increasing spectrin's inherent stiffness and from those of ankyrin and 4.1 binding sites. Recent development of mice with inactivation of there by leaving the network topology unaltered. To answer this question, monoclonal antibody BRAC18 genes by homologous recombination has enabled generation of red cells with either partial binding to band 3 was targeted as a model system because transmembrane protein band 3 is a major (heterozygote) or complete (homozygote) deficiency of protein 4.1, protein 4.2 or band 3. These tethering point between the skeletal network and the bilayer and it regulates membrane rigidity through its red cells enable us to establish the importance of these proteins in regulating red cell membrane interaction with the spectrin based membrane skeleton. Membrane rigidity was measured by micropipette mechanical properties. The techniques of Fluorescence-imaged Microdeformation (FIMD) was aspiration and fluorescence imaged microdeformation (FIMD) was employed because it measures used to evaluate the membrane-skeletal associations in red cells from normal and heterozygote component molecular density in stretched networks and would be sensitive to topological alterations 4.1, 4.2 and band 3 mice. The integrity of the membrane-skeleton was measured by following the involving altered contour length. Cells were prepared by fluorescently labeling erythrocyte band 3 and redistribution paftem of actin. The FIMD experiments showed changes in molecular redistribution actin, in situ, with fluorescein-5-maleimide (FMA) and rhodamine-phalloidin (RhPh), respectively. Actin of the vertically interacting proteins (i.e. membrane-skeletal interaction) but not in the laterally was chosen because it is an ideal topological marker of spectrin end-to-end length and network density. interacting proteins (i.e. skeletal protein interactions) as seen by the unchanged redistribution of Appropriateness of the model was confirmed because with increasing incubation concentrations of actin following deformation of red cells wHh partial deficiencies in 4.1, 4.2 and band 3. Elastic BRAC18 there was a dose-dependent increase in membrane rigidity. This increased rigidity was associated shear rigidity measurements using single cell aspiration and ektacytometry showed decreases in with increased interaction of band 3 with spectrin as observed by FIMD, following the addition of shear rigidity of red cells from heterozygotes compared to normals. Shear rigidity of red cells from BRAC18, which showed an increased slope of entrance density minus the cap density (Pe-Pc), of FMA- homozygotes showed an even greater decrease. These results imply that the network is able to labeled band 3, versus aspiration length nornalized by pipene radius (IJRp). However, and surprisingly, maintain a certain level of membrane deformability while the overall stability of the cells has BRAC18 binding had no measurable effect on the density difference for RhPh-labeled actin. These results decreased as seen by decreased surface area. Furthermore, these data imply that the topology of indicated that the binding of BRAC18 antibody to band 3, which produced a marked increase in membrane the membrane-skeletal network in these cells parfially deficient in various membrane protein is sfill rigidity, did not alter the topology of the skeletal network. These data imply that the molecular mechanism normal. These findings in combination with previous data obtained with ankyrin deficient murine for rigidification is increased inherent stiffness of spectrin rather than topological crosslinking resulting in red cells provide a belter understanding of how mechanical properties of red cells are regulated by a shortening of the contour length of spectfin. the molecular interactions involving various proteins.

Th-Pos276 Th-Pos277 SELF-ASSOCIATION OF HUMAN ERYTHROCYTE BAND 3 IN SITU: A cDNA CLONING OF K-CL COTRANSPORTER AND AQUAPORIN-CHIP FROM SIMPLE FLUORESCENCE ASSAY BASED ON RESONANCE ENERGY ERYTHROID PROGENITOR CELLS IN DOG. ((H. Fujise'2, H. Ochiai'2 and K. Higa')) HOMOTRANSFER ((S.Nl. Blackman. D\V. Piston. A.H. Beth.)) Dept. of Molecular 'Department of Pathobiochemistry, School of Veterinary Medicine, and 2Department of Physiology & Biophysics, Vanderbilt Iniversityv Nashville. TN, 37232 Radiobiology, hIstitute of Biosciences, Azabu University, Fuchinobe, Sagamihara, Kanagawa 229 Japan The high overall concentration of integral membrane proteins in most membranes suggests that a distribution of low-affinity oligomeric states may exist in situ. The erythrocyte anion exchange protein, band 3, is thought to exist as stable dimers which may further associate Water extrusion dunng regulative volume decrease (RVD) mediated by K-Cl to form tetramers or larger oligomers. Oligomerization in situ can be inferred from rota- cotransporter (KCC) may be connected with water channel, aquaporin (AQU)-CHIP. To tional dynamics measurements, but optical and EPR results disagree over whether a large identify existence of KCC and AQU-CHIP in dog red blood cells (DRBCs), mRNA of fraction of band 3 is highly aggregated. Resonance energy homotransfer. occurring between KCCand AQU-CHIP in erythroid progenitorin dog was investigated. Mononuclear cells like is an alternative fluorophores, svhich reports self-association by sensing the interaction obtained from the peripheral blood was firstly cuitured in IMDM medium with fetal calf between labeled subunits in an Homotransfer has been to occur in eosin- oligomer. shown serum, transferrin and phytohemagglutinin stimulated-leukocyte conditioned medium, 5-maleimide-labeled band 3. and reports on its oligomeric state. This study compares the complementary results from rotational dynamics and homotransfer. as measured by fluores- then the cells were cultured secondary in the medium with recombinant erythropoietin cence anisotropy: (A) Addition of Zn++. known from cross-linking and rotational dynamics after removal of phagocytic cells. The mature erythroblast were collected at 3rd day of experiments to aggregate band 3. greatly increases the amount of eosin homotransfer in second phase culture, and the mRNA was extracted. RT-PCR was perfomied by using ghost membranes. (B) Proteolytic removal of the cytoplasmic domain of band 3 is knoswn to oligonucleotide primers which were designed according to the reported cDNA increase rotational mobilitv, but has no effect on homotransfer. consistent with the release sequences in other animals. Both in erythroid progenitor cells and kidney from dog, of a mobility restriction without a in significant change oligomeric state. (C) Addition of cDNA fragments of KCC and AQU-CHP were detected. Both KCC and AQU-CHIP in up to 2% ether, a membrane increases band 3 rotational mobility. (v/v) fluidizing agent. dog, the identity of the cDNA sequences between erythroid progenitor cells and but does not alter homotransfer,suggesting that the mobility change arises solely from the decreased effective viscosity of the bilaver. Also, these experiments can generally be adapted kidney were perfect. The deduced amino acid sequence of dog KCC was 95%, 91 and to a real-time kinetic examination of self-association changes. Studies are also being per- 92% identical to human, rabbit and rat KCC, respectively. The deduced amino acid formed on intact erythrocytes. and on mutant forms of band 3 such as in Southeast Asian sequence of dog AQU-CHIP was 93, 92 and 90% identical to bovine, human and rat ovalocytosis. Supported by HL34 737 and DKO 7363. ones, respectively. Thus, mRNA of KCC and AQU-CHIP existed in the erythroid progenitor cells in dog, and it was 100% identical to that in kidney. So, in mature red blood cells, RVD might perform due to co-operated interaction of KCC and AQU-CHIP. A394 ERYTHROCYTE MEMBRANESMEMEBRANES Th-Pos278 Th-Pos279 COMPARATIVE DIFFERENCES BETWEEN IRON TRANSPORT KINETICS OF LOW OSMOTIC WATER PERMEABILITY IN ERYTHROCYTES FROM ENDOSOM_5S FROM R1ABBIT AND RAT RETICUL?CYTES (((M. Ramano2, J.1A TRANSGENIC MICE LACKING WATER CHANNEL AQUAPORIN-1. ((Tonghul Watkins i K. Yeh , M. Yehl, H. Garrick , L. Garrick, C-Y. Li J. Glass )) Cancer Center, LSUMC-S, Shreveport, LA 71l3O-39321 Ma, Baoxue Yang and A.S. Verkman)) UCSF, CA 94143 (Spon. by H.R Kao) and Dept. of Biochemistry, SUNY Buffalo, NY 14214-3000 . Water channel aquaporin-1 (AQP1) is expressed strongly in erythrocytes and Reticulocyte iron (Fe) absorption via transferrin (Tf) endocytosis is a major factor in Fe metabolism. Although the plasma membranes of epithelia and/or endothelia in kidney, lung, brain, eye and exact sequence of events following endocytosis of Tf are not other organs. A transgenic AQP1 null mouse was generated by targeted gene fully resolved, there are several aspects of the intracellular disruption. The AQP1 gene was isolated from a mouse genomic library. A handling of Tf, ultimately leading to Fe translocation across targeting vector for homologous recombination was constructed using an 8 kb the endosomal membrane, which can be established for rabbits. Overall, the sequence includes (1) dissociation of Fe(III) AQP1 DNA fragment containing exon 1. Part of exon 1 (bp 360-384) and -1.4 kb from Tf which is directly coupled to acidification of the of intron 1 were replaced by a Pol2neobpA cassette for positive selection; a endosome interior, (2) Fe(III) binding to acceptors on the PGKtk cassette was inserted at the 5'-end of the AQP1 targeting sequence for interior surface of the endosome (one of the acceptors may be stem were the H+-ATPase), (3) Reduction of Fe(II) by internal ascorbate negative selection. Embryonic cells (CB1-4) transfected, screened and/or an NADH dehydrogenase oxidoreductase, (4) translocation and implanted to produce the AQP1 knockout mice according to established of Fe(II) across the membrane by an iron transporter, (5) procedures. Genotype analysis of the first 94 mice born by intercrossing of mobilization of Fe(II) by a small molecular weight iron heterozygote founders showed 26 wildtype, 46 heterozygote, and 22 knockout binding pool or a specific Fe carrier. We have compared the iron transport properties of endosomes isolated from normal mice. The knockout mice expressed no detectable AQP1 protein or full-length Wistar rats (+/+) and homozygous (b/b) and heterozygous (+/b) transcnpt in any tissue; AQP1 protein and transcript expression were decreased Belgrade rats which have a mutation in iron absorption. The by -50% in heterozygotes compared to wildtype mice. By stopped-flow light defect occurs in an intracellular step of the transferrin osmotic water in from mice was cycle. Minimal acidification (if not alkalinization) was scattering, permeability (Pf) erythrocytes wildtype observed for b/b and +/b endosomes while +/+ endosomes high (0.015 cm/s, 10 OC), weakly temperature-dependent (3-4 kcal/mol), and acidified to pH 6.7. Nevertheless in intact reticulocytes mercurial inhibitable. In knock-out mice P was decreased by 12-fold and some iron is taken up suggesting that acidification is not the became more temperature-dependent (>8 kcal mol). Urea permeability remained only requirement for Fe dissociation from Tf. In contrast, the after deletion. These studies establish an AQP1 null mouse and isolated endosomes would not mobilize iron unless the pH was high AQP1 artificially lowered. Models addressing these differences indicate that AQP1 is the major erythrocyte water transporting protein. between species as well as mutants will be discussed.

Th-Pos280 Th-Pos281 DIFFERENCES BETWEEEN ACIDIFICATION, IONIC FLUXES, AND MEMBRANE VASODILATORS ACTIVATE ERYTHROCYTE K-CL COTRANSPORT. ((N.C. POTENTIAL OF CLATHRIN COATED AND UNCOATED ENDOSOMES. ((R.D, Adragma, H. Ahmed, and P.K. Lauf)), WSU, School of Medicine, Dayton, OH 45435. Hardison, A. Hoffpauir, J.S. Weeks, E.J. Besser, T.C. Moore, K. Tyler, B. Lindenmayer, J.A. Watkins, C-Y. Li, and J. Glass)) Cancer Center, LSUMC-S, Shreveport, LA 71130-3932. The mechanism ofaction ofvasodilators at the cellular and membrane level is not clearly understood. K-Cl cotransport (COT), the coupled movement of Cl and K ions, plays an Differences between the functional properties of vesicles important role in regulatory volume decrease (RVD). Recent findings from our during the transitions involved in various processes of laboratory indicate that nitrite (NO2 -, a derivative of the vasodilator nitric oxide (NO)) endocytosis have been poorly understood. The relationships K-Cl COT. we studied the effect of used vasodilators on K- between vesicular acidification (ApH), generation of membrane stimulates Here, commonly potential (AT), and ionic fluxes for isolated clathrin coated Cl COT to understand their mechanism of action at the membrane level. Isosorbide (early) endosomes have been examined in order for quantitative mononitrate (ISSB), hydralazine (HYZ), pentaerythritol (PE) and nitroprusside (NP) were comparisons with uncoated endosomes and to constrain general tested on Cl-dependent K efflux in low K sheep red blood cells (RBCs). K-Cl COT models for the control of acidification. Upon the addition of was estimated as the difference between the rate constant of K efflux in Cl minus that 1 mM ATP or 1 mM GTP the apparent rate constants for flux in NO3, and at different osmolalities (240-450 mOsM) and drug concentrations. Our kinetics are shown below. Clathrin Coated Uncoated Endosome main findings are: All vasodilators tested (in hypotonic medium) activated K-Cl COT. ATP GTP ATP GTP The sequence of activation was: HYZ > ISSB > PE > hypotonic medium > isotonic Flux 10 3s-1 10-38s1 10-3s-1 10-38-1 medium (290 mOsM, control = 100 %). Furthermore, the mechanism of action of HYZ appears to be non-additive with respect to cell-swelling at lower concentrations whereas ApH 7.7±1.2 4.6±0.06 24.5±4.0 13.5±2.5 those of HYZ at high concentrations and of ISSB and NP appear to be additive. The AT 0.54±0.06 0.17±0.06 4.5±0.8 9.0±0.9 mechanism of action of PE appears to require cell-swelling suggesting a role of the cytoskeleton on PE action. Since NO derivatives are oxidants, the effect of vasodilators Na+ 2.2±0.2 3.80.5 3.5±0.3 2.5±0.4 on cellular glutathione (GSH) and ATP was determined. The RBC GSH concentration decreased by 100 % with increasing HYZ concentration but was not affected by ISSB, K+ 0.5t0.1 N.D. 2.1±0.2 3.0±0.3 NP or PE. Thus, HYZ's mechanism of activation seems to differ from that of the other Cl 2.1±0.3 N.D. 4.2±1.0 3.8±0.1 vasodilators tested and appears to involve oxidation of cellular metabolites. RBC ATP Differences between cyclic nucleotide effects, phosphorylation was not different from the controls. Our findings suggest that vasodilators, in addition states, and nucleotide kinetics are also observed suggesting to affecting ion channels, may decrease total peripheral resistance by activation of K-Cl that these transport properties are due to differences in cotransport and cell volume reduction. (Supported by NIH and NAHA and AHAOH). phosphorylation states.

Th-Pos282 Th-Pos283 ATP DIRECTLY BLOCKS A Ca2+-ACTIVATED K+ CHANNEL ON MECHANISM OF GLUCOSE-STIMULATED OXIDATION OF HUMAN BULLFROG ERYTHROCYTES. ((NI. Shindo, Y. Sohma. Y. Imai.)) Dept. ERYTHROCYTE PHOSPHOLIPIDS ((NM J. Wilson. T. Jose. NI. Farley. Z. of Physiology, Osaka Medical College. Takatsuki. Osaka 569. JAPAN Saz-Parkinson. A Trudel, D L Daleke.)) Department of Biochemistrv and Mlolecular Biology, Mledical Sciences Program, Indiana University, Bloomington. IN 47405 Using the patch-clamp technique. we have identified an intermediate conductance Hyperglycemia in diabetes induces alterations in blood cell membrane structure that Ca2+_activated K+ channel on bullfrog (Rana catesbeiana) erythrocytes and have may contribute to the cardiovascular complications commonly associated with this dis- investigated the regulation of channel activitv bv cytosolic ATP. The channel was ease In vitro hyperglycemic treatment of non-diabetic erythrocytes mimics these effects highly selective for K+ over Na+. gave a linear I-V relationship with symmetrical and induces an oxidation-dependent increase in passive lipid flip-flop resulting in surface 117.5 mM K+ solutions and had a single channel conductance of 56 +/- 6 pS (n=6). exposure of phosphatidylserine. In the present work, we test two potential mechanisms The channel usually showed brief opening events of msec duration divided by rela- for hyperglycemia-induced lipid oxidation: glucose-stimulated metal radical recycling and metabolic activation of oxidases by increased flux through the pentose tively long closed periods of 10 100 msec duration. Channel activity was dependent NADPH-dependent phosphate pathway. The effect of glucose on Fe2+-ascorbate-induced oxidation of polyunsat- on Ca2+ concentration (K1/2 = 600 nM) but voltage-independent. These basic char- urated phosphatidylcholine liposomes was measured using three indicators of lipid oxidation. acteristics are similar to those of human and frog erythrocvte Ca2+_activated K+ Although Fe2+-ascorbate induced a linear increase in conjugated diene formation. hydroper- (Gardos) channels previously reported. However. exposing the channel to ATP. in oxy fatty acid content, and malondialdehyde production, the addition of glucose (up to 50 the absence of MIg +. altered channel activity in a unique way. ATP reduced channel mM) had no effect on these markers of lipid oxidation. Similar results were obtained using activity with block exhibiting a novel bell-shaped concentration-dependence. The heme in place of Fe2+-ascorbate. These data argue against a role for glucose-stimulated trace metal recycling in hyperglycemia-induced lipid oxidation. Hyperglycemia has also channel was inhibited most by 10 MAi ATP (Po reduced to 5 +/- 2% of control. been shown to produce transient increases in NADPH levels in intact erythrocytes through n = 5) but less blocked by lower (50 20% at 1 iMM. n = 6) and higher (15 +/- the pentose phosphate pathwav. Preliminary studies indicate that in vitro hyperglycemia +/ 2% at 100 yIM. n=8: 110 +/- 17% at 1 m.M. n=8. and 121 +/ at 5 mM. causes a concentration-dependent inhibition of glucose-6-phosphate dehydrogenase, the first n=2) concentrations of ATP. 'Moreover, the effect of ATP could be mimicked by a step in the pathway. Thus, hyperglycemia may stimulate oxidation both by activating non-hydrolysable ATP analogue. AMP-PNP. This suggests that ATP affects chan- NADPH-dependent oxidases and, at later stages. by crippling the ability of the cell to nel activitv by binding to the channel and not by phosphorylation of the channel or maintain adequate glutathione levels. Supported by the NIH (GM47230), American Heart associated control sites. Association. and American Diabetes Association. ERYTHROCYTE MEMBRANESMEMBRANES A395 Th-Pos284 Th-Pos285 C02 PERMEABILITY OF THE RED BLOOD CELL MEMBRANE INDUCIBLE EXPRESSION OF HAE1 (BAND3) IN HEK293 CELLS: ((R.E.Forster, G.Gros,L.Lin, Y.Ono. and M.Wunder.)) Univ. of Penna., School FUNCTIONAL AND BIOCHEMICAL CHARACTERIZATION ((R.T. of Medicine and Zentrum Physiologie. Medizinsche Hochschule Hannover, Timmer. R.B. Gunn.)) Dept. of Physiology. Emory Universitv. Atlanta. GA 30322 Germany. The human anion exchange protein, hAE1 (Band3) of human red cells has been expressed in HEK293 cells under the control of an inducible promoter (a modified It has long been assumed that natural lipid bilayer membranes, as of the red blood ecdysone promoter) that can be activated in cells that express a chimeric ecdysone cell, are highly permeable to gases because these molecules are small, uncharged receptor (Invitrogen. Carlsbad, CA). Permanent cell lines have been established that to a level that can both concentration of and more soluble in lipids than water. The disappearance of singly labelled 18 express hAEI high be controlled bh the 0.5 - and time to from a red cell as the between labelled inducing agent ( 5 yg/mL muristerone A) of exposure inducing oxygen C02 suspension 18 oxygen exchanges agent (24-96 h). Our initial estimates using Western analvsis indicate that 1.0 C02/HC03- and unlabelled HOH provides a measure of the membrane permeability pg hAEI / 106 cells o 48 h is produced when cells are induced using of 0.5 pg to HC03- and the carbonic anhydrase (CA) activity inside the cell (Itada et al., / mL muristerone A. The efficacv of other inducing agents (e.g. ecdysone and J.Biol.Chem. 252:3881-3890.1977). NVe have recently found that adding sufficient ecdysterone) has also been examined. The hAE1 that is produced under these 4,4' diisothiocyano 2,2' stilbene disulfonate (DIDS) to bind all of Band III protein in conditions is a population of fully glycosvlated forms (MNIW 110-130 kD) presumably a red cell suspension, reduces P HCO3 and appears to reduce cellular CA activity, at the plasma membrane and a form that is consistent with unglycosylated or core although it has no effect on the enzyme in free solution. A significant membrane glycosvlated hAE1 (MIW 100 kD) that is perhaps retained in either the ER or Golgi. diffusion resistance to C02 would explain this finding, but the model, on which our Immunofluorescence has been carried out on induced cells to provide additional computations have been based, considers this resistance zero. When our data were information on the subcellular distribution of the expressed hAE1. The functional interpreted using a more advanced computational model developed in Hannover, properties of the induced hAE1 have been examined using 36C1 flux measurements which solves for membrane permeabilities to C02 and to HC03-, as well as CA (influx and efflux) of cells that are attached to culture plates or in suspension. activity, we find that DIDS decreases the membrane permeability to both HC03- Expression in induced cells was compared to Cl fluxes in either uninduced cells or and C02. Preliminary data show: in control red cells (n=2) P HCO3 = 0.0012 cells that did not contain the hAE1 construct. Functional expression is increased cm/sec, P C02= 1.1 cm/sec; in red cells exposed to DIDS (n=6), P HCO3 = from a background of 94 mmol / kg protein 6 min to 450 mmol / kg protein 6 min 0.0002 cm/sec, P C02 = 0.05 cm/sec. The mechanism by which DIDS acts on the in induced cells. The background Cl flux was <10% DNDS inhibitable. whereas the C02 permeability is not clear, but may be through membrane transport proteins induced flux is 80-100% inhibited by 250 pM DNDS. The kinetics of activation have rather than through the lipid structure itself. also been examined. (This work supported in part by grants HL28674 to RBG and HL08989 to RTT).

CF]rR Th-Pos286 Th-Pos287 GATING OF CFTR CHLORIDE CHANNELS BY NUCLEOSIDE TRIPHOSPHATES. ARG352 IS A MAJOR DETERMINANT OF CHARGE ((S.D. Zeltwanger, F. Wang, and T.C. Hwang)) Department of Physiology, Dalton SELECTIVITY IN THE CFTR CHLORIDE CHANNEL ((Romain Cardiovascular Research Center, University of Missouri, Columbia, MO 65211. Guinamard and Myles H. Akabas.)) Ctr. for Molec. Recog.. Columbia Univ.. New York. NY, 10032 Nucleotide binding and hydrolysis at two different nucleotide binding domains, NBDI and NBD2, controls the gating cycle of CFTR chloride channels. A previous report has shown The cvstic fibrosis transmembrane conductance regulator (CFTR)forms an anion- on current a differential effect of 1 mM ATP, GTP, UTP, and CTP macroscopic CFTR selective channel. We previously showed that charge selectivity occurs at the cyto- et the mechanism for this difference is unknown. (Anderson al., 1991), but underlying plasmic end of the channel. The molecular determinants of charge selectivity are Gating behavior in the presence of different nucleotides was studied in excised patches from unknown. We now show that mutation of Arg352. a residue flanking the cvtoplasmic NIH3T3 cells stabely expressing wild-type CFTR. After excision, channels were first phosphorylated using cAMP-dependent protein kinase (PKA). Different concentrations of end of the M6 segment, to cysteine reduced the Cl- to Na+ permeability ratio from various nucleotides were then perfused to the cytoplasmic surface of the membrane. Dose- 37:1 (wild-type) to 6:1 (mutant). Mutation of the adjacent residue Gln353 had no response relationships were obtained by normalizing current obtained with different effect on the Cl- to Na+ permeability ratio (36:1). We then infer that Arg352 is an concentrations of ATP, GTP, UTP and CTP to the current obtained with 2.75 mM ATP. important determinant of charge selectivity in the CFTR channel. In symmetrical The current elicited by I mM ATP, GTP, UTP, and CTP was 0.91 ± 0.06 (n = 8), 0.71 ± NaCl solutions both wild-type and R352C mutant have a similar single channel con- 0.07 (n = 16), 0.64 ± 0.06 (n = 7), and 0.45 ± 0.05 (n = 16) -fold of 2.75 mM ATP, ductance, about 6 pS, and linear current-voltage relationships. In a 10-fold NaCI respectively. This sequence (ATP > GTP > UTP > CTP) is in agreement with the gradient the current-voltage relationship of wild-type channels displays Goldmann previous report. However when higher concentrations of nucleotides were used (5 mM) the rectification, however, the R352C mutant does not. We infer that the mutation in- current elicited by ATP, GTP, UTP, and CTP was 1.01 ± 0.03 (n = 5), 0.98 + 0.04 (n = creases the conductance to Na+ and decreases the conductance to Cl-. WVe suggest 6), 0.88 ± 0.09 (n = 4), and 0.43 ± 0.11 (n = 4) -fold of 2.75 mM ATP, respectively. that in the wild-type channel the positive charge on Arg352 creates an electrostatic This sequence (ATP = GTP = UTP > CTP) suggest that the difference seen between ATP, potential in the channel that forms a barrier to cation permeation while facilitating GTP, and UTP at lower concentrations is likely due to a difference in binding to the anion permeation. We examined the effects of the R352C mutation on other aspects NBD's. CTP is unique in that even at supersaturaing concentrations it can only elicit of ion conduction in CFTR. The R352C mutation eliminated the SCN- induced a in -43% of the current that can be generated with 2.75 mM ATP, suggesting difference anomalous mole-fraction effects but did not alter the halide selectivity sequence. some to the NBD's Results from channel step following binding (e.g. hydrolysis). single Thus, Arg352 is at or near a SCN- binding site but it may not form the site that kinetic analysis, performed to further investigate the interaction of nucleotides with the NBD's,willbepresentedanddiscussed. (Supported by NIH and CF Foundation) determines the halide permeabilitv sequence. We are making additional mutations at the position of Arg352 to further investigate its role in conduction and selectivitv. (Supported by NIH, CFF. AHA-NYC Affiliate)

Th-Pos288 Th-Pos289 RESIDUES NEAR THE EXTRACELLULAR END OF TM6 AND TM12 IN CFTR CONTRIBUTE TO VOLTAGE-JUMP RELAXATIONS IN A CFTR MUTANT ((McDonough, S.', Z.-R. Zhangt, ANION SELECTIVITY ((McCarty, Nael A., and Zhi-Ren Zhang)) Depts. of Physiology and Pediatrics, N. Davidson', H.A. Lester', and N.A. McCarty2)) 'Div. of Biology, Caltech, Pasadena, CA Emory University School of Medicine, Atlanta, GA 30322. (Spon. by J.F. White) 91125; 2Depts. of Physiology and Pediatrics, Emory Univ., Atlanta, GA 30322. Several lines of study have implicated transmembrane domain 6 (TM6) in contributing to the walls of the pore of the CFTR chloride channel. We have also shown that TM 12 contributes to the pore along with The currents carried by CFTR are characteristically time-independent, whether due to TM6, by virtue of the interaction of residues in these helices with the voltage-dependent pore-blocking endogenous or heterologous expression. We found that mutation of serine 1 18 to drug DPC (diphenylamine-2-carboxylate) (McDonough et al. (1994) Neuron 13:623-634). This analysis phenylalanine in transmembrane domain (TM) number led to voltage-jump relaxations when suggested two cross-sectional domains within the pore of CFTR: a binding domain, at the level of S34 1, Membrane was held at -45 then to a and a modifying domain two tures of the helix toward the extracellular end, at the level of K335 and expressed in Xenopus oocytes. potential mV, stepped TI 134. We recently began to ask whether residues in these two domains contribute to the patters of anion prepulse of +80 mV for 200 ms, and then stepped to various test potentials between +80 mVand selectivity for CFTR channels. Here, we report measurements from three mutations in these domains. -140 mV. For negative test potentials, this protocol induced relaxations to larger whole-cell Oocytes were injected with S to 19 ng of cRNA along with 0.4 ng of cRNA for the 112-adrenergic receptor conductances (see Fig., left panel, dashed line shows zero current level). In contrast, after a and studied using two-electrode voltage clamp. Upon activation with isoproterenol, voltages were ramped prepulse to -140 mV, there were relaxations to lower conductances at all test potentials (right between -80 and +60 mV in both directions. For each of the 10 test anions, relative permcabilities at more than Both also led to (PX:PCl) were calculated from the measured reversal potentials (Vrev) using the Goldman-Hodgkin-Katz panel), which became prominent voltages positive Ec. protocols equation; relative conductances (GX:GCI) for anion entry were measured from Vrev to (Vrev + 25 mV). the appearance of tail currents following return to the holding potential. These kinetics are not Sequences were as follows (abbrev's: Iseth, Isethionate; Glut, Glutamate; Gluc, Gluconate; Ac, Acetate): seen in wildtype (WT) or any ofthe CFTR mutants described thus far. Relaxations at potentials WT (PX:PCI): SCN > N03 > Br > Cl > I > Iseth = Glut > Gluc > Ac = C104 > S204 between -140 and -80 mV were fit with a time constant (T) of26 ms. Between +40 and +80 (n=16) (GX:GCI): Cl > NO3 > Br > Glut = Gluc = Ac = Iseth > S204 > I > C104 = SCN mV, 'r was slightly voltage-dependent, ranging from 26 22 ms over these potentials. Single-

channels in excised, inside-out patches exhibited inward rectification , as well as more rapid K335F (PX:PCI): SCN > NO3 >Br > Cl = I > Glut = C104 - Gluc = Iseth > Ac > S204 gating kinetics than WT. The mutation (n=7) (GX:GCI): NO3 > Cl > Br > SCN > Glut = S204 = I = Gluc = Iseth = Ac > C104 made the voltage dependence of DPC S341A (PX:PCl): SCN >NO3 >Br > Cl > I > Glut = Gluc = C104 = Iseth = Ac > S204 blockade more shallow; at -100 mV, (n=5) (GX:GCI): NO3 > Br > Cl > Gluc = Glut = Ac > Iseth = SCN > I = S204 > C104 affinity for DPC equalled that of WT.

- - TI 134F (PX:PCI): SCN > NO3 >Br > Cl > I > Glut > C104 = Gluc = Ac - Iseth > S204 t ------Th- ese findings suggest a functional (n=5) (GX:GCI): Cl > Br = NO3 > Ac > SCN = Gluc = Iseth = S204 > Glut > C104 > I role for TM I I in the CFTR channel. These results suggest: (I) the selectivity filter is distributed along the length of the CFTR pore, notjust at the cytoplasmic end, (2) residues in TM 12 also contribute to selectivity, and (3) as others have suggested, S1118F (Supported in part by NIH DK47027.) relative conductances are more sensitive to mutations in the pore than are relative permeabilities. A396 4CFTR Th-Pos290 Th-Pos291 INTERACTION BETWEEN PERMEATION AND GATING STUDIED IN A PORE- PKA- AND CA2+1-DEPENDENT REGULATION OF WT AND S768A DOMAIN MUTANT IN CFTR. ((McCarty, N.A. and Z.-R. Zhang)) Depts. of Physiology and MUTANT CFTR CL- CHANNELS IN XENOPUS OOCYTES. ((K.W.Chan'. Pediatrics, Emory Univ., Atlanta, GA 30322. (Spon. by D.C. Eaton) S.S.Smith2. L.Csanadyl. A.C.Nairnt. D.C.Dawson2. D.C.Gadsbvt.)) 'Rockefeller t niversity. New York. NY 10021 & 2Universitt ofMlichigan Mledical School. Ann Arbor. We have described a mutation in transmembrane domain (TM) number I I ofCFTR which led to .il 48109. voltage-jump relaxations when expressed in Xenopus oocytes (abstract by McDonough et al., this meeting). This mutation, S11 18F, affects both pore characteristics and gating S768. an excellent in vitro substrate for PKA. seems to have inhibitorv influence on characteristics. To determine the underlying mechanism, we have further studied permeation CFTR channel activation because, in Xenopus oocytes. mutant S768A CFTR channels are characteristics using two-electrode voltage clamp. Relative permeabilities (PX:PCI) and relative more sensitive than WVT CFTR channels to activation by IBMIX plus forskolin. Resting. conductances (Gx:Gcl) for Cl- and 10 test anions were calculated using instantaneous IN unstimulated oocstes expressing WVT or S768A CFTR display a basal Cl conductance. not relations from potentials ranging between -140 and +80 in 20 mV steps lasting 75 ms. By seen in control (H20-injected) oocytes, that represents 10% (NN'T) or 80% (S768A) of the comparing currents at the beginning of the voltage step with those at the end ofthe step, we can maximal CFTR Cl conductance activated by 50 pMAl forskolin plus 1 mMl IBMIX. Single assay for time-dependent changes in Px:PCt and Gx:Gcl during the course ofthe relaxations. channel recordings confirm that CFTR channels underlie this Cl conductance. Assuming Compared to wildtype (WT), SI I 18F showed: !.PSCN:PCI. 4PAC:Pcl, tPaC:4PcI, and tPS204:Pct- WVT and S768A CFTR channels to be identical when dephosphorylated. the much larger (Abbrev's: Ac, acetate; C104, perchlorate; S204, thiosulfate). No time-dependent changes in basal Cl- conductance associated with S768A channels strongly suggests that S768 on WVT Px:PcI were found for WT or SlI 18F. The mutant also showed: -Glseth:Gcl, CFTR is phosphorylated in resting oocytes, and that this phosphorvlation does indeed GS204:Ga, inhibit channel function. basal Cl- conductance in S768A oocytes means that .IGAc:GCI, and TGSCN:GcI. Further, GGIut ,at,:GCI and GGco,,ate:Gcl were lower at the end ofthe But,the large voltage step than at the beginning, suggesting that the apparent diameter ofthe pore changes at least one stimulatory site must be phosphorylated on S768A CFTR. Rapid disappearence during the voltage-jump relaxation. This time-dependent change in Gx:Gcl was not found for of that Cl- conductance on injection of the PKA inhibitor RpcAMIPs. or with BAPTA, WT channels. To investigate an interaction between these effects on gating and on permeation, demonstrates that sustained PKA activity and a Ca2+,-dependent process are both required we asked whether the time-course of the relaxation differs between permeating anions. Oocytes to maintain basal activation of the S768A channels. The similarly rapid disappearence of were held at -30 mV, stepped to a prepulse potential of-140 mV for 100 ms, and then stepped to WVT CFTR Cl- conductance (maintained bv continuous PKA stimulation) upon injection various test potentials between +80 mV and -140 mV. The time-constants ofthe relaxations of RpcANIPs argues that the inhibitory influence of S768 phosphorylation does not reflect (X) on that at +40, +60, and +80 mV were longer by roughly 80% in the presence of SCN than in Cl or Br. accelerated dephosphorvlation of stimulatorv PKA sites CFTR. Since inhibition can be overcome by maximal stimulation of PKA, we infer that phosphorvlation of S768 These data suggest that: (I) TM I I lines the pore in the CFTR channel, and (2) the TM domains slows otherwise but does not PKA its of CFTR may play a role in the conformational changes associated with opening/closing the (or impairs) abolish. subsequent phosphorylation (or consequence) of key stimulatory sites on CFTR. (Support: NIH DK51767 (DCG & ACN) pore - SI I 18F may act as a reporter group, slowing the gating process to a quantifiable rate. and DK45880 (DCD)).

Th-Pos292 Th-Pos293 TEMPERATURE DEPENDENCE OF CFTR SINGLE CHANNEL DELETION OF THE SECOND NUCLEOTIDE BINDING FOLD GATING ((A.A.Aleksandrov, L.A.Aleksandrova, J.R.Riordan.)) Biochemistry (NBF2) FROM CFTR ALTERS PROCESSING AND FUNCTIONAL and Mlolecular Biology, Mayo Foundation, Mayo Clinic Arizona, Scottsdale, AZ, PROPERTIES OF THE CFTR CHANNEL. ((B. Zerhusen and J. Mla.)) 85259 Dept. of Physiol. & Biophvs., Case WNestern Res Univ. Cleveland. OH.44106 Single-channel currents of wild-type CFTR reconstituted in lipid bilayers were To study the function of NBF2 and its interaction with the R domain in the recorded to study the temperature dependence of channel gating between +20C regulation of the CFTR channel. three deletion mutants of the human epithelial and +40C.Arrhenius plots were prepared to estimate the activation energy for the CFTR were constructed: bR(708-835). 6NBF2(1185-1349). and 6R-6N'BF2. The direct and reverse transitions between the closed and open state, in order to evaluate mutant CFTR proteins were expressed in HEK 293 cells. and their single channel whether the energy of ATP hydrolysis is necessary for channel gating. The opening functions were examined using the bilayer reconstitution svstem. GFP-CFTR fu- of the channel was highly temperature dependent and required an activation energy sion constructs wvere used for localization of the mutant CFTR proteins expressed in of about 100 kJ/mol. If we assume the energy of ATP hydrolysis is 38 kJ/mol, HEK 293 cells. Cells transfected with GFP-wtCFTR and GFP-6R-6NBF2 exhibited then hydrolysis of two ATP molecules is insufficient to open the channel. Closing of fluorescence at both plasma and intracellular membranes: whereas cells transfected the channel was only weakly temperature dependent with an activation energy not with GFP-6N-BF2 and GFP-6F508 exhibited fluorescence predominantly in intra- more than the activation of diffusion in water. We found no significant difference cellular membranes. indicating that b6NBF2 does not process properly to the plasma in free energy between the open and closed states. Taken together, these observa- membrane of HEK 293 cells. Both b6\BF2 and bR-bN'BF2 form functional chloride tions suggest that the activation energy required to open the channel comes from channels in the lipid bilayer membrane. but they open mainly to the subconduc- ATP binding followed by an additional large change in structure as the CFTR-ATP tance states ( 6 pS and 3 pS) unlike the wt-CFTR channel which exhibited a full complex passes to the transition state for hydrolysis. The energy of ATP hydrolysis conductance state of 8 pS in 200 mMNl CsCl. The bR-6N\BF2 channel. similar to the is not used as a source of energy for channel gating. The energy of CFTR-ATP 6R-CFTR channel. does not require PKA-phosphorylation to open. In addition. interaction in the transition state is responsible. ATP hydrolysis itself is needed bR-6NBF2 exhibited prolonged open state compared with the wt-CFTR channel. to make the gating process reversible. The energy of ATP hydrolysis is the price Our data is consistent with the hypothesis that NBF2 has an inhibitorv role in the for the reveisibility of the channel opening. The channel is closed, and the initial gating of the CFTR channel. Deletion of NtBF2 from CFTR affects both gating closed state structure restored when the products of hydrolysis are released. With kinetics and conduction property of the chloride channel. and also the processing this ligand gating model it is possible to explain the high activation energy for the behavior of CFTR. channel opening. (supported by NIH-NIDDK)

NA CHANNELS Th-Pos294 Th-Pos295 CARDIAC SODIUM CHANNEL MUTATIONS UNDERLYING IONIC BASIS FOR THE EFFECT OF TTX ON SPONTANEOUS LONG QT-3 ALTER THE KINETICS OF FASTI INACTIVATION ACTIVITY IN NEONATAL RABBIT SA NODE MYOCYTES ((M. Baruscotti, AND DEACTIVATION ((P.C. Ruben', C. Prince3, Q. Wang4 and J.E. C. Altomare, D. DiFrancesco, R.B. Robinson.)) Dept. of Physiol., U. of Milan, Milan Italy Richmond2.)) iDept. Biology, Utah State Univ., Logan. UT. 3Dept. Biology and and Dept. of Pharmacology, Columbia U., New York, NY 10032 3School of Medicine, Univ. of Utah, Salt Lake City, UT, and 4Baylor Col. of Medicine. TX. We have previously reported that neonatal SA node myocytes exhibit a TTX-sensitive Houston, Na current which is important for automaticity, in that TTX slows spontaneous rate in neonatal but not adult myocytes isolated from the SA node (J Physiol 492:21-30, 1996). Long-QT3 syndrome is a form of cardiac arrythmia linked to three mutations in the We tested for the presence of a non-inactivating window current by imposing a slow ramp cardiac sodium channel. To characterize the biophysical substrates of this disease. clamp. Experiments were performed on isolated sinoatrial node cells using perforated patch wildtype and long-QT3 mutant human cardiac sodium channels were expressed in and a ramp clamp from -60 to -20 mV at a rate of 0.035 V/sec to match the normal rate of Xenopus oocytes and currents measured from on-cell macropatches. The rate of fast diastolic depolarization in these cells at room temperature. No adult myocytes (n=4) show inactivation onset was measured during prepulse experiments and from the decay a TTX-sensitive current with this protocol, but 18 of 25 neonatal cells (from 3-14 day old of sodium current during depolarizations. Both N1325S and a three amino acid animals) exhibited such a current during the ramp. The peak current occurred at -40+1 deletion in the domain III-IV linker (AKPQ) resulted in slowver open-state fast inac- mV and peak density was 0.9+0.2 pA/pF. However, the magnitude of the TTX-sensitive tivation, whereas R1644H had no effect on the rate of open-state inactivation. These current was dependent on the rate of change of the ramp, with larger depolarizations in- data are consistent with a model for hyperexcitability and differ from a previous re- creasing the current and slower depolarizations reducing or abolishing it. Thus, using a port of accelerated fast inactivation in AKPQ (Bennett et al., 1995). In contrast to ramp clamp we found no evidence of a steady-state TTX-sensitive current at voltages in the our all three mutations rate diastolic potential range. We therefore conducted additional step clamp protocols, using data for open-state inactivation. increased the of closed- longer depolarizations than the 40 msec steps of our original study. We found that for po- state inactivation compared to wildtype cardiac sodium channels. Recovery from tentials near the maximum diastolic potential the current fully inactivates (consistent with fast inactivation was measured from double pulse experiments. N1325S, R1644H the ramp clamp data), but with a time constant of hundreds of msec (for 5 cells, the time and AKPQ were all found to significantly increase the rate of recoverv from fast constant for a step to -55/-60 mV ranged from 100 to 300 msec). This slow time course of inactivation. Deactivation kinetics were measured from tail currents evoked bv hv- the macroscopic current inactivation for steps near threshold could account for the TTX- perpolarizing steps to voltages ranging from -120 to 0 mV following depolarizations sensitivity of spontaneous rate and diastolic potential slope, without the need for invoking to +50 mV for 0.25 ms. All three sodium channel mutations known to cause long- a non-inactivating component to the TTX-sensitive Na current. QT3 significantly prolonged the rate of deactivation over the range of voltages from -120 to -80 mV). Prolonged deactivation provides an additional biophvsical expla- nation for the hyperexcitability of cardiac sodium channels that leads to long-QT3 syndrome. ilaNA CUANNF.M..RI-ElkLI14114irLla L'S-7.7A3971 Th-Pos296 Th-Pos297 CARDIAC SODIUM CHANNEL MARKOV MODEL WITH RECOVERY HIGH AFFINITY ANTIBODIES TO SODIUM CHANNEL ,-I AND P-2 FROM INACTIVATION ((L.A. Irvine, M.S. Jafri, and R.L. Winslow)) SUBUNITS DEMONSTRATE VARIABILITY IN SUBUNIT COMPOSITION Department of Biomedical Engineering, The Johns Hopkins University School IN THE CENTRAL NERVOUS SYSTEM. ((A.M. Corbett)) Dept. of ofMedicine, Baltimore, MD 21205 Biochemistry & Molecular Biology, Wright State University, Dayton, OH 45435. Several Markov models exist for the cardiac sodium channel. Unfortunately, High affinity polyclonal antibodies were produced in rabbit against peptides these models cannot be used as the basis for modeling antiarrhythmic drug corresponding to unique amino acid sequences from the N-terminal segment of action because they treat the inactivated state as absorbing, and thus cannot sodium channel P-1 and 13-2 subunits. High titer antibodies were developed predict drug effects in response to trains of stimuli (ie. use-dependence). In against the P1-1 peptide within a single month, while antibody titer peaked for the addition, model rate constants are not explicit functions of either voltage or P-2 peptide at two months. P-1 and P-2 antibodies showed: 1) no cross reaction temperature. Therefore, we have formulated a new Markov model of cardiac with a non-specific peptide, 2) half-maximal block of specific interaction with sodium channels. The model is similar to the CAl hippocampal sodium channel 3-10 nM peptide, 3) specific interaction with a 39K dalton band (p1-1 antibody) model of Kuo and Bean (Neuron, 12:819-829) with modified rate constants. and 31K dalton band (13-2 antibody) on Westem blots ofcrude brain membranes, Most significantly the open to inactivated state transitions are made voltage- and 4) immunoprecipitation of 65% of saxitoxin-labelled solubilized sodium dependent. Model rate constants are both temperature- and voltage-dependent channels with P1-1 antibody, while the P-2 antibodies only immunoprecipitated and are determined by fitting model responses to whole cell and single channel 42% ofthese sodium channels. Preliminary immunolocalization studies show experimental data using a simulated annealing minimization algorithm. In coincident P1-1 and P-2 staining only in neuronal somas. Previous response to voltage-clamp stimuli, the model accurately reproduces gating and immunlocalization studies have shown that the RI sodium channel subtype a- ionic currents, the steady-state inactivation curve, the time course of recovery subunit is uniformly expressed in neuronal somas in all regions ofthe brain from inactivation, and the single channel mean open time. The model thus except the cerebellum. This data suggests that RI sodium channel subtype has provides a more complete representation ofcardiac sodium channels and can be both beta subunits associated with it, but that sodium channel subtypes localized used as the basis for modeling antiarrhythmic drug action. to other parts ofthe neuron may have a different subunit composition. Supported by NIH NS28377.

Th-Pos298 Th-Pos299 EFFECTS OF EXTRACELLULAR SEQUENCES ON SODIUM CHANNEL THE SODIUM CHANNEL INACTIVATION PARTICLE INACTIVATION AND MODULATION BY P1 SUBUNITS ((Y. Qu, S-F. Chen, DELIMITED: STRUCTURE/FUNCTION RELATIONSHIP OF J. Rogers, T. Scheuer and W.A. Catterall)) Dept. of Pharmacology, U. of RESIDUES SURROUNDING THE IFM MOTIF. ((F.A. Boeckman, Washington, Seattle, WA 98195-7280 C.A. Rohl2, T. Scheuer', R.E. Klevit2 and W.A. Catterallt.)) 'Dept. of Pharmacology and the 2Biochemistry and Biomolecular Structure Center, Univ. of Na channels consist of a, 131, and, in neurons, P2 subunits. Rat brain a subunits Washington, Seattle, WA 98195 (rIIA) inactivate slowly when expressed alone, and coexpression with 01 accelerates inactivation. Cardiac a subunits (rHl) inactivate more rapidly than rItA alone, but coexpression of 11 has little effect. Chimeras were studied in The intracellular loop between homologous domains III and IV of the Na+ chan- which each extracellular loop of rHl was substituted into rItA. Insertion of rHl nel o subunit has been proposed to operate as a hinged cytoplasmic gate in which loops connecting S5 to SS1 in domain I (IS5-SS1), ISS2-S6, IISS2-S6, IIISl-S2, the hydrophobic IFM motif (residues 1488 to 1490) acts as an inactivation particle. and IVSI-S2 had little effect on the kinetics of inactivation but accelerated Previously, cysteine substitution of F1489 demonstrated that this residue was inac- recovery from inactivation in the absence of 11 subunits. In the presence of 11, cessible to sulfhydryl modification once the channel was inactivated but was com- recovery of these chimeras from inactivation was similar to wild-type rIIA. pletely susceptible to the modifying reagents from the resting state. We used the Evidently, these rHI extracellular loops destabilize the inactivated state of rIIA at substituted cysteine accessibility method (SCAM) to analyze the proposed inactiva- negative potentials in the absence of P1, making rIIA behave more like rHI. In tion particle and surrounding residues for their accessibility to sulfhydryl reagents contrast, a chimera with rHl loop IVS3-S4 had slowed recovery from inactivation in the process of fast inactivation. Na+ channel a subunits in which each residue relative to rIlA in either the absence or presence of P1. This loop also contains from 1485 to 1491 had been individually substituted with cysteine were expressed binding determinants for a-scorpion and anemone toxins which slow inactivation. in conjunction with /31 subunits in Xenopus oocytes. Inside-out macropatches were These results indicate that substitution of several rHl loops affect conformational exposed to 50 nM MTSET and analyzed for effects on fast inactivation. Mutants changes required for recovery from inactivation. In contrast, only substitution of at residues 1485, 1487 and 1488 were unaffected in the presence of the sulfhydryl loop IVSS2-S6 reduced the effectiveness of P1 in accelerating both inacdvation of Na channels and from The of reagent MTSET while channels with cysteines at positions 1486, 1489, 1490 and open recovery inactivation. importance this 1491 were exquisitely sensitive to the modifying reagent. In these modified channels single extracellular segment in the effects of 11 on rIlA contrasts with our data demonstradng important determinants of inactivation in several extracellular inactivation during pulses from -100 to -20 mV was blocked almost completely. In loops. Altogether, our results suggest that multiple extraceliular loops influence the absence of MTSET, fast inactivation during depolarizations was similar to the the inactivation properties of rIIA and rHI, with IVS3-S4 playing the most wild-type channel with the exception of mutant G1486C in which inactivation was important role in inactivation and IVSS2-S6 in moduladon by P1. significantly slowed. These results will be considered in the context of the structure of the inactivation particle as deduced by NMR analysis.

Th-Pos300 Th-Pos301 INHIBMON OF NATIVE SKELETAL MUSCLE SODIUM CHANNELS BY A PKA PHYSIOLOGICAL FUNCTION OF THE VOLTAGE-DEPENDENT SODIUM ACTIVATOR IN RAT FAST-TWITCH MUSCLE FIBERS. ((J.-F. Desaphy. A. De Luca and CHANNEL REQUIRES MECHANICAL STABILIZATION. ((A.Shcherbatko D. Conte Camerino)) Unit of Pharmacology, Dept. of Pharmacobiology, Faculty of Phannacy. and P.Brehm)) Dept. of Neurobiol.& Behav., SUNY, Stony Brook, NY, 11794. Univ. ofBari, Bari, Italy. When expressed in Xenopus oocytes, the a subunits of the rat skeletal muscle (A1) Although the rat skeletal muscle sodium (rSkMI) channel can be phosphorylated by cyclic AMP- and human peripheral nerve (PN1) sodium channels require coexpression of a ,B dependent kinase (PKA) both in vitro and in vivo, no change in its biophysical properties has subunit for physiologically appropriate fast inactivation. We find that the slow been observed in response to PKA activation when it was heterogously expressed. Thus, we have inactivation typically observed for a subunit function can be converted to pure fast investigated the effect of a membrane-permeant PKA activator l8-(4 chlorophenylthio)-cyclic inactivation without the requirement of a 13 subunit. Cell-attached macropatch (tip AMP, CPT-cAMPI on native rSkMI channels by means of the cell-attached patch clamp OD-l5gM) measurements from a subunits of both Na channel types reveal a technique performed on freshly dissociated fibers offlexor digitorum brevis muscles ofadult rats. transition from slow inactivation (TlSms; Vtest >40 mV) to pure fast inactivation Fibers were bathed in a 145 mM-CsCI solution so that membrane potential was about -5 mV, as (t=200gs) over a 20' recording period (see Fleig et al., 1994. Pflugers Arch. measured by intracellular microclectrode. Pipettes. filled with a 150 mM-NaCI solution, had 427:399). Patch excision greatly accelerated this process. The midpoints for resistances of about 3.5 MD and allowed the registration of macroscopic-like sodium currents. activation (-45+3mV) and steady-state inactivation (-93+7mV) for macropatch Extenal application of 0.5 mM CPT-cAMP induced a decrease in sodium current elicited by 30- recordings were shifted negative compared to those obtained using the cut-open ms depolarizations apphed every second from -100 to -20 mV. Inhibition initiated 254 ± 41 s oocyte technique (-21±4mV for activation and -47+2mV for inactivation). after applying the compound, then attained 500/o of control current in 432 ± 48 s and reached its Importantly, no conversion to fast inactivation was observed using the cut-open maximum (81 ± 3 °%) in 729 ± 47 s (n=8). Voltage dependence of conductance (deducted from I- oocyte technique. The fact that micropatch (tip OD<5wM) recordings also reflected V curves) was not modified but the maximal conductance Gnmax was reduced by 63 ± 4 % in 591 little conversion indicated that macropatch findings were not a simple consequence ± 36s (n=5). In the same period, the steady-state inactivation was shifted toward the left by 7.2± of the cell-attached recording technique. A role of cytoplasmic factors in the 3.2 mV (from -1.3 to -18.6 mV; n=5). However, reduction of Gmax occurred before the shift of conversion process is suggested by the observation that treatment with a steady-state inactivation. Current kinetics were affected in only 2 out of 8 cells, which showed microtubule disrupter, nocodazole, caused abrupt transition to fast inactivation slower inactivation. The inhibition was not use-dependent, occurring with the same rate at -100 kinetics. We propose that the functional conversion associated with macropatch mV (frequency 0) as during repetitive depolarizations to -20 mV (frequency I or 0.5 Hz), The recording results from a dissociation of the a subunit from an as yet unidentified time constants for the recovery from inactivation were not significantly altered (n=2). The cytoskeletal component. Furthermore, we find that coexpression of Na channel a nucleotide did not modifv single channel conductance (n=8). Whether these effects result and P1 subunits resulted in fast inactivation with time constants corresponding to effectively from activation of PKA and can be reproduced by stimulation of a physiological those observed following time-dependent conversion of a subunit alone. The receptor ia under study. Such a regulation may have a therapeutic potential in situation of musicl similarity between the effects of 1 subunit and mechanical disruption on over-excitability as in some forms of inherited myotonia and paralysis. (Italian Telethon #901) inactivation kinetics of the a subunit suggests a common mode of action. A398AVI-~R NA CHANNELSHANEL Th-Pos3O2 Th-Pos3O3 ELECTROSTATIC EFFECTS ON Na CHANNEL GATING ((J.R. Miller, M.K. Patel, J.P. P-SEGMENT RESIDUES OF THE NA+ CHANNEL INFLUENCE USE-DEPENDENT Mounsey, J.E. John, J.R. Moorman)) University of Virginia BLOCK BY LOCAL ANESTHETICS. ((T. Yamagishi, J. R. Balser, E. Marban, G.F. Tomaselli)) Johns Hopkins University, Baltimore, MD 21205 Na channel inactivation is largely the work of the cytoplasmic domain 12 The P segments of the Na+ channel contribute to formafion of the outer vestibule of .wild the ion-selective We have demonstrated that linking domains Ill and IV. While * A KK 1317118 NN pore. by cysteine mutagenesis hydrophobic and aromatic residues \ v KKK 1321/2212 NNN residues in both SS1 and SS2 are accessible to extemally-applied thiol-avid and phosphorylation of a conserved reagents, e.g. Cd2+ and methanethiosulfonates (MTS). However, we found no serine all play a large role, the impact residues in the P segments that are accessible to Cd2+ or MTS when applied to the 6. cytoplasmic face of the channel. Local anesthetics bind to the inner mouth of the of the 12 positively charged residues E has been less well-studied. To test the pore of the Na+ channel, and a permanently charged derivative of lidocaine, QX-314, R was used to probe the intemal accessibility of P-segment residues. Nine cysteine hypothesis that the positively charged side of the filter this substitution mutants on the N-terminal putative selectivity (D400, lysine residues flanking E755, K1237, A1529) in the rat skeletal muscle ml channel were expressed in phosphorylation site have an 0- HEK293 cells and studied by whole-cell patch clamp. Mutations in both domains electrostatic effect on inactivation -120 -60 0 (Q399C, L396C) and IlIl (F1236C) P segments alter use-dependent block of the gating, we prepared two mutant Voltage channel by QX-314, decreasing the magnitude and hastening recovery from block skeletal muscle channels with reduced compared to the WT channel. In the absence of drug, the mutations in domain shift positive charge; KK 1317/18 NN and KKK 1322/23/25 NN. We studied inactivation gating the steady-state inactivation curve +5 and +10 mV (Q399C & L396C respectively) using macroscopic currents in RNA-injected Xenopus oocytes recorded using cut-open oocyte and hasten recovery from inactivation. In contrast, F1236C does not shift the steady clamping, and microscopic currents in cell-attached patches. The plot is the Hodgkin-Huxley T, state inactivation curve and slows recovery from inactivation compared with the Wr parameter as a function of voltage combining data from recovery from inactivation, two-pulse channel. The attenuation of use-dependent block by domain mutations can be inactivation and decay of macroscopic currents during depolarizing steps. The smooth lines are explained by changes in inactivation gating. In contrast, it is difficult to reconcile the fits to HH theory. The effect of charge reduction was to reduce the shorten T, at all potentials, gating changes of the F1236C mutant with a reduction in use-dependent block, and to shift the peak to more depolarized potentials. Single channel recording showed that suggesting that this residue may be involved directly in drug binding. Our results mutant channels had reduced burst duration compared with control but no change in mean open emphasize that gating changes can produce apparent changes in use-dependent time or waiting time. We conclude that the charged residues near the phosphorylation site have drug binding, and implicate portions of the channel other than IV-S6 in local a role in determining Na channel inactivation gating. anesthetic block.

Th-Pos3O4 Th-Pos3O5 FUNCTIONAL ROLE OF RESIDUES LINKING THE P SEGMENT TO A MUTATION OF THE SODIUM CHANNEL SUGGESTING THAT SODIUM CHANNEL BY DOMAIN IV S6 CONTRIBUTES TO THE OUTER VESTIBULE ((A. Sunami, S6 IN THE PROBED CYSTEINE S.C. Dudley, G. Lipkind, H.A. Fozzard.)) Cardiac Electrophysiology Labs, University of MUTAGENESIS. ((P. Vilez, T. Yamagishi, G.F. Tomaselli, E. Marban.)) Chicago, Chicago, IL 60637 Johns Hopkins University, Baltimore MD 21205 Transmembrane S6 segments have been shown to contribute to formation of the intracellu- lar pore of the various channels. On the other mutation near the extracellular our and others hand, 11760A, Recent results from laboratory (Neuron 16:1037, 1996) end of domain IV S6 in rat brain IIA Na+ channel created an access pathway to the inter- unexpectedly implicate "superficial' portions of various P segments in the nal binding site for quaternary membrane-impermeant derivative of lidocaine (QX314) from determination of Na' channel selectivity. We have extended the the outside (Ragsdale et al., Science 265, 1724-8, 1994). Consistent with this, the Lipkind- characterization of this region by performing serial cysteine mutagenesis on Fozzard model of the outer vestibule (Biophys. J. 66, 1-13, 1994) allows room to put the S6 structures between the P-loops. We tested the possibility that amino terminal end of the S6 residues G1533 through P1537 in the gtl rat skeletal muscle Na channel. segments may form part of the outer vestibule using mutagenesis of the adult rat skeletal These positions lie just C-terminal to SS2, on the way to S6 in domain IV, the muscle Na+ channel (pl). The mutation 11575A expressed in Xenopus oocytes, which is domain in which the surprising changes in selectivity appear to be most equivalent to 11760A in the rat brain channel, allowed block by externally applied QX314 0.05 in the p1 which is resistant to external prominent. Ionic currents were measured using whole-cell patch clamp in (500 pM, Hz) channel, normally QX314. Also, I1575A changed the reversal potential from 50 ± 1 to 42 ± 2 mV (Na+ 94, Ca2+ 0.5, Mg2+ transiently transfected HEK cells. The following phenotypic features were 1 mM), suggesting that I1575 affected selectivity and might be part of the outer vestibule. characterized: gating, selectivity, Cd2" block, TTX block and accessibility to Similar negative shifts of the reversal potential were observed in mutants of the putative se- MTS reagents. G1533C and L1534C alter selectivity; channels become lectivity filter (E755A: 33 ± 1, K1237A: -4 ± 1, A1529D: 43 ± 3 mV). Nevertheless, 11575A had little effect on binding affinity of tetrodotoxin, saxitoxin and p-conotoxin and on K+ permeable to K' and NH4'. Gl533C mutant also becomes Cd20-sensitive and permeability, whereas E755A and K1237A increased K+ permeability. K1237A enhanced TTX-resistant. In contrast, its neighbor LI 534C remains TTX-sensitive while Cs+ selectivity, but 11575A and E755A showed no change. Currently, we are measuring the gaining Cd20-sensitivity. G1533C is MTS-modifiable, while L1534C is not. conductance and the relative ion permeability in 11575A and selectivity filter mutants to determine if in the reversal and seen with 11575A are The results of new mutants that further the notion changes potential QX314 permeation provide examples support the result of direct participation of this residue in formation of the outer vestibule. that regions of the channel previously believed to be superficial and functionally unimportant instead participate intimately in the process of ion permeation.

Th-Pos3O6 Th-Pos307 A NOVEL APPROACH TO THE STUDY OF STRUCTURE/FUNCTION IN INTERACTIONS BETWEEN Na+-CHANNEL BLOCKER THE SODIUM ION CHANNEL ((A.A. Yanagihara. NI.F. Henteleff. MI.D. Rayner.)) DIBUCAINE AND Na+ CHANNEL INACTIVATION GATE Bekesy Laboratory of Neurobiology. University of Hawaii. Honolulu. Hawaii 96822 PEPTIDES AS STUDIED BY 'H-NMR SPECTROSCOPY ((Y. Kuroda, J. Ishikawa, Y. Tanaka, K. Tanaka, A. Otaka, N. Fujii, T. Nakagawa.)) Compared with the homotetrameric K channel, the complex nature of the heterotetrameric Na channel makes functional analysis of alterations by site-directed mutagenesis difficult Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, 606-01, because of potential masking effects of heterologous domains. To determine whether a Japan homotetramer composed of a single Na channel domain would be independently functional. a novel strategy was devised based on the hypothesis that flanking regions of the K channel Three hydrophobic amino acid residues (isoleucine-phenylalanine-methionine: (wvhich have been demonstrated to direct its tetrameric assembly) wvould do so wvhen flanking IFM) in the intracellular linker between domains III and IV of the Na+-channel a Na channel domain. Domain I (DI) coding region from the voltage-gated Na channel of a-subunit are known to work as inactivation particles for occluding the intracellu- rat skeletal muscle (SkXlpl) was ligated between flanking N- and C-terminal coding regions lar mouth of the channel pore in the fast inactivation process. The Na+-channel of an inactivation-removed voltage-gated K Shaker B 29-4 IR (Sh D) to yield a channel, blocker dibucaine the inactivated state and thus is more or chimeric construct (Sh-SkMImDI-Sh). Microinjection of the construct mRNA into Xenopus prolongs expected, less. laevis oocytes resulted in an intriguing exogenous current. This rapidly (< lms) activating to interact with the intracellular linker. In our previous publication (Kuroda et current showed no inactivation at 50 ms and closes with no visible tail current. Comparable al., 1996, Biophys. J. 71:1191-1207), we noted that there are negatively charged net outward currents and I-V curves were observed by TEV clamp recording despite bath amino acids only on both sides of the IFM amino acid residues in the III-IV linker changes from K+, Na+, Cs+, NH4+, Cs+ and Tris Ringer solutions suggesting that this (Asp-1487, Glu- 1492, and Glu-1493), which may play an important role in attract- net outward current may instead be due to an inward anion flux of the more permeant ing the channel blocker, especially for the tertiary amine local anesthetics, to or Cl ion. Low Cl and Cl free solutions reduced or abolished. respectively. this novel current near the inactivation particles. Presently, we have synthesized the fragment peptide suggesting that this construct channel conducts Cl ions. Functionalitv of a DI homotetramer which includes the IFM residues and demonstrates for the first time that the molecular properties required for functional gating, (GGQDIFMTEEQK; MP-lA) some related are and for ion selectivity, remain inherent in this domain. These findings open the way to peptides which mutated at the acidic amino acid residues (D, E) into the cor- more detailed studies of selectivitv and gating properties of this DI construct, as well as responding neutral amino acid residues (N, Q). We have investigated the secondary equivalent constructs from other channel domains. Supported by NIH RCMI grant 5G12 structure of MP-lA in a trifluoroethanol solution and also investigated the inter- RR/A103061 and AHA awards. actions of these fragment peptides with dibucaine in sonicated phosphatidylserine (PS) liposome solutions by 'H-NMR spectroscopy. We concluded that the proto- nated quaternary ammonium of dibucaine is interacting electrostatically with the negative charge arising from the Glu-1492. -~ ~~ ~ ~ ~~~~~~NNA CHIANNELS A399 Th-Pos308 Th-Pos3O9 HINDRANCE ON THE CHIRAL CARBON ATOM OF lb ANTIARRHYTHMICS EFFECTS OF CLINICAL CONCENTRATIONS OF ISOFLURANE ON ENHANCES THE USE-DEPENDENT BLOCK OF MUSCLE Na+ CURRENTS AND SODIUM CURRENTS IN RAT CORTICAL NEURONS. ((T.N.Vysotskaya, THE ANTIMYOTONIC ACTIVITY EVALUATED IN VITRO AND IN VIVO IN Ph.D., D.S.Duch, Ph.D.)) Cornell University Medical College, New York, NY 10021. ADR/ADR MOUSE. ((A. De Luca, S. Piemo, F. Natuzzi, A. Durantil, G. Lentinil, C. Franchinil, V. Tortorellal, H. Jockusch2, and D. Conte Camerino)) Unit of It has been reported recently that rat brain-Ila sodium channels expressed in a mammalian Pharmacology, Dept. of Pharmacobiology and 'Dept. of Medicinal Chemistry, Univ. cell line and in DRG neurons are much more sensitive to general anesthetics than earlier of Bari, Italy, and 7Developmental Biology Unit, Univ. of Bielefeld, Germany. studies with non-mammalian sodium channels predicted. These studies are in accord with In searching for more potent antimyotonic drugs, we previously found that chemical cellular studies which have revealed that volatile anestheticsat clinical concentrations alter modifications at the chiral carbon atom of lb antiarrhythmics mexiletine (Mex) and the discharge pattern and AP properties of CNS and peripheral neurons in mammalian tocainide (Toc) account for drug potency on muscle sodium channels (De Luca et al., preparations. Additionally anesthetics have been found to alter signal propagation in thin, N.S. Arch Pharmacol. in press). Thus, the effects of an increased hindrance on the unmyelinated fibers (intracortical fibers,sensory afferents). The purpose of the present study chiral center by the introduction of an isopropyl (Me5 and Tol) or a phenyl group was to examine the sensitivity of sodium channels in cortical neurons to general anesthetics. (Me4) were investigated in vitro on Na+ currents of single frog muscle fibers with the Primary cultures of rat frontal cortex neurons were obtained after enzymatic dissociation. Hille-Campbell voltage clamp. The new compounds as (-)-R enantiomers produced a Currents were measured using the whole cell patch clamp configuration. K+ and Ca++ tonic block, evaluated with single 10 ms test pulses from the holding potential of -100 currents were blocked with intracellular Cs (140mM) and extracellular LaCl 0.005mM. Sim- mV to -20 mV, with the following potency: Me4 (IC50 = 12 ± 0.8 JIM) > Me5 (18 ± 2 ilar to results with the rat brain Ila sodium channels,isoflurane suppressed sodium currents p.M) > Mex (55 ± 12 FM) > Tol (209 ± 9 gM) > Toc (583 ± 11 gM). Me4, MeS and through at least two mechanisms:1)A voltage-independent suppression of resting or open Tol were highly use-dependent blockers as the IC50 decreased by 3-8fold during a channels athyperpolarized potentials. At MAC for isoflurane,(0.28 mM), average peak cur- 10 Hz stimulation, vs. the twofold decrease observed with Mex and Toc. Accordingly, rent suppression was 46%(+/-17%sem, n=6 cells). 2)A hyperpolarizing shift in the voltage- Me4, MeS and Tol were also more potent than parent compounds in reducing in dependence of channel inactivation.The control midpoint of sodium channel inactivation was vitro the spontaneous discharges of action potentials and the high frequency firing of about -50 mV. Similar to rat brain sodium channels, 0.27 mM isoflurane shifted the inacti- intercostal muscle fibers of myotonic adr/adr mouse recorded with current clamp vation midpoint by about -20 mV (+/-4mV sem) in 4 examined cells. Conclusions: Clinical intracellular technique. Acute s.c. administration to adr mice of close-to-therapeutic concentrations of isoflurane blocked a significant fraction of sodium currents in brain cor- doses of Mex (l0pg/g) and Toc (20gg/g) significantly reduced the time of righting tex. These results support the hypothesis that at least some mammalian neuronal sodium reflex (TRR; 8.6 ± 0.63 s n = 19 in adr vs. 0.5 s of healthy mice) by 57% and 67%, channels are sensitive to clinical concentrations of volatile anesthetics in situ. respectively. Comparable effects on TRR were observed with 7pg/g Tol (80%), 2.5pg/g Me4 (50%) and 2.5gjg/g MeS (64%), without manifest side effects. Thus the increased hindrance on the chiral center may favour the interaction with an hydrophobic domain of the drug receptor enhancing drug potency at channel level, use-dependent behaviour and antimyotonic activity (Telethon-Italy, project # 901).

Th-Pos31O Th-Pos311 FAST AND SLOW INACTIVATION OF THE HUMAN CARDIAC SODIUM A NOVEL QUATERNARY AMMONIUM DERIVATIVE OF LIDOCAINE CHANNEL a-SUBUNIT IS DIFFERENTALLY ALTERED BY VOLATILE (TONICAINE) AS A POTENT NA+ CHANNEL BLOCKER. ANESTHETICS. ((A. Stadnicka, W.M. Kwok, H.A. Hartmann* and ZJ Bosnjak)) ((P. Gerner, K.D. Auktor, C.F. Quan, G.K. Wang)), Brigham & Women's Dept. Anesthesiol., Medical College of Wisconsin, Milwaukee, WI 53226, and *Dept. Hospital, Harvard Medical School, Boston, MA 02115. Molec. Physiol. & Biophys., Baylor College of Medicine, Houston, Texas 77030. We investigated the effects of halothane and isoflurane on the human cardiac (hHla) We have previously described that a N-3-phenylethyl lidocaine quaternary Na+ channel a-subunit stably expressed in HEK293 cells. Peak Na+ current was ammonium bromide (tonicaine) can be used as a long acting local anesthetic in decreased by 51±10% with 1.3 mM halothane and 60±12 % with 1.0 mM isoflurane. vivo (Anesthesiology 83:1293-1301, 1995). To determine its potency relative to Activation kinetics and the steady-state activation were not changed. The rate of lidocaine and QX-314 in vitro, we constructed dose-response curves for Nae inactivation time constant decreased by 15.6±2 % with halothane and 19±3 % with current inhibition under whole cell voltage clamp conditions in cultured rat isoflurane. With halothane, the V,2s of the steady-state inactivation induced by 50 ms neuronal GH3 cells. Tonicaine and QX-314 were applied through internal and 500 ms prepulses, showed hyperpolarized shifts from -68.6±4 to -79.1±5 mV, and perfusion whereas lidocaine was applied externally. Internal perfusion of QX- from -82.2±2 to -90.1±3 mV, respectively. With isoflurane, V,0 shifted from -70.1±5 314 is necessary because it is a permanently charged molecule and does not to -81.4±5 mV and from -84.8±4 to -90.3±6 mV, respectively. The k values were not readily cross the membrane. Tonicaine, also a charged molecule, does cross the significantly changed. During slow inactivation induced by 1Os prepulses, V,0 shifted membrane but the rate is too slow to reach its steady state inhibition when from -41.5±7 to -65.3±4 mV with halothane, and from -44.1±14 to -67.2±5 mV with applied externally. We found that the potency of internally applied tonicaine isoflurane. The k decreased from 23.1±0.4 to 18±0.4 with halothane, and from was 80 times greater than externally applied lidocaine and 2.5 times greater than 27.2±0.8 to 19.6±1.4 with isoflurane. The rate of recovery from inactivation time internally applied QX-314 (Kd of 7, 569 and 18 pM, respectively). The Hill constant increased from 5.4+0.4 to 9.1+0.8 ms with halothane, but was not coefficient was measured about 1 for all three drugs. Additional use-dependent significantly changed by isoflurane (5.3±0.5 to 5.8±0.8 ms). While the effects on peak block ofNa0 currents was found these We current and slow inactivation were reversible anesthetics the during repetitive pulses using drugs. completely upon washout, a fast and intermediate inactivation, and recovery from inactivation were only partially concluded that permanently charged amphipathic molecule like tonicaine is a recovered. These results indicate that volatile anesthetics profoundly alter inactivation potent Nae channel blocker and can be trapped within the cytoplasm which in of human cardiac Na0 channel a-subunit. turn may contribute to its prolonged mode oflocal anesthetic action in vivo. (Supported by NIH grant GM 48090)

Th-Pos312 Th-Pos313 RELATIONSHIPS BETWEEN STEADY-STATE INACTIVATION, COCAINE BINDING, TWO RESIDUES IN SEGMENT IVS6 OF THE RAT BRAIN TYPE AND COCAINE AFFINITY AT VOLTAGE-GATED Na CHANNELS. (('S.N. Wright, IIA Nae CHANNEL a SUBUNIT FORM PART OF THE 2S.-Y. Wang, 3Y.-F. Xiao, and 'G.K. Wang)) 'Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115; 2SUNY, Albany, NY 12222; 3Beth Israel Hospital, RECEPTOR SITE FOR LAMOTRIGINE. ((Guoying Liu, Todd Harvard Medical School, Boston, MA 02215 Scheuer, and Wiffiam A. Catterall)) Dept. ofPharmacology, Univ. of Washington, Seattle, WA 98195-7280 We transiently expressed voltage-gated Na clsannels in HEK 293t cells to examine cocaine block under whole-cell voltage-clamp conditions. We investigated how changes in steady-state inactivation affected both channel affinity for cocaine and the voltage dependence of cocaine Voltage-dependent Na0 channels are targets for local anesthetic and binding. We determined the extent of steady-state cocaine block by delivering 10-sec anticonvulsant drugs. Previous studies ofdrug block have identified conditioning pulses of various amplitudes. After the conditioning pulse, the membrane potential was stepped back to the holding potential (-140 mV) for 100 ms, and the percentage residues F1764 and Y1771 in transmembrane segment IVS6 ofthe rat of block was measured at a subsequent test pulse. The 100 ms interval at -140 mV allowed brain type IIA Na+ channel a subunit as forming part ofoverlapping drug-free channels to recover from fast inactivation. The midpoint of steady-state inactivation receptor sites for local anesthetics as well as for the anticonvulsant, for Na channels from human heart (hHl) was -15 mV more negative than was the midpoint of inactivation for rat skeletal muscle (j.d) Na channels. We produced shifts in the midpoints of phenytoin. We have extended our study ofthese mutant channels to steady-state inactivation of hHl and pl (1) by creating IHIl/pl channel chimeras; and (2) by the anticonvulsant lamotrigine and three other structurally related coexpressing the a subuniits of IsHI and pl with the rat brain 11 subunit. The midpoints of investigational compounds. Each compound preferentially blocks and steady-state inactivation of the chiimeras were intennediate to those of hHl and pal, whereas the midpoints of inactivation of lsHl-P1 and pl-p1 were shifted to less negative voltages as stabilizes the inactivated state ofthe wild-type channel a subunit when compared to the midpoints of inactivation of lilH and ±1. We found that differences in steady- expressed with the subunit in Xenopus oocytes and studied by two state cocaine block among these channels correlated well with the differences in steady-state 031 inactivation. As with hHl and pl, cocaine block of the chiimeras and of p1-P1 and hHl-pl microelectrode voltage clamp. In addition, the compounds give increased as the amplitude of the conditioning pulse became less negative. The cocaine varying degrees ofuse-dependent block. When block ofmutants affinities were similar for all of the channels. Cocaine block of resting channels was relatively F1764A and Y1771A was studied, block ofinactivated channels by weak (Kd = -250 paM), whereas block of inactivated channels was -25-fold greater (Kd = -10 pM). Therefore, shifts in the midpoint voltage of steady-state inactivation produced by either each compound was reduced 10-20 fold. In addition, use-dependent method caused parallel shifts itl cocaine binding, by altering the proportion of resting to block was virtually abolished. These findings indicate that amino acids iniactivated chaninlels at a given voltage, but did not cause changes in cocaine affinity at either F1764 and Y1771 are key determinants ofthe receptor site for resting or inactivated channels. This work was supported by NIH fellowship GM18760 (SNW) and NIH grant GM35401 (GKW). lamotrigine and related compounds. A400A400NACANLNA CHANNELS Th-Pos314 Th-Pos315 A POINT MUTATION IN DOMAIN III OF A DrosophilaNEURONAL Na CHANNEL A SPECIFIC INTERACTION BETWEEN THE NA CHANNEL AND CONFERS RESISTANCE TO TYPE I PYRETHROIDS. SITE-3 TOXIN ANTHOPLEURIN B ((G. R. Benzinger. J. Vi. Kyle, K. M. ((R.L. Martin, B. Pittendrigh, J. Liu, R. Reenan, B. Ganetzky, R. ffrench-Constant, and Blumenthal', and D.A. Hanck.)) The University of Chicago, Chicago IL 60637 and D.A. Hanck)) University ofChicago and University of Wisconsin-Madison. 'The University of Cincinnati, Cincinnati OH 45267 Voltage-gated sodium channels are the presumed site ofaction ofpyrethroid insecticides. The polypeptide neurotoxin Anthopleurin B (ApB) isolated from the venom of We screened several Drosophila lines that had Na channel mutations for resistance to the sea-anemone Anthopleura xanthogrammzca is one of a family of toxins that bind allethrin, a type I pyrethroid. In insecticidal bioassays thepara'4 fly line showed greater to the extracellular face of voltage-dependent sodium channels and retard channel than 4-fold resistance to allethrin. The amino acid substittution associateu withpara74 lies inactivation. Because most regions of the sodium channel known to contribute to within the S6 transmembrane region ofdomain III. This site is analogous to the mutation inactivation are located intracellularly or within the membrane bilayer. identifica- in domain II underlying knockdown resistance (kdr), a naturally occurring form of tion of the toxin/channel binding site is of obvious interest. Recently, mutation of a pyrethroid resistance found in houseflies and other insects. Electrophysiological studies glutamic acid residue on the extracellular face of the fourth domain of the rat neu- were performed on isolated embryonic Drosophila neurons placed in primary culture for ronal sodium channel (rBr2a) was shown to disrupt toxin/channel binding (Rogers 3 days to one month. para74 sodium channels appeared qualitatively similar to wild type et al.. J. Biol. Chem. 271:15950). A negative charge at this position is highly con- channels. Application of 500 nM allethrin caused removal ofinactivation and prolonged served between mammalian Na channel isoforms. We have constructed mutations tail currents in wild type Na channels but had little or no effect on para74 mutant Na of the corresponding residue (Asp-1612) in the rat cardiac channel isoform (rH1) channels (n=3). and shown that the lowered affinity occurs primarily through an increase in the toxin/channel dissociation rate koff. Further, we have used thermodynamic mutant cycle analysis to quantify the specific interaction between this anionic amino acid and Lys-37 of ApB (Az G = 1.5 kcal/mol), a residue that is conserved among mans sea anemone toxins. Reversal of the charge at Asp-1612, as in the mutant Asp-1612- Arg, also affects channel inactivation independent of toxin (-14 mV shift in channel availability). suggesting that Asp-1612 may contribute both to toxin/channel affin- itv and to transduction of the toxin's effects on channel kinetics. The interaction between Asp-1612 and toxin residue Lys-37 provides a defined interaction between Wild type P74 mutant the channel and a molecule with a known three-dimensional structure. (V,= -30 mV, Vh= -130 mV 150 ms test pulse)

Th-Pos316 Th-Pos317 EFFECTS OF AN INACTIVATION GATE PEPTIDE ON TWO IDENTIFICATION OF MOLECULAR DETERMINANTS SLOWLY INACTIVATING NA+ CHANNEL MUTANTS ((H. Lerche, RESPONSIBLE FOR DIFFERENCES IN d-SCORPION TOXIN W. Peter, N. MIitrovic, R. Fleischhauer, M. Schiebe, F. I,ehmann-Horn.)) BINDING BETWEEN BRAIN AND CARDIAC SODIUM Departiitent of Applied Physiology, University of Urm, D-89069 Ultim, Germany CHANNELS. ((S. Cestele, Y. Qtu, J. Rogers, H. Rochat#, T. Scheuer and W. A. Catterall.)) Dept. of Pharmacology. U. of Washington, Seattle, WA 98195, Two human skeletal irlilscle Na+ channel rntutations cauising paraitiyotonia con- USA arid #CNRS UMR6560, 1:3916 Marseille, France. genita (R1448P, located in the voltage sensor IV/S4) or potassiiim-aggravated my- otonia (GI306E, located in the proposed inactivation gate. the III/IV-linuker), were The 3-scorpioni toxin CssIV, isolated from venom of the scorpion Centruroides studied by heterologous expression in HEK293 cells tising whole cell patch clamping suffusus suffusus. binds with higher affinity to brain sodium channels than car- and a pentapeptide (KI 'NIK) contaitiing the proposed inacitvation particle. Mutant diac sodiuni chaninels. To identify amino acid residues responsible for this differ- Na+ currents decayed 5-fold (R1448P) and 3-fold (G1306E) slower compared with ence, chimiieric sodium channels were constructed using site-directed rnutagentesis, in wild-type (XV'T). KII' IK (0.3-ImM) accelerated the cuirrent decay when applied which the amino acid residues of the extracellular loops of the rat brain Ila channel intracellularly: The peptide introduced a fast decaying component to the slowly a subunit were converted to those in the cardiac rH1I isofornm. Results of binding ex- inactivating muitants that increased in amplitude with depolarization. J'ail currents periosienits indicate that conversionis in loops IS5-SS1, IISI-S2, 1IS3-S4 and IIISS2-S6 of iiiutant and WT channels recorded in the presence of peptide had larger am- may account for the different affinities of CssIV for brain and cardiac sodium chan- plituides and the decay of the peak tail anuplitride with prolonged depolarizations nels. The largest effect was observed with the loop S3-S4 vhich contained a single was markedly slowed suiggesting an open chanutel block by KIFMIK. The peak tail amino acid change (G845N) and reduced binding affinity 13 fold. Electrophysiolog- decay was voltage-dependent, indicating that the dissociation rate of KIFMK de- ical analysis showved that CssIV (4 nM) induces a shift of the voltage dependence creased with depolarization. Both. the effects oit inacitvation and tail curreints were of activation toward more negative potential values and reduces the currelnt ampli- concentration-dependetit. Increasing [Na+]± antagonized peptide block cornpatible tude in the brain isoform, whereas in the cardiac isoform CssIV has no effect on the wsith a pore-blocking mechanism. S'he results are compatible with a litlear three voltage dependence of activation. In the G845N chimera (loop IIS3-S4) up to 1 PM state model for an inactivated, open and blocked state, I-O-B, with an absorbing CssIV produced no detectable effect on the voltage dependeince of activation, and inactivated state, a voltage-independent on rate and a voltage-dependeint off rate for its electrophysiological effects were similar to those of the cardiac isoform. TIhese KIFMK binding. T he effects of the peptide were the same for both mutants which results indicate that at least four extracellular loops are involved in the formation of are located its different regions of the Na+ chatinel, suggesting that either KIFMK 3-scorpion toxini receptor site and that G845 may not only account for the different does not bind to the sattue site as the natural iitactivation particle or - if not - that affinity of CssIV on brain and cardiac sodium channel but may also have a critical the puttative docking site for the inactivation particle has not been modified by the role in the effects of this toxin on sodium channel activation. R1448P mrutationi.

Th-Pos318 Th-Pos319 RESTORING RAT SKELETAL MUSCLE-LIKE p-CONOTOXIN SENSITIVITY Ca-DEPENDENT SHIFTS OF Na CHANNEL ACTIVATION TO THE CLONED HUMAN SKELETAL MUSCLE SODIUM CHANNEL.((J. St- ARISING FROM STATE-DEPENDENCE OF Ca-BINDING IN THE Pierre, J. Sirois, E. Trottier and M. Chahine)) Laval University, Laval Hospital PORE ((Anna Boccaccio. Oscar Moran and Franco Contti.)) ICB-CNR. X ia De Research Center, St-Foy, Quebec, Cansada. Marini. 6. 1516149 Genova. Italv

Voltage-gated sodium channels (NaChs) are membrane proteins that play an essential The blockage by Ca of the open Na channel and the antagoniism by Ca of TTX role in establishing cell excitability and maintaining its conduction properties. Many binding to closed channels occur in very different ranges of [Ca]. WNe measured NaChs have been cloned, sequenced, expressed in heterologous systems such as the open-state block in channels expressed in Xenopus oocytes bv the a-subunits Xenopus laevis oocytes or mammalian cell lines, and characterized from brain (IIA) or adult skeletal muscle (p1) of rat. Recordings of instantaneous electrophysiologically and pharmacologically. A variety of natural toxins are known tail-currents from cell-attached macro patches show that the binding of Ca to the to act on voltage-gated NaChs by binding at specific high-affinity sites. We have blocking site has a dissociation constant of 20 mM at 0 mX' and senses 27% of the previously showed that human (hSkMl) and rat (rSkMI) skeletal muscle NaChs membrane potential drop. The IC,5 for Ca-inhibition of TTX-binding is. instead. present electrophysiological and pharmacological similarities. For example, both smaller than 1 mMI and rather voltage-insensitive. Assuming a single Ca-binding rSkMI and hSkMI are sensitive to tetrodotoxin (TrX). However, hSkMI is less site in the outer pore-vestibule, the state-dependency of Ca-binding implies a direct effect of Ca on sensitive to the block by p-CTX (IC5o=1760±40 nM) than rSkMl (IC5o=51.4+2.2nM) gating (Armstrong and Cota, PNAS 88: 6528531. 1991) and entails (22-fold lower). Putative extracellular regions of hSkMI and rSkMI differ by 22 Ca-dependent shifts of activation that we have estimated according to a simple model. In the we measured amino acids located in S5-S6 region known as the channel pore. In domain 11 S5-S6 range 0.2<[Ca]<10 mM, for IIA shifts similar to those of Pusch (Eur. Biophys. J 18:327-33, 1990) and slightly smaller shifts for p1. one amino acids differs between 1 region, only hSkMI (leucine735) and rSkM Their size 12 mX1 from 0.2 to Here in this (< 5 mM-Ca) is very close to that expected from (serine729). study we replaced by site-directed mutagenesis leucine735) in state-dependent binding. WVe have also analysed with that model the data by Pusch hSkMI by a serine. The affinity of p-CTX on hSkMI/L735S was found to be 9-fold (1990) on the IIA-mutant K226Q, which has a markedly reduced voltage-sensitivitv higher (IC5o=185.6±6.6nM) than wild type hSkMI due to a 3.1-fold decrease in ko,. and shows much larger shifts. The model does predict the observed roughlv inverse and a 1.4-fold increase in k,,. This mutation did not completely restored the p-CTX relation between the shifts and the steepness of the activation curve, which is hard sensitivity. Our results suggest that the serine residue in rSkMl play an important role to explain with theories attributing all shifts to the screening of surface charges by in p-CTX binding. Other amino acids may count for the difference between hSkM I Ca. The latter effect maydominate the of Na channels in native and rSkMI in of phenomenology terms their sensitivity to p-CTX. membranes, where larger shifts suggest a fixed negative charge density on the outer surface higher than in the oocy-te membrane. Partially supported by Telethon Grant \N 926. 114MNA CHANNELSILIJLJLtxilqllqjpljli3 tt-tulA401 Th-Pos320 Th-Pos321 REPULSIVE INTERACTIONS BETWEEN INTERNAL AND p-CONOTOXIN BLOCK OF Na CHANNELS - A FOCAL EXTERNAL BLOCKERS OF A SODIUM CHANNEL. ((I.E. Sierralta, ELECTROSTATIC MECHANISM? ((K. Hui, I.E. Sierralta, R.J. French)) G.W. Zamponi*, and R.J. French)) Depts. Physiol. & Biophys. and *Pharm. & Dept. Physiol. & Biophys., Univ. Calgary, Calgary, Alberta, Canada. Therap., Univ Calgary, Calgary, AB, Canada, T2N 4NI. (Spon. S. Barnes) The 22 amino-acid peptide, W-conotoxin (gCTX, net charge, +6) blocks Na channels of adult rat skeletal muscle (rSkMI), with a 1:1 stoichiometry, Internally applied diethylammonium (DEA) or tetrapropylammonium (TPrA), at apparently in an all-or-none manner. Unlike previously examined derivatives oft- millimolar concentrations, induce a rapid block of single Na channel currents. CTX, R13Q, in which Arg-13 is replaced by neutral Gln, only partially blocks the Depolarization enhances block. Block by DEA appears as a reduction in current through single batrachotoxin-activated channels in neutral bilayers, apparent single-channel current, whereas TPrA is seen as brief leaving block by (ms) -28% residual current (Becker et al., Biochem. 31:8229; 1992, French et al., discrete events. Both agents have a charge of +1, and block the channel at an R1 Neuron 16:407, 1996). We have now measured the relative conductance (ys/yo) apparent electrical position -55% of the way from the cytoplasmic side. 3Q of the 'blocked' state with several R13 (net charge +5), derived from the 22amino-acid peptide p-conotoxin GIIIA derivatives: R13K, R13A, and R13D. Doubly substituted toxin derivatives (D2N/R13Q and D12N/R13Q), with the (pCTX) by replacement of Arg-13 with Gln, blocks the channel incompletely same net charge as native allowed with a residual current of -28% of the normal single channel amplitude (Becker IICTX, about the same residual current as the singly substituted derivatives, R13Q and R13A, in which only the charge at et al., 1992, Biochemistry 31:8229; French et al., 1996, Neuron 16:407). Mean residue 13 in was neutralized. blocked times for the discrete R13Q blocking events are several seconds The results indicate that the Derivative Charge, duration, allowing us to study block of the R13Q-bound channels by DEA and y1/Ymn n TPrA. For both intracellular blockers, potency of block is reduced by R13Q, degree of block by g-CTX R13D Residue 13 meansd. 4 is dependent on the charge - indicated by positive shifts in plots of fractional block vs voltage, compared to R13D 0.56±0.02 4 values obtained when R13Q was not bound. Shifts in voltage for 50% block at residue-13, but is R l3A 0 0.32±0.02 4 were: DEA, 23 mV and TPrA, 18 mV. The similar values for the two internal uncorrelated with side-chain D2N/R13Q 0 0.30±0.03 3 volume or with 0 blockers of different structure and size suggest that the interaction with RI3Q is the net D12N/R13Q 0.26±0.07 3 charge on the toxin. 1 largely electrostatic. On that basis, we estimate that the centres of charge of the R3K 0 08±0 06 4 simultaneously bound RI3Q and amine blockers are separated by -3.5nm.

Th-Pos322 Th-Pos323 S4 TRANSLOCATION DURING SODIUM CHANNEL DEACTIVATION IS INHIBITION OF OPEN TO INACTIVATED CHARGE MOVEMENT BY AN ORDERED PROCESS ((J. Groomel, E. Fujimoto2, J. Olsen2. A.L. George. Jr. SITE-3 TOXINS IS LOCALIZED TO THE FIRST ARGININE OF DOMAIN and P.C. Ruben2)) 'Dept. Biol., Harvey Mludd College, Claremont. CA. Dept. Biol.. IV S4 IN VOLTAGE-GATED SODIUM CHANNELS ((M.F. Sheets, J.W. Kyle, Utah State Univ., Logan, UT, 3Dept. NIed. and Pharmacol., Vanderbilt Lniv.. Nashville, and D.A. Ilanck.)) University of Utah, Salt Lake City, UT (84112) and University of TN Chicago, Chicago IL (60637) Classical concepts of sodium channel gating predict that the order of events associated wvith Site-3 toxitis bind to the extracellular surface of voltage-gated Na channels and inhibit iti- charge movement (e.g. S4 translocation) leading to channel activation and deactivation is activation. Otir gating current studies have identified the priticipal action to be inhibition of random. Hoswever. putative channel structure suggests non-equivalent roles of S4 s in chan- charge associated with inactivatioti from the open state, which accounts for approxitiiately nel gating. based on unequal numbers of charged residues. To experimentally distinguish 33% of the total charge) (JGP 106:617. 95). Localization studies from a number of labora- betsween biophvsical theorv and molecular structure, wvildtype and mutant human muscle tories have itiiplicated extracellular amino acid residues on domain and domain IV of the sodium channels were expressed in Xenopus oocvtes. Mlacroscopic currents 'vererrmeasured channel in channel/toxin interactiotts. We, therefore, tested the hypothesis that toxin bitid- from on-cell macropatches. Deactisation kinetics were measured from first-order exponen- ing inhibits the charge movement associated with domain IV, S4 by constructing a rnutant of tial functions fit to tail currents evoked by hyperpolarizing steps to voltages ranging from the humaii heart Na channel (hHbla) in which the most extracellitlar charged residue (R1622) -200 to -70 mi' following depolarizations to 0 or 50 mV for 0.25 or 0.3 ms. Replacement was replaced with a cysteine. This charged residue has been shown previously to participate of the outermost charged residue with cvsteine in the S4 membrane-spanning segments in in Na channel gating (Neuron 15:213, 95). We expressed this niutant in fused tsA201 cells, each domain wvere compared to svildtvpe channels. Cysteine replacement in the first. third using a previotisly published protocol (AJP 269:C1001, 96) and compared the effect of the and fourth domains (R219C. K1126C and R1448C) significantly slowred deactivation wvith site 3 toxin from Anthopleura ranthogrammica, ApA, on charge associated with activation the order of effect at -110 mi' to -70 mV being R1448C > K1126C > R219C. By contrast, (Q-a,) in six cells with a database of wild-type cells. Although macroscopic current decay cysteine replacement of the outermost charge in domain II (R669C) had no significant ef- was slowed in the mutant compared with wild-type, binding and complete modification by fect on the rate of channel deactivation at anv voltage. A double mutation of domaitis III ApA toxin could be confirmed as an additional inhibition of current decay. A comparison of and IV (K1126C/R1448C) slowved the deactivation rate over the range-170 mi' to -70 mi', total gatitig charge (Qwat) to chantiel density (estinmated from iottic curretit) indicated that and had a greater effect than either of the twco mutations alone. These data suiggest that the mutant moved less charge than wild-type. Moreover, when channels were modified with deactivation at physiological voltages is most likely' produced by DIVS4 translocation. and ApA toxiti, in contrast to wild-type channels, only a small charge reduction was detected. (1) that dolwnstream" reactions indirectlv affect the rate of channel deactivation, or (2) We concltide that the major effect of site 3 toxins on Na channel gating is to inhibit the that DIIIS4 and DIS4 translocations can also deactivate the channel, but wcitli decreased movement of the most extracellular charged amino acid on domain IV, S4 of voltage-gated probabilities relative to DIVS4. Na channels.

Th-Pos324 Th-Pos325 DIFFERENTIAL EFFECTS ON FAST INACTIVATION OF DOMAIN THE ROLE OF THE PORE IN MODULATION OF SODIUM IV S4 CHARGE NEUTRALIZATIONS ((J.L. Abbruzzese. E. F'tijitiioto CHANNEL GATING BY EXTERNAL PERMEANT IONS ((C. atil l'.('. Itubetn.)) I)epartmietnt of Biologs. titahi State University. Logati. U'l' Townsend, H.A. Hartmann, and R. Horn.)) The Institute of Ilyperexcitabibty, 8 :122-5:105 Dept. of Physiology, Thomas Jefferson University. Philadelphia, PA 19107. and Dept. of Nlolectular Physiology and Biophysics, Baylor College of Nlediciiie, 'revionis sttudies of l)otnaint IR' S-1 charge tneuitralizatiotn alil rese'rsal ill sodiurii Houston, ''X 77030. channite'ls hase foctused oni tlie otitermost charge's. llossever, DIX'SI iticltides the greate'st nulmer of charged residtlies atid is. thetettfore a uttique subject il wliiclt to Low concentrations of extracellular pertueant ions reduice the probability for a itivestigate the contributionts of each charged residute to the biophysical behiasior of sodiuni channel to open at large depolarized voltages [1]. To determine whether sodiliiiti (-hatin(ls..('oisi('a'ietitlIs. fouir n3titants ssere cotistriucted, siibstitutitig a glti- the pore region of the channel plays a role in the modulation of gating by external tattitiie for tthe fotirthl tltrotigi tthe seetlth argittities foiiiid in DAI'S-1 itt rat skeletal permeant ions, wce examuitted the efrects of pore blockers, cysteine modification, and ntti'scle sodititii chatintels. lihe' itititatts wsere coexpressed swith the 3 stibtittit in X'cti- inutations withiti the channel pore. Humnan cardiac sodium channels (hHla) were pits ooc%tes. atid chianinel attisitv swas recorle(il froin cell-attached niatropatclhes. transiently expressed in both titammalan tsA201 cells and Xenopus oocytes. Intra- TIlie effects of the differetit tharge netitralizatiotis Ott fast inactisation suggest that cellular pore blockers such as tetraethylammonium (17.5-35 mNM) and diethylantine tie ititerior cliarge(l residtles hase icldividtializetl roles that cati iiot be readily pre- (20 mM) abolish the effect of external sodium concentration ([Na]I) on peak open dictedl It positiott. It I19Q sigtlificantlv stabiliz"s the fast ittactivatedl state; onset probability. Similarly, modification of the native pore-lining residue cysteine 37:3 kittetits are faster aild recovery. is tip to teit titites slower than swildtype. 1y contrast, by the thiol reagent methanethiostulfonate ethylsulfonate (MITSES) diminishes the Rl 152Q slows the o'.set of fast ittactisatiotn atId actelerates its recovery. It 1455Q effect of [Na]5. Furthermore, a mutation in the pore of the channel that profoundly apptears to htave inttermitediate effects on fast ittactivatioti. ssith somewhat faster oti- alters selectivity (K1418C) also annuls the effect of [Na]5. In contrast, mutating s'et atti( recoerv. I'inalls. Ill1:58Q does not sigttifitantlI alter chatinel kittetics. btit ait aspartic acid residue located more externally in the pore region (D1422) to a presents ftll closed-state fast ittactivatioti at phtysiologicaly itiiportatit voltages (i.e. cysteine has no effect on the response to los [Na]5. Taken together these results -90 niX'). Sloss itactivation is signtificantly redtlttedi in IR1l I19Q, withi less than 50Oc, suggest that interactions between extracellular permeant iotis and the deepest re- of Ihattntels inactivatitig followisng I minute prepl'.ses (cottipared to 85t/c ofwil(ltvpe gion of the channel pore modulate channel open probability. [1] Townsend et al., chatittels inactisatitig). 'Ithe cotitbination of stabilized fast ittactivation antI desta- J.Gen.Physiol.110:11-21; 1997. tilized slos intactivatiotn protides further esidetie of interaction betweert these two btiophysical processes: fast ittattisatioti appears to lititit slow iitactivatioti. AimAAW IMAMA %-J3SThIVUANNlF.IS#O Th-Pos326 Th-Pos327 GLYCOSYLATION SHIFTS THE VOLTAGE DEPENDENCY OF CHANGE OF SURFACE POTENTIAL INDUCED BY CARDIAC SODIUM CHANNEL GATING ((Y Zhang, HA Hartmann, J INTRACELLULAR DIGESTION OF MEMBRANE PROTEINS ((Jing Satin.)) Dept of Physiology, Univ of Kentucky, Lexington, KY 40536 and Baylor Zhang, Mei-de Wei, and Leslie M. Loew.)) Department of Physiology, University Univ Coll of Medicine of Connecticut Health Center, Farmington, CT 06030 The voltage dependence of activation and inactivation for the cardiac Na channel We have reported that some of the membrane electric properties, such as the are hyperpolarized relative to that for the adult skeletal muscle isoform. To eval- transmembrane potential and the intramembrane dipole potential, can be measured uate the contribution of sugar residues to gating we pretreated mammalian cells with accuracy by combining whole cell patch clamp with fluorescence ratio imag- expressing either hHla or ul with glycosidases. In hHla cells, castanospermine ing of a potentiometric dye (di-8-ANEPPS) in cultured cells. In the present study, pretreatment caused a depolarizing shift of V1/2 for steady-state activation. In the same technique was used to evaluate the change of the surface potential asso- comparison, the endoglycosidases castanospermine and swainsonine as well as ex- ciated with the membrane proteins in cultured NlE-115 neuroblastoma cells. The oglycosidase neuraminidase also elicited about a 6 mV depolarizing shift of V1/2 for steady-state activation in ul groups. For steady-state inactivation, endoglycosi- membrane proteins on the inner surface of the cell were enzymatically degraded by dase pretreatment resulted in a depolarizing shift of V1/2 for hHla, but a small dialysis of papain (0.5-2mg/ml) through a whole cell patch micropipette and the and significant shift of V1/2 to a hyperpolarizing direction for ul. The possible degree of degradation was evaluated by the removal of inactivation of voltage-gated role of a charge-screening effect in glycosidase-induced depolarizing shift of V1/2 sodium channels. The change of the fluorescence ratio was simultaneously moni- for steady-state activation was tested with addition of extracellular divalent cation. tored by taking pairs of images at the excitation wavelength of 440nm and 530nm, High extracellular Mg concentration masked the castanospermine-elicited depolar- respectively, while clamping the transmembrane potential of the cell at OmV. The izing shift of V1/2 for steady-state activation in ul transfected cells. Glycosidase degradation of the membrane proteins decreased the fluorescence ratio; the average did not alter the single channel conductance, the mean opening time or the opening decrease was 5% ± 1% (Mean ± SEM; n = 9). To estimate the absolute value of the probability. These data suggest that glycosylation of hHla and ul could 1) regulate change of the surface potential, the fluorescence ratio was calibrated by clamping cell excitability by differentially altering voltage dependency of channel gating in the membrane voltages at various levels. The ratio was then normalized to that these two sodium channel isoforms; 2) the effects of glycosylation on shifts of volt- obtained from OmV and plotted against the membrane potential. This calibration age dependency of channel gating in cardiac and skeletal muscle sodium channels procedure was similar to that used previously to measure the resting potential of the could not completely explain the 20 mV difference of voltage dependency for both cell and indicated that the surface potential change induced by dialysis of papain activation and inactivation between these two channel isoforms, and 3) the effect of was approximately 33mV. We are currently studying the dipole potential profile of glycosylation on activation kinetics is at least partly mediated by charge-screening the cell and its possible effect on voltage-gated ion channels. (Supported by USPHS mechanism. Grant GM35063).

Th-Pos328 Th-Pos329 OPTIMIZATION OF A TWO-ELECTRODE VOLTAGE CLAMP FOR GATING CURRENT IN RAT BRAIN IIA SODIUM CHANNELS: RECORDING OF SODIUM IONIC AND GATING CURRENTS FROM CHANGES IN RATIO BETWEEN MEASURED GATING AND XEkNOPLS OOCYTES. ((N.G. Greeff and H.R. Polder.)) Physiologisches Institut. CORRESPONDING IONIC CURRENTS ((N.G.Greeff, F.J.P. Kuhn and Universitat Ziirich-Irchel. CH-8057 Zuirich: npi electronic GmbH. D-TI732 Tamm. W. Kathe.)) Physiologisches Institut, Universitat Ziirich-Irchel. CH-8057 Zurich. Sodium channel gating is a fast process: gating current (I,) occurs within less than one millisecond during the activation phase. If a two-electrode voltage clamp is used to record I. As gating current (I.) signals in sodium channels usually are very small, we cloned the membrane capacitance (C,,,) of about 200 nF has to be charged through a microelectrode ca- and d-subunits of rat brain IIA sodium channel into a high expression vector. with a resistance of 200-700 kOhm. This resistance and C, form a low-pass filter reducing Recorded macroscopic ionic currents after expression in Xenopus oocytes showed clamp speed. The clamp is further slowed dosvn by the series resistance (R,) between the a 10-fold increase from 4-6 to 50-60 pA inward peak ionic current (average val- sensing points of the clamp control in the cytoplasm and the bath in series to C, of the ues; pulses at -10 mV, resting potential -100 mV). WN'e successfully optimized a cell membrane. since C, has to be charged through R,. To overcome these constraints. a TEVC clamp to speed up capacitance transients (settling time of 100-150 j.s) and voltage clamp with a voltage controlled current source (VCCS) output and a compliance of to make currents visible. Total block of ionic current 2 ±220V was developed allowing to drive several hundred pA through electrodes of 300-600 gating macroscopic by pM kOhm. Therefore. the initial time constant of about 10 ms (determined bv C,,) is reduced to tetrodotoxin (TTX) at 15 IC and recording of recovery from fast inactivation (pre- the microsecond range, depending on the stray capacitv around the electrode. Based upon pulse to 0 mV for 20 ms followed by a 20 ms test pulse, with a varied recovery our experience in voltage clamp designs for the squid giant axon (I.C. Forster and N.G. period (1-60 ms) between prepulse and test pulse) were used to exclude possible Greeff. J. Neurosci. NMeth. 1990, 33: 185-205) we adapted conventional R, compensation distortion of gating current signals by ionic current or asymmetry signals. Recovery to this circuitry. The clamp has to be linear: otherwise. the capacitv transients of 100-300 from TTX block of ionic current, and pulses to the reversal potential revealed that pA cannot be subtracted by pulse protocols (P/N and analogs). Therefore. wve had to find the expected ratio of 1:100 between gating current and measurable corresponding a compromise between speed vs. linearity: in a model cell incorporating an R, of 1 kOhm. ionic current was not constant but shifted to values of between 1:50 and 1:5 de- 200 nF could be charged within 200 ps to a 100 mV step through an electrode of .500 kOhm and the capacity transient was suppressed bv 1000:1. Two strategies have been adapted pending on the time of expression. Through days 1-3 of incubation ionic currents to record sodium channel I in Xenopus oocytes: either the settling time at the membrane increased reaching persisting peak values between days 4 and 5. Before day 4, only was speeded up to about 60 ps and the appearing asymmetry was blanked out. or a slower small gating current signals of less than 1 pA were recorded. The signals regularly clamp setting with a better linearity and less asymmetry was used. increased to about 2-3 1.A peak gating current on days 4-5. These findings suggest that the actual number of sodium channels expressed in Xenopus oocytes is up to 10 times higher than calculated from recorded ionic currents. Supported by SNF grant Nr. 31-37987.93.

Th-Pos330 SUBUNIT STOICHIOMETRY OF A CORE CONDUCTION ELEMENT IN A CLONED EPITHELIAL AMILORIDE-SENSITIVE Na CHANNEL ((°B.K. Berdiev, 'K.H. Karlson, *F. Kosar, 'B.A. Stanton, 'T.R. Kleyman, 1.l. Ismallov)) "Dept. of Physiol. & Biophys., Univ. Alabama @ Birmingham, Birmingham, AL 35294, *Dept. of Med., Univ. of Pennsylvania, Phyladelphia, PA 19104, 'Dept. of Physiol., Dartmouth Medical School, Hanover, NH 03755. Stoichiometry of a core conduction element in cloned epithelial Na+ channels (ENaC) was studied in combinatorial planar lipid bilayer experinments. Single a-rENaC and the acy-rENaC were disassembled with 50 pM dflhiotreitol into protochannels with nearly identical biophysical properties: 13 pS conductance, double exponentially distributed closed times (T,1=18*8 ms, T1=l52*31 ms, (a-rENaC); and Tcj=24*9 ms, T12=167*33 ms (axy-rENaC,)), and single open iffme (T,=,157±43 ma (a-rENaC), and T,o=171±39 ma (apy-rENaC). Two pairs of in vitro translated proteins were employed: 1) wild type (WT) and the 278WYRFHY283-tract deleted a-rENaC, which has a greatly diminished (250-fold) sensitivity to amiloride; and 2) WT and the N-terminally truncated A1 09T- Ma-rENaC which displays accelerated kinetics: T,=37*13 ms, Tc=42*1 I ms, and T,=49*19 ms, TC = 52±17 ms, for a-and apy-rENaC, respectively. The channels found in a awr:amN mixture (with or without the WT J- and y-) formed two groups, one with To and T, which correspond to the fully mutant N-terminally truncated channels, and another, with two closed states and one open state, which correspond to the fully Wr a-rENaC, or a,By-rENaC distrubuted depending of the ratio of the WT and N-terminally truncated protein. Comparison of the statistical distributions of channel phenotypes with the theoretically expected probabilities of random association of two elements suggests tetrameric organizaton of a-subunits as a minimal model for the core conduction element in either a-, or apy-ENaC. Five channel subtypes with distinct sensitivities to amiloride were found in a 1a:1aw,:,,,2,,0 protein mixture support this condusion. Supported by NIH grant DK 37206. EPITHELIALEPITHELIAL PHYSIOLOGY A403 Th-Pos331 Th-Pos332 SPATLkL RESOLUTION OF THE EFFECTS OF CHOLESTEROl. 1I 'Il'RIVATION AQUAPORIN 1 (AQP-1) SELECTIVITY FILTER ((G. Whittembury, E. AND/OR ENRICHMENT ON OK CELL MEMBRANES BY LAU 1X 1) \N GP TWO- Gonzalez, A. M. Gutierrez, M. Echevarria and C. S. Hernandez.)) Instituto PHOTON FLUORESCENCE MICROSCOPY. ((H. Zajicek, W. Yu, I l. Wang, E. Venezolano de Investigaciones Cientificas, IVIC, Caracas, Venezuela Gratton, T. Parasassi, and M. Levi)) The University of Texas Southwestern Med Ctr and VAMC, Dallas, TX; LFD University of Illinois, Urbana-Champaign. Molecular sieving experiments have led to conclude that the selectivity filter of is a in AQP-1 in the proximal straight tubule, PST, basolateral cell membrane single To detemine the effects of alterations cholesterol content on membrane lipid file structure with a diameter of 4.5 A (J Membrane Biol 143:189. 1995). Here we dynamics opossum kidney (OK) cells were grown either a) in a lipoprotein deficient evaluate its length, AX, using Hill's eqns (Int Rev Cytol 163: 1.1995) which require serum (LPDS), which resulted in a decrease in cell cholesterol content, or b) in LPDS measurements of osmotic permeabilities and reflection coefficients (P,.iPos,, 0,s, which was then supplemented with LDL, which resulted in an increase in cell Usp) of impermeant (i) and permeant (p) solutes. respectively. PST were dissected cholesterol content. The cells were labeled with the membrane fluorescent probe 2- from rabbit kidneys, held with crimping pipettes in a chamber bathed in a buffered dimethylamino-6-lauroylnaphthalene (LAURDAN) and imaged by a two-photon mannitol (isosmotic) basic solution (MBS, 295 mOsm/kg). Then changes in tubule excitation microscope. From the fluorescence intensity images, LAURDAN cell volume with time (dV/Adt) were monitored on line (in an oscilloscope) with generalized polarization (GP) images were calculated. Both in control and in treated an inverted microscope, a TV camera and an image processor, after hyperosmotic cells, a broad GP distribution was observed, with higher GP values in the plasma steps in MIBS osmolality (AC,i and AC,p). To measure P,,,i and a,i PST were membrane, intermediate values in the cytoplasm and lower values in the nuclear exposed for 20 s to MBS made hyperosmotic by addition of a AC,i of 35 mOsm/kg membrane. In agreement with previous cuvette-based fluorescence spectroscopy of mannitol (M), acetamide (A) and urea (U). Cells shrunk within 500 ms of t=0 to studies, cholesterol deprivation resulted in an average decrease of the GP value. From their osmometric volume and remained shrunk for the 20 s of the osmotic challenge. the images, this decrease in GP appeared mainly localized in the plasma membrane. Po,,i = 3000 +/- 25 sm/s with M, A and U. a,i = 1.00 +/- 0.01 for MI. A and U. After treatment of the cells with LDL, the average GP reached a vsalue above that of the These solutes do not penetrate AQP-1. By contrast, the shrinking curve with a ACsp control cells. This increase of the average GP value was due to both the plasma of 35 mOsm/kg formamide (F, ACs,Formamid,) was about 1/6th slower and smaller membrane and the cytoplasmic membranes. Indications of a morphological alteration (i.e subosmometric) than that produced by ACsi = 35 mOsm/kg. With F cells did of the apical brush border membrane following all above treatments were also not remain shrunk but recovered their original volume within 3 s. Pos,Formamide = obtained. 480 +/- 30 am/s. O,,Formamide = 0.16 +/- 0.01. These values lead to an AX of 6 - 9 A which agrees with the hourglass model of AQP-1 (Annu Rev Physiol 58:619, 1996) and are lower than previous ones of 13 - 18 calculated from the Po,/P; ratio.

Th-Pos333 Th-Pos334 EFFECT OF THE CARBOXYTERMINUS OF THE, SUBUNIT OF REGULATORY INTERACTIONS BETWEEN CFTR AND EPITHELIAL ENAC ON CHANNEL OPEN PROBABILITY ((G.K. Fyfe and C.. SODIUM CHANNEL CO-EXPRESSED IN XENOPUS OOCYTES. Canessa (Spon. by I. Favre).)) Department of Cellular and MIolecular Physiology. ((H. Chabot, M.F. Vives, A. Dagenais, Y. Berthiaume and R. Grygorczyk)) Yale University, Neiv Haven, CT 06520 H6tel Dieu Research Center, Montreal, QC, Canada. Epithelial sodium channels (EN'aC) with truncations of the carboxvtermini of Defective regulatory interactions between the cystic fibrosis conductance the 3(3T) or -,(-,T) subunits exhibit a 3 to .S-fold larger amiloride-sensitive whole- regulator (CFTR) and the epithelial sodium channel (ENaC) have been cell current than wild-type channels (Schild et al.. P.N.A.S 92:5699-5703. 199.5). implicated in the elevated Na transport rates across cystic fibrosis airway Similarly. the current expressed by Q^T is .5-fold larger than the one expressed by cko;. To investigate whether the increase in current is due to an increase in the epitheliumn. To study the mechanisms of these interactions we have measured open probability (Po) of channels bearing truncated subunits. wce examined the amiloride-inhibitable Na current (I,,*) in oocytes co-expressing rat ENaC and Po of o, and Q^yT channels expressed in Xenopus oocytes using the cell-attached human wild-type CFTR. Stimulating CFTR with 1 mM IBMX reduced I,,i, by up configuration of the patch clamp technique. XN-e found that the conductanice of a, to 70%. In oocytes expressing ENaC alone, IBMX had only a small (<15%) and a"jT. with 150 mMI Li+ in the pipette. was about 6 pS for both. The Po of inhibitory effect on I,,,,i, and thus could not fully explain the large inhibitory a, as 0.80 and for OaT was 0.60. Howvever. the incidence of patches containing effects observed in the presence of CFTR. To test if local changes in Cl channels swas higher in oocytes expressing oITi. We conclude that truncation of the concentration due to activation of CFTR might play a role in the inhibition ofNa carboxyterminus of the subunit does not increase Po and therefore cannot account were for an increase in wvhole-cell current. These results agree swith our recent finding that current, oocytes voltage clamped at the Cl reversal potential (-20 mV) to the carboxstermini of the 3 and, subunits contain internalization signals that swhen keep net Cl flux close to 0. This maneuver did not prevent the reduction of I,,,*, deleted increase the number of channels expressed at the cell surface (Shimkets et after IBMX stimulation. Interestingly, I,,,,i was not reduced when other al.. J. Biol. Chem. 272:25537-25541. 1997) maneuvers, not involving IBMX, were used to stimulate CFTR such as directly injecting cAMP into the oocyte (100 ±M final concentration). These data indicate that the inhibitory effect of CFTR on ENaC expressed in Xenopus oocytes does not require Cl flux through CFTR and may be a specific action of IBMX. (Supported by Canadian Cystic Fibrosis Foundation).

Th-Pos335 Th-Pos336 EVALUATION OF METHODS TO ANALYZE CONTINUOUS PHYSIOLOGICAL ATP RELEASE FROM CULTURED SHARK RECTAL CILIARY BEAT FREQUENCY RECORDINGS ((AI. Salathe, R. J. GLAND CELLS ((C. F. Jones, L. J. Roberts, G. R. Jackson, Jr., A. G. Prat, Blookman.)) Dept. of MIol. &: Cell. Pharmacology and Dept. of Medicine, and H. F. Cantiello)) Renal Unit, Harvard Med. Sch., Massachusetts Universitv of Mliadni School of Mledicine. NMiami, Fl, 33101 General Hospital, Charlestown, MA 02129. The ability to isioniitor ciliary beat frequency (CBF) continuously with millisec- onid time resolutioni is critical for the examination of molecular mechanisms that Recent studies from our laboratory determined the presence of regulate ciliarv motility. W'e have developed a videomicroscopy-based photometric electrodiffusional ATP-permeable pathways in cultured shark rectal method for estimating beat frequency of a single cilium, based on recording light gland (SRG) cells from the spiny dogfish, Squalus acanthias (Cantiello intensity changes that occur as a single cilium's basal body moves in and out of et al, Am. J. Physiol. 272:C466-C475, 1997). In this study, the luciferin- a single digitized image pixel ( 180 x 180 nm). Ilere, we analyze the advantages luciferase assay was used to determine the release of ATP in both and disadvantages of several methods to estimate CBF from such intensity signals, both simulated and recorded. These methods either used beat-to-beat analyses cultured SRG cells, and fresh gland under physiological (1/time betwveen zero-crossings or peak-to-peak interval) or fast fourier transforms electrochemical conditions. Primary cultures of SRG cells released ATP (FFT). The beat-to-beat methods were applied to simulated data, yielding a CBF- spontaneously, but did not increase after either cholera toxin dependent time resolution (e.g. at 7 Hz 143 ms). Applied to recorded data, however, stimulation or addition of a cAMP cocktail. In addition, spontaneous these methods suffered from unacceptable noise (shifts of zero line and poor peak ATP release was largely dependent on the presence of extracellular Cl. definition). FFT analysis, uising a sliding window, was applied to data sets-with 32, Replacement of extracellular Cl with gluconate inhibited the release of 64, or 128 samples and the sample window was moved through the intensity records, ATP, and increased intracellular ATP content. isolated tissue advancinrg it by one frarne. This analysis provides a frequency resolution of 0.11 Hz Freshly (using resolution enhancement), and a CBF time resolution at the sampling rate samples also showed a spontaneous release of ATP similar to that of (33 ms with RS- 170 video). For simulated data, windows of 64 or 128 samples/FFT cultured SRG cells. This ATP release, however, was increased by were able to delineate rapid CBF changes, but tunderestimated the true duration of hypotonic shock. The data indicate that SRG cells present molecular a gradtual CBF change. A 32 sample FFT window was able to track actual changes pathways for the spontaneous release of pericellular ATP, which in in C(F accurately whether they occurred instantaneously or gradually. The same turn acts as an autocoid. Spontaneous ATP release in SRG may be observations were made with recorded intensity data. This analysis sets the stage cAMP-independent. for evaluating true kinetic relationships between rapid changes in cytoplasmic Ca2+ and CBF. (Supported by HHMI, NHLBI 20989 & 55341). This work was supported by NIH grant EHS-P30-ES03828.