365-369 POSTERS Sunda

as a cold-sensitive allele in vivo. Our earlier analyses have the relaxed fiber at an ionic strength of 30 mM. Despite differences revealed that: (1) G680V exhibits reduced basal and actin-activated in magnitude, in all cases the frequency dependent stiffness ATPase activities; (2) it cannot move actin filaments and inhibits showed similar characteristics. The stiffness of the relaxed fiber at movement by wild type myosin in mixed assays; (3) it cosediments low ionic strength and the rigor stiffness after unloading at 30 mM with actin even in the presence of ATP, but not in the presence of closely coincided. No indication of attachment and/or detachment ATPyS; (4) its defects in vivo are suppressed by combining with a of cross bridges was observed in the spectrum of stiffness of the second mutation that accelerates Pi release. Here we report that relaxed fiber (in agreement with Bagni et al, G680V SI was unable to quench fluorescence of pyrene-actin in J.Electromyogr.Kinesiol.,1999). The present results indicate that the presence of ATP. In contrast, it quenched the pyrene the remainder of stiffness of the unloaded rigor is carried by fluorescence in the presence of ADP, indicating that an extended unloaded cross bridges. ADP-bound state cannot account for its excessive actin binding in the presence of ATP. Taken together, it was suggested that the Muscle Regulatory Proteins ATPase cycle of acto-G680V is blocked in a strongly bound 1 A.S .ADP.Pi state, or the "A state" of the 3G model (Geeves et al., 368-Pos Board # B224 1984). Negatively stained acto-G680V SI crossbridges in the THE presence of ATP appeared similar to DIFFERENCES IN MECHANISMS BY WHICH typical rigor, except that they TROPONIN C EXTRACTION AND THE TNI-IP INHIBIT were more disordered. Further structural analyses on acto-G680V THIN FILAMENT S1 should shed light on the nature of the yet elusive prestroke ACTIVATION conformation of myosin. Fred Schachate, Philip W Brandt2; 'Duke University Medical Center, 353 Sands Building, Durham, NC 27710, Columbia University Medical School, 630 W. 168th Street, New York, NY 366-Pos Board # B222 10032 WHERE DOES THE ENERGY GO IN MUSCLE DURING A RAMP STRETCH? Troponin C (TnC) plays a central role in both Ca2+ and rigor crossbridge (RXB) activation. Partial TnC extraction reduces the Marco Linaril, Roger C. Woledge2, Nancy A. Curtin3; 'University maximum tension of Florence, Viale G.B. Morgagni, 63, Florence, 50134 and cooperativity of activation by disrupting the Italy, cooperative unit on the thin filament. To determine whether any 2University College London, Brockley Hill, Stanmore, HA7 4LP inhibition of United Kingdom, 3Imperial College School of Medicine, London, TnC function has similar consequences, we SW7 2AZ United Kingdom investigated the effect of the troponin 1-inhibitory peptide (TnI-ip). TnI-ip inhibits RXB activation by shifting the pS/tension During stretch muscles absorb more energy than they release (Hill relationship to higher activator concentrations, without reducing its and Howarth, Proc. R. Soc. London Ser B 151:169, 1959). We cooperativity or maximum tension. This is simply explained by reinvestigated this effect on single frog muscle fibers (1 C, 2.1 Sun stabilization of the relaxed state. Tnl-ip's effect on Ca2+-activation sarcomere length). Fibers were mounted between a motor and a is also consistent with stabilization of the relaxed state, as TnI-ip force transducer in a drop of Ringer solution on a metal-film increases the (Ca2+] required for maximal tension, but does not thermopile, which has an unprecedented spatial resolution. reduce the cooperativity of Ca2+ activation. Interestingly, Tnl-ip Constant velocity stretches were applied at the plateau of the also reduces the maximal tension of Ca2+ activation. These isometric tetanus. During the stretch there was a net energy observations, as well as studies on the effect of TnI-ip following absorption by the fibers. Two types of fibers could be TnC extraction, demonstrate that TnI-ip does not disrupt the distinguished on the basis of how the heat rate and the energy (E=- cooperative unit. Moreover, because TnI-ip inhibits maximal Ca2- work+heat) depended on stretch. in the fibers of 18' type, for tension without reducing cooperativity, it suggests that tension moderate lengthening velocities, an initial decrease in E was generation is not essential for the transmission of cooperativity followed by no further change. In the fibers of 2nd type, E along the thin filament. continued to decrease during the entire period of lengthening. These results can be explained assuming that during lengthening, following the initial energy storage due to cross-bridges dragged 369-Pos Board # B225 into a high energy state (Piazzesi et al., J. Physiol. 445:659, 1992), MYOCARDIAL ISCHEMIA UNCOUPLES THE (1) in the fibers of I" type work is fully converted into heat as if FORCEIATPASE RELATION THROUGH MYOFILAMENT cross-bridges acted as a brake; (2) in the fibers of 2nd type energy PROTEIN ALTERATIONS OTHER THAN TNI continued to be stored presumably as the elasticity in parallel with DEGRADATION. weak sarcomeres is loaded. Supported by the Wellcome Trust. Jason Leo McDonough, Jennifer E Van Eyk; Queen's University, Rm 414 Botterell Hall, Kingston, Ontario K7L 3N6 Canada 367-Pos Board # B223 Brief periods of ischemia(l)/reperfusion(R) produce myocardial FREQUENCY DEPENDENT STIFFNESS OF THE stunning, in which the maximum force of triton-skinned muscle UNSTRAINED RIGOR AND OF THE WEAKLY BOUND fiber bundles is decreased compared to control perfused or CROSS BRIDGE CLOSELY COINCIDES. ischeniic only hearts. While TnI degradation during stunning may Evert de Beer', Ben Treijtel2, Tugenhold Blang62; 'University account for this contractile dysfunction, its effect on the economy Utrecht, PO Box 85060, Utrecht, 3508 AB Netherlands, of force production is not known. Hence, myofibrillar ATPase 2Academic Medical Cemter, University of Amsterdam, PO Box activity and TnI degradation were assessed in isolated rat hearts 22700, Amsterdam, 1100 DA Netherlands following control perfusion (45"), ischemia alone (15"I), and stunning Recently we proved that (15"V45"R). Ischemia alone produced depressed unloading of skeletal muscle in rigor maximal and minimal myofibillar ATPase activities compared to results in a fall of frequency dependent stiffness. After unloading control the fiber by a (83±2 vs 125±2, and 62±1 vs 91±1 nmolPiWmin/mg, shortening length adjustment of 0.6 %, a remaining respectively), without substantial TnI degradation (7+2%), despite stiffness was observed, while the cross bridges stayed attached. To maintained explore the nature of force production. Stunned myocardium demonstrated a this stiffness we measured the in-phase and similar depression in maximal and minimal myofibrillar ATPase out-of-phase stiffness under three experimental conditions by activities (87±2 and 68+1 releasing and stretching step changes in length of 0.025 %. From nmolPi/min/mg, respectively), as well as the induced the expected degradation of Tnl (21±t6%). The presence of other tension transients the normalized (for length and modified myofilament proteins that may diameter) stiffness was estimated from 10 Hz to 50 kHz. We account for this behavior compared is being assessed using a proteomic approach. These results rigor stiffness before and after unloading, at an ionic suggest that myocardial ischemia strength of 160 mM and at an ionic strength of 30 mM, and that of increases the economy of the

82a SundavLO LI&I 1%&464 y POSThERSX %., U.0 A. '169_174I IT

force/ATPase relation, while subsequent reperfusion re-couples the TROPONIN-I REDUCES THE TROPONIN-T MEDIATED relationship at a lower economy. ACTIVATION OF MYOSIN S1-THIN FILAMENT ATPASE ACTIVITY. 370-Pos Board # B226 Yin Luo, Guang Yang, Knut Langsetmo, John Gergely, Terence BIOCHEMICAL STUDIES OF HUMAN CARDIAC TNI 1- Tao; Boston Biomedical Research Institute, 64 Grove St., 192: IMPLICATIONS FOR THE MECHANISM OF Watertown, MA 02472 MYOCARDIAL STUNNING The actin-activated skeletal myosin S1 ATPase activity is Ca(2+)- D. Brian Foster', Ann M. Murphy2, Jennifer E. Van Eyk'; regulated via the thin filament components tropomyosin (Tm) and 'Queen's University, Kingston, Ontario K7L 3N6 Canada, 2Johns troponin (Tn), which is composed of troponin-C (TnC), troponin-I Hopkins University, Baltimore, Maryland (TnI) and troponin-T (TnT). TnI alone can fully inhibit SloTmoF- Contractility of the heart is depressed following bouts of ischemia actin ATPase activity (inhibition); Ca2+-bound TnC reverses this (X) and subsequent reperfusion (RP) and the primary lesion occurs inhibition (deinhibition); the presence of TnT further elevates the within the myofilament proteins. Specifically, in the isolated rat activity (activation). We found that stoichiometric labeling of one heart, the first protein changes upon I/RP are phosphorylation and of the three endogenous Cys of Tnl, Cys64, using a mono-Cys TnI proteolysis of Tnl. Triton-skinned muscle fibers from the I/RP mutant (ThI64), with iodoacetamide or 4-benzophenone- hearts display decreased maximum force production and altered iodoacetamide reduced the TnT-mediated activation by 73 or 87%, Ca2+-sensitivity. The primary proteolytic product, TnI residues 1- respectively, without affecting the inhibition and the deinhibition. 193, lacks 17 amino acids at its C-terminus. To determine how There was no measurable effect on the ability of the labeled TnI64 truncated TnI contributes to contractile dysfunction, we have to form the ternary Tn complex. Labeling at other Cys of TnI did undertaken in vitro analysis of recombinant human cardiacTnll- not have any effect. Since Cys64 is in the TnT-binding region of 192. TnII-192 and wtcTnl bind to actin or actin-tropomyosin with Tnl, our results suggest that the attached probes disrupt the equivalent affinity. Both proteins inhibit actoTMSl ATPase conformation of Tn at the TnlI-TnT interface that is critical for activity to a similar extent and potency. The TnIl-192-mediated TnTs function in mediating the activation of muscle contraction. inhibition of actoSlTM ATPase is neutralized by cTnC(+Ca2e) (Supported by NIH AR21673) with slightly increased potency. However, TnIl-192 elutes from a cTnC-Sepharose affinity column (+ Ca2+) at the same buffer 373-Pos Board # B229 stringency as wtcTnl (6M urea,lmM EDTA). TnT-Sepharose OVEREXPRESSION OF FAST SKELETAL MUSCLE chromatography yielded similar results using urea gradient elution TROPONIN T ALTERS CA2-REGULATED in 6M urea. As well, synthetic TnI peptides corresponding to the CONTRACTILITY OF TRANSGENIC MOUSE extreme C-terminus of Tnl (residues 193-209 or 188-209) do not CARDIOMYOCYTES bind TnT or TnC affinity columns. This suggests that although the Z.-B. Yu, J.-P. Jin; Case Westrem Reserve University, 10900 C-terminus of Tnl does not contain direct binding sites for TnT nor Euclid Ave., Cleveland, Ohio 44106-4970 TnC, it may conduct the Ca2e activation signal to ultimately influence Tn-TM and allow maximum force Expression of cardiac troponin T (TnT) splicing variants and development. mutations has been found in cardiomyopathy and heart failure. To investigate the and effects of TnT 371-Pos # physiological pathological Board B227 structure alterations on cardiac muscle contraction, we constructed THE EFFECT OF HCM MUTATIONS IN TROPONIN T a transgenic mouse model over-expressing a fast skeletal muscle AND TROPONIN I ON THE CALCIUM BINDING OF TnT in the heart (Huang, Q.-Q., Brozovich, F.V., & Jin, J.-P., J. HUMAN CARDIAC TROPONIN Physiol. 520:231-242, 1999). The incorporation of heterogeneous Charles S Redwood, Kathryn Elliott, Giovanna M Esposito, Hugh TnT isoforms in the transgenic cardiac myofibrils resulted in Watkins; University of Oxford, WTCHG, Roosevelt Drive, changes in shortening and relaxing velocity of isolated Oxford, OX3 7BN United Kingdom cardiomyocytes. Ca2+ transient was analyzed simultaneously via Familial hypertrophic cardiomyopathy (HCM) is a disease of the fura-2AM fluorescence for changes in the Ca2+ dynamics in the sarcomere and is caused by mutations in genes encoding transgenic cardiomyocytes over-expressing fast skeletal muscle components of both the thick and thin filaments. In order to TnT. The isoproteoenol-induced enhancement of the Ca2+ analyse the functional effects of HCM mutations in troponin T and transient and contractility was reduced in the transgenic troponin I we have overexpressed wild type and mutant human cardiomyocytes. The results suggest that the presence of cardiac troponin subunits in E.coli and have used the purified heterogeneous troponin complex in the cardiac muscle resulting recombinant proteins to reconstitute human cardiac troponin in from the incorporation of difference TnT isoforms causes vitro. In actin-tropomyosin-activated myosin ATPase assays, alterations in the Ca2+ signaling pathway. This is due to the complex reconstituted using certain troponin T or troponin I HCM changes in Ca2-troponin interactions and is not overridden by the mutants gave increased Ca2+ sensitivity of ATPase regulation enhancement of SR Ca2e pump activity. The effect of TnT compared with wild type troponin; complex containing a HCM structural variation may play a pathogenic role in cardiomyopathy troponin T mutant lacking the 28 C-terminal amino acids had and heart failure when abnormal cardiac TnT isoforms or mutants ApCaso of +0.43 compared with wild type troponin, whereas are expressed. incorporation of missense troponin I mutants, Argl45Gly or Argl62Trp, gave ApCa5o of +0.56 and +0.13 respectively. In order 374-Pos Board # B230 to test whether the increased Ca2+ sensitivity is caused by a direct ANALYSIS OF CARDIAC TROPONIN I change in the Ca2+affinity of the regulatory site in cardiac troponin PHOSPHORYLATION IN FAILING AND NONFAILING we have reconstituted troponin using dansyl-troponin C and HUMAN HEARTS USING ION TRAP MASS measured the Ca2+ affinity of troponin by fluorescence titration SPECTROMETRY (Ka=5x1061M-'). The effect of HCM mutations in troponin I and Cristan Ion Ruse', Michael Kinter', Belinda Willard', Thomas troponin T on the Ca2+affinity will be reported. (Supported by the Hans', J.-P. Jin2,Meredith Bond'; 'Cleveland Clinic, 2CWRU British Heart Foundation and the Medical Research Council) Phosphorylation of cTnlI by PKA and PKC regulates Ca dependence of force development. The N-terminus of cTnlI 372-Pos Board # B228 contains two adjacent serine residues that form a unique dual site CHEMICAL MODIFICATION AT RESIDUE 64 IN THE of PKA-dependent phosphorylation. We previously showed that TROPONIN-T BINDING REGION OF RABBIT SKELETAL phosphorylation of the PKA-specific sites on human cTn (hcTnl)

83a 374-379-.1 I T--.# I -0 L %ol Li A J-j.Ll%Lx ~.j * POSTIRS cZimcinvLILII%J447

is decreased by 25% in dilated cardiomyopathy. However, the myocytes (24.3±1.0 ms (48) on day 2 and 25.6±1.4 ms (45) on day extent of phosphorylation of the PKC-specific sites remains to be 4), t,,R increased in ssTnI expressing myocytes from 32.2±1.0 ms determined. We are investigating changes in PKAIPKC (48) to 45.9±6.9 ms (23) between days 2 and 4 following gene phosphorylation of human cThI at the amino acid level in failing transfer. No change in tl,2R was detected on day 4 in myocytes and non-failing heart, using ion trap mass spectrometry (lTMS). after cTnI gene transfer. These results suggest that the ratio of TnI HcTnI together with TnC/TnT was effectively isolated in one-step isoform expression has a proportional influence on relaxation rates by immunoaffinity chromatography using a TnI mAb from failing in cardiac myocytes. and non-failing human heart. In-gel digestion of eluted hcTnl with trypsin and Asp-N yielded 91% sequence coverage, including the 377-Pos Board # B233 PKA and PKC phosphorylatable residues. Upon back- THE EFFECT OF TROPONIN T ISOFORMS ON phosphorylation of pure hcTnl (from Sigma) by PKA, a number of REGULATED THIN FILAMENT MECHANICAL short, polar peptides including PKA/PKC phosphorylation sites, FUNCTION IN VITRO. e.g.: RSpSpNYR, RPTpLR, ISpADAMMQALLGAR, and AKESpLDLR were detected P. VanBuren, S.L. Alix, K.J. Begin, M.M. LeWinter, N.R. Alpert; by high sensitivity 1TMS. Univ of Experiments are underway to identify the phosphorylation status Vermont, Burlington VT 05405 and to characterize stoichiometry of these sites in explanted human Alteration of troponin T ( TnT ) isoform expression has been hearts. reported in human and animal models of myocardial failure. An increase in the expression of the TnT fetal isoform is correlated 375-Pos Board # B231 with a reduction in myofibrillar ATPase. We isolated the two adult beef cardiac TnT ABNORMAL SPLICING OF CARDIAC TROPONIN T IN isoforms which have similar differences when DILATED CARDIOMYOPATHY AND HEART FAILURE compared to the two human TnT isoforms expressed in myocardial failure. Thin filaments were Brandon reconstituted containing pure James Biesiadeckdi, J.-P. Jin2; 'Case Westem Reserve of the isoforms. The in vitro was School of populations motility assay used to University, Medicine, 10900 Euclid, Cleveland, Ohio the effect of the two TnT isoforms on 44106, 2Case Western Reserve directly compare force and University unloaded shortening as a function of free calcium. We found no Troponin T (TnT) is an essential element in the thin filament significant differences between the two isoforms for either velocity regulatory system of striated muscle. Developmentally regulated or force at both maximal and submaximal calcium activation. To alternative RNA splicing generates TnT isoforms differing in their assess whether the extent of myosin strong binding could N-terminal region. Normally mature cardiac muscle expresses only differentially affect thin filament activation, myosin density on the the adult cardiac TnT isoform. However, we have found that motility surface was serially reduced. No difference in motile turkey and dog hearts with inherited dilated cardiomyopathy and function was detected for the thin filaments containing either of the failure express multiple TnT isoforms. cDNA cloning and two TnT isoforms. In light of the small magnitude of the TnT sequencing revealed that unusual splicing resulted in abnormal isoforms shifts detected in myocardial failure and the lack of exon exclusion from and/or inclusion in the N-terminal region of significant mechanical effect detected in the motility assay, TnT the cardiac TnT. Wild-type and a low molecular weight turkey isoform variation probably does not affect contractile performance cardiac TnT variant were expressed in E.coli for structural and in heart failure. functional characterization. Monoclonal antibody epitope analysis showed that the abnormal splicing in the N-terminal region 378-Pos produced Board # B234 conformational changes in the C-terminal domain of REGULATION OF CARDIAC TWITCH KINETICS BY cardiac TnT. Protein binding assays demonstrated that the TROPONIN I PHOSPHORYLATION abnormally spliced cardiac TnT had significantly higher binding affinities to tropomyosin and troponin I. The results indicate that YeQing Pi, Kara R. Kemnitz, Evangelia G. Kranias, Jeffery W. the abnormal splicing of the N-terminal region altered the Walker; University of Wisconsin, Madison, WI 53706 conformation and function of the central and C-terminal regions of To investigate the role of cardiac troponin I (cTnI) cardiac TnT. The expression of multiple TnT variants exhibiting phosphorylation in contractile regulation, we generated transgenic functional differences desynchronizes the myocardial contractile mice expressing a mutated cTnI protein (cTnlI-Alas) lacking units and may contribute to the pathogenesis of cardiomyopathy phosphorylation sites for PKC (Ser43,45 and Thrl44 mutated to and heart failure. Ala) and PKA (Ser23,24 mutated to Ala) on a cTnI null background. In wild-type (WT) cardiac myocytes paced at 0.4 Hz, 376-Pos Board # B232 endothelin-1 (ET-1) increased the time to 90% relaxation (tso) by RELAXATION KINETICS IN SINGLE ADULT CARDIAC 39% and increased the relaxation time constant (r) by 62%. hI MYOCYTES ARE DECREASED BY SLOW SKELETAL cTnI-Ala5 myocytes, ET-1 only increased t9o by 8% and r by 15% TROPONIN I EXPRESSION IN A DOSE DEPENDENT indicating that cTnI phosphorylation plays a key role in twitch MANNER. broadening seen in response to ET-1. cTnI-Ala5 myocytes lacking Philip A. Wahr, Margaret V. Westfall, Joseph M. Metzger; phospholamban (cTnI-Alas x PLB-KO) behaved similarly University of Michigan, 7730 Medical Science II, Ann Arbor, MI suggesting that PLB plays no role in the actions of ET-1 on twitch 48109-0622 kinetics. The j-agonist, Iso, decreased X by 40% in WT, 11% in To better cTnI-Ala5, and 4% in cTnI-Ala5 x PLB-KO myocytes, consistent understand the impact of TnI on the relaxation rate, with roles for adenoviral gene transfer was used to express either slow skeletal major cThI and PLB phosphorylation in the [- TnI adrenergic response. The results indicate that ET-1 acting through (ssTnl) or cardiac TnI (cTnl) in isolated, intact adult cardiac PKC myocytes. Western blot analysis was used to determine the extent phosphorylation of cTnI slows cardiac myocyte relaxation of replacement of native cTnI by ssTnl on days 2-4 of culture. thereby increasing twitch duration and the efficiency of Following gene transfer, ssTnl expression ranged from 30±4.7% contraction. (Supported by NIH). (6) on day 2 to 92.6+3.0% (4) on day 4. No ssTnl was detected in control myocytes. Laser diffraction was used to estimate the 379-Pos Board # B235 sarcomere shortening rate by measuring the time from stimulation MODULATORS OF TROPONIN C (TNC) AFFINITY FOR to peak shortening (tp,gj) and relaxation rate by the time from peak THIN FILAMENTS (TF) IN SKINNED FIBERS OF shortening to 1/2 re-lengthening (ti,r). No difference in tpeak was SKELETAL AND CARDIAC MUSCLE. detected at any time point studied in control or transduced Valeria Pereira de Sousa, Jose Renato Pinto, Martha M. myocytes. In contrast, although t,,R was unchanged in control Sorenson; Universidade Federal do Rio de Janeiro, Cidade

84a Sundav POSTERS 379-383

Universitaria, CCS, bl. H, UFRJ, Rio de Janeiro, RJ 21941-590 terminal deletion mutant of cTnT (cTnTDEL), respectively. Brazil Normalized pCa-tension relationships of mutant fibers TnC binding to TFs in vertebrate muscle has long been known to demonstrated a significant increase in Ca2+-sensitivity at short and depend on Ca2+/Mg2e in C-terminal sites. Other factors affecting long sarcomere lengths (SL). At short SL, the pCa5O values, TnC binding to the regulatory complex have received less representing the mid-points of pCa-tension relations, were attention. Here we report how changes in H+ (pH 6-8), 17/2 (30- 5.69+0.01, 5.96±0.01 and 5.81±0.01 for WT, R92Q and 250mM K propionate) and redox state (0-2mM DTT) affect TnC- cTnTDEL fibers, respectively. At long SL, the pCa5O values were TF association in 0-2mM Mg, using skinned fibers reconstituted 5.81±0.01, 6.08±fi0.01 and 5.95± 0.01 for WT, R92Q and with chicken rTnC and measuring decay of standardized Po cTnTDEL fibers, respectively. The fiber bundles reconstituted tensions (pCa 4.4) following exposure to test solutions. Tension with the recombinant mutant cTnTDEL protein developed only loss is reversed by adding rTnC. In 0 Mg, only pH alters TnC 37% of Ca-activated maximal force developed by the wild type dissociation. With Mg, TnC dissociates faster at pH 8 or in high cTnT reconstituted fiber bundles, with no apparent changes in Ca ['/2; its affinity for TF is less than in pH 6, low r1/2 or D'TT, sensitivity. At short SL, Ca-activated maximal tension in both respectively. When the 3 "best" conditions are combined, the R92Q and cTnTDEL fibers declined disproportionately to Ca- apparent affinity of TnC for TF is -3X higher than for the activated maximal ATPase activity, which led to a 30% (P < combination that favors TnC loss (Ko.54.8 vs 13.2y/ml).Mg 0.0001) increase in tension cost. Thus, the mutation-linked effects dependence of F154W fluorescence in solution suggests no large on cardiac myofilament function depend not only on the nature of changes in Mg affinity. In skinned trabeculae, rTnC loss is slower; the mutation, but also on the concentration of the mutant in the at pH 8 and (unlike skeletal TFs) in DTT the rate increases. Thus sarcomere. pH, 17/2 and DTT modulate Mg-induced conformational changes in the C-domain, and we identify 2 different combinations that favor 382-Pos Board # B238 TnC removal or TnC reconstitution/storage of skeletal and cardiac RESCUE OF A TRANSGENIC PHENOTYPE OF skinned fibers. Support: FAPERJ, CNPq, PADCT (Brazil). FAMILIAL HYPERTROPHIC CARDIOMYOPATHY (FHC) Fatima deFreitas, Jiaju Zhao, Todd E Miller, Aldrin V Gomes, 380-Pos Board # B236 Danuta Szczesna, James D Potter; University of Miami, 1600 MECHANO-CHEMISTRY OF CROSS-LINKED TROPONIN N.W. 10th Ave., Miami, FL 33136 C FILMS. We have made a transgenic (TG) mouse model of human FHC by Anna E Bukatinal, Victor N Morozov2, Nikolai B Gusev3, Gary C overexpressing the 179N mutation of human cardiac troponin T Sieck'; lMayo Foundation, 200 SW First St, Rochester, Minnesota (HCTnT) in mouse hearts, using the mouse alpha myosin heavy 55905, 2Russian Academy of Sciences I1TEB, Russian Federation, chain (a-MHC) promoter (Miller et al., J Biol Chem, 2000, in 3Moscow State University, Dept. of Biochemistry, School of press). Cardiac fibers from mice expressing the mutant protein Biology, Russian Federation exhibited increased sensitivity to calcium, relative to non- Troponin C (TnC) films (2-5 gm thickness) were prepared by transgenic (NTG) littermates and mice overexpressing the wild- drying or electrospray deposition of TnC solutions, cross-linked type HCTnT. To test the hypothesis that this altered calcium with glutaraldehyde and tested mechanically according to sensitivity was due to the I79N mutation, we used propyl-thio- (Morozov & Morozova, 1992, 1999). A decrease in pCa from 9 to uracil (PTU) to inhibit thyroid hormone synthesis, and to down- 5.8 caused a reversible increase in isometric tension of TnC films regulate the a-MHC promoter. Three month old animals were fed with no significant change in stiffness. The mechanical effect of this diet for 1 to 2 months, and hearts were collected for fiber low [Ca2+] in the absence of Mg2+ could be attributed to binding assays, RNA and protein analysis. PTU induced hypothyroidism, with an apparent binding constant (Kca) of - 107 M l. However, the evidenced by elevated levels of beta myosin heavy chain RNA. total Ca2+effect could not be described by a single Kca. In contrast, PTU also essentially abolished the transgenic RNA; this effect was with 2 mM Mg2+ these data were well fitted by one Kca - 106 M-'. also observed at the protein level. Fiber studies revealed that the A further decrease in pCa to 4 caused some increase in stiffness altered calcium sensitivity seen in the 179N TG mice was and an additional increase in tension (- 2.5 times in both cardiac completely restored to normal with PTU treatment, while no and skeletal TnC films). The estimated shrinkage of unloaded TnC changes were observed in NTG littermates and wild-type TG mice. filmupon change in pCa from 9 to 4 was 2-10 % and could be seen These data are consistent with the notion that the increased calcium under disecting microscope. 2 mM Mg2' reduced this shrinkage by sensitivity in the 179N TG mice is due to the 179N mutation. only - 15% indicating to Ca2+ specific effects. Ca2+ caused Supported by NIH HL 42325 mechanical changes in troponin films as well (Kca - 7x106 M'lin the presence of 2 mM Mg2e). Conclusion: Several processes of 383-Pos Board # B239 different origin are accountable for the mechanical response. These VARIATIONS IN SECONDARY STRUCTURE CAUSED BY results showing a large scale mechanical response suggest that the A SINGLE AMINO ACID SUBSTITUTION IN HUMAN mechano-chemiical method may be used as a sensitive probe of CARDIAC TROPONIN T MUTANTS FOUND IN TnC state. FAMILIAL HYPERTROPHIC CARDIOMYOPATHY. Keita Harada, Aldrin V Gomes, James D Potter; University of 381-Pos Board # B237 Miami, 1600 N.W. 10th Ave., Miami, FL 33136 CARDIAC TROPONIN T MUTATIONS: THE Troponin T (TnT) is the tropomyosin (TM)-binding subunit of CORRELATION BETWEEN THE TYPE OF MUTATION which mediates the AND THE troponin, Ca2+regulation of vertebrate striated NATURE OF MYOFILAMENT DYSFUNCTION. muscle contraction together with TM. The a-helical content of David E. Montgomery', Jil Tardiff2, Murali Chandra'; TnT (especially the N-terminal region, TnTl) is very high, and this 'University of Illinois at Chicago, 835 S. Wolcott, Chicago, IL a-helical region might be important for the interaction with TM. 60612, 2Albert Einsein College of Medicine, Bronx, NY 10461 We have previously demonstrated that the human cardiac TnT Several mutations in cardiac troponin T (cTnT) are known to cause (HCTnT) mutants, I79N and Fl 10, found in familial hypertrophic familial hypertrophic cardiomyopathy (FHC) in humans. We cardiomyopathy (FHC), increase the Ca2" sensitivity of isometric measured steady-state isometric force and ATPase activity in force generation in skinned cardiac muscle. Recently, we have detergent skinned fiber bundles from three transgenic (TG) mouse observed that 179N has an increased a-helical content compared hearts in which 50%, 92% and 6% of the total cTnT was replaced with that of wild-type HCTnT. On the other hand, FHC HCTnT by the wild type cTnT (WTD, R92Q mutant cTnT (R92Q) or a C- Fl 10I did not demonstrate a significant change in secondary

85a 383-388 .Ctllntlov.lOiL31 I

structure. We have further investigated the secondary structural 386-Pos Board # B242 variations and the functional consequences of seven FHC HCTnT FUNCTIONAL EFFECTS OF MODIFICATIONS OF mutations, R92W, R92L, R94L, A104V, R130C, E244D and CARDIAC TNT AT PKC PHOSPHORYLATION SITES E163R. Preliminary results show that R92W and R92L do not have Marius P. Sumandea, R. John Solaro; University of Illinois at an altered secondary structure. Supported by NIH and AR45391 HLA2325 Chicago, 835 S. Wolcott Ave, Chicago, Illinois 60612 There is compelling evidence that modifications by PKC 384-Pos Board B240 phosphorylation of cardiac TnT and cardiac T7n, may be important # in the transition from hypertrophy to failure. Recent studies in our BINDING OF TROPONIN T AND I PEPTIDES TO TROPONIN C laboratory have indicated that pan-activation of PKC results in phosphorylation of cTnT and cTnI and induces a 30% reduction of Tharin M. A. Blumcnschein, Brian P. Tripet, Robert S. Hodges, maximum force of mouse cardiac myofilaments. When cTnT was Brian D. Sykes; University of Alberta, 474 Medical Sciences partially replaced with a fast skeletal isoform (which does not have Building, Edmonton, Alberta T6G 2H7 Canada the phosphorylation sites Thr 194 and Thr 291), this effect of PKC The troponin complex has a fundamental role in muscle activation was no longer evident. In this case there was no contraction regulation, and is constituted of 3 proteins, troponins T, phosphorylation of fsTnT and a diminished incorporation of C and I. Troponin C (TnC) structure has been determined by both phosphate into cTnI. These results indicate that TnT X-ray crystallography (Herzberg and James, J. M. B. 203, 761; phosphorylation may be pivotal for the PKC-induced depression of Satyshur et al., J. B. C. 263, 1628; Houdusse et al. Structure 5, tension in the myofilament. In the present study, we generated 1695) and NMR (Slupsky and Sykes, Biochemistry 34, 15953). cTnT mutants in which Asp and Ala residues were placed at the There is also some structural information on the interaction PKC phosphorylation sites. The adult mouse cTnT was selectively between troponins C and I (Mercier et al., Biochemistry 39, 2902; mutated, culminating with the triple mutants T194/203/291A and Vassylyev et al., P.N.A.S. USA 95, 4847; Tung et al., Protein Sci. T194/203/291E. Charge modified mutants were compared with 9, 1312). On the other hand, very little is known about troponin T phosphorylations induced by PKC in vitro, using myofilament structure, isolated or in the complex. We used non-denaturing gels reconstitution experiments and a novel CM-PS bead assay. The and NMR to study the interactions of 4 TnT peptides with skeletal relative significance of cTnT PKC sites on Ca2I-dependent TnC, and its competition with TnI peptides. From rabbit fast activation of myofilament force, crossbridge kinetics and skeletal muscle peptides TnTlr7.176. TnT,66-", TnT,87221 and economy, and in the modulation of interaction among the TnT2i5s249, only TnTi"_," binds to TnC. TnI6t13s and TnI56,,5 heterotrimeric troponin complex, is presented. compete with TnT,66.199, while TnI,4o can bind at the same time. Using HSQC-NMR to follow a titration, we determined that the 387-Pos Board # B243 dissociation constant for TnTi661,99 is 34.9±6.4 PM. A second EFFECT OF DIFFERENT ISOFORMS OF TROPONIN T peptide binds to TnC at higher concentrations, with a dissociation ON ACTIN-TM ACTIVATED MYOSIN ATPASE ACTIVITY constant higher than 600 pM. We determined the approximate in Aldrin V Gomes, Georgianna Guzman, Michelle Jones, James D region TnC to which the TnT peptide is bound, by chemical Potter; University of Miami, 1600 N.W. 10th Ave., Miami, FL shift mapping techniques. 33136 Troponin T (TnT) is the largest of three proteins that form the 385-Pos Board # B241 troponin complex, and plays a critical role in the activation of the STRUCTURE OF THE C-DOMAIN OF CARDIAC actomyosin ATPase activity. TnT isoforms can be from TROPONIN C IN generated COMPLEX WITH CA2+ SENSITIZER a minimum of 10 different mRNA's and potentially 32, in a EMD57033. developmentally regulated and tissue specific manner. To Xu Wang', Monica X. Li', Leo Spyracopoulos', Norbert Beier2, determine if the different N-terminal TnT isoforms are functionally Murali Chandra3, R. John Solaro3, Brian D. Sykes'; 'University of distinct with respect to the activation of actin-Tm activated myosin Alberta, CIHR Group in Protein Structure and Function, ATPase, different TnT isoforms, TnTlf (has all exons), ThT2f Edmonton, AB T6G 2H7 Canada, 2E. Merck, Frankfurter Strasse (missing exon 4) and TnT3f (missing exons x,6 and 7), were 250, Darmstadt, 64271 Germany, 3University of Illinois-Chicago, expressed, purified and reconstituted in actomyosin ATPase College of Medicine, Chicago, Illinois 60612-7342 assays. The TnTlf isoformhad greater maximal activity than both Binding of Ca2e to cardiac troponin C (cTnC) triggers contraction the TnT2f and TnT3f isoforms. C-termninal TnT isoforms, TnT3fa in cardiac muscle. In diseased heart, myocardium is desensitized (containing exon 16 and no exon 17) and TnT3$D(containing exon to Ca2+,leading to weak cardiac contractility. Compounds that can 17 and no exon 16) and a C-terminal mutant, 3fAl (in which both sensitize cardiac muscle to Ca2e would have potential therapeutic exons 16 and 17 are deleted) were also purified and reconstituted value in treating heart failure. EMD 57033 is an identified 'Ca2e in actomyosin ATPase assays. The maximal level of ATPase sensitizer' and cTnC is a potential target of the drug. We have activation was not significantly different for the various C-terminal recently shown that the EMD 57033 binding site resides in the C- TnT isoforms and mutant investigated. These results suggest that domain of cardiac TnC (cCTnC) [Li et al.(2000) Biochemistry 39, the N-terminal of TnT may be important for maximal ATPase 8782-8790]. In this work, we present the NMR solution structure activity. Supported by NIH AR45391 and HL42325. of a complex between the Ca2+-saturated C-domain of cTuC and EMD 57033. Hydrophobic interactions between the drug and the 388-Pos Board # B244 protein keep EMD 57033 bound in the hydrophobic core of the CHARGE CHANGE AT PROTEIN KINASE C SITES OF protein. The drug molecule is orientated such that the chiral group of EMD PHOSPHORYLATION IN CARDIAC TROPONIN I 57033 fits in the hydrophobic pocket. This stereospecific AFFECTS THIN FILAMENT REGULATION. interaction may explain why the (-) enantiomer of EMD 57033 is inactive. Titration of cCTnCo2Ca2+*EMD 57033 complex with Eileen M. Burkart', Richard Song', Karen H. Hales', Natosha two regions of cardiac TnI and reveals Fmnley2, Paul R. Rosevear2, Anne F. Martin', R. John Solaro'; (cTnI3471 cTnli2s-143) that of Illinois the drug does not share common binding epitope with cTnl,281,47 'University at Chicago, Chicago, IL 60612, 2University but is displaced by cTn34,71 completely. Implication of these of Cincinnati, Cincinnati, OH 45267 results will be discussed. There is strong evidence demonstrating that phosphorylation of cardiac troponin I (cTnI) is increased during heart failure. We examined how two PKC phosphorylation sites (ser-43,45) in cTnI are involved in the regulation of myofilament activity. Our

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approach involved determining the relative effects of charge to 50-100% of the control value while kca and k,u were unchanged. change, induced by substitution at both ser-43 and ser-45 with Reconstitution with cTnC restored force to a lesser extent (44 ± 6 glutamic acid or aspartic acid (cTnI S43E/S45E or cTnI % n = 14) and reduced by two-fold both kca and ku, (kr = 4.04 ± S43D/S45D). We used detergent-skinned mouse cardiac fiber 0.23 s.'). These results suggest that cTnC modifies the dynamics of bundles to measure changes in myofilament Ca2+ sensitivity and fast skeletal thin filament activation. Experiments are currently in maximum Ca2+-dependent force in fibers reconstituted with cTnl progress to investigate the effects of cTnC reconstitution on the S43E/S45E or cTnI S43D/S45D. Compared to controls with a force transients induced by sudden changes in [Pi]. [Supported by pCaso of 5.55 ± 0.004, fibers reconstituted with cTnI S43E/S45E or NIH grants AR 30988 (E.H.) and HL 38834 (L.T.) and AHA cTnI S43D/S45D were desensitized to Ca2' (pCaso = 5.34 ± 0.003 9650128N (L.T.)] or pCa5o = 5.44 ± 0.008 respectively). Maximum Ca2e-dependent force in fibers reconstituted with cTnI S43E/S45E or cTnI 391-Pos Board # B247 S43D/S45D was approximately 25% lower compared to controls. SPECTROSCOPIC MAPPING OF TROPONIN T REVEALS This decrease in maximum tension is similar to that induced by SITE-SPECIFIC CONFORMATIONAL PERTURBATIONS phosphorylation of cTnI, which indicates that this effect can be BY THIN FILAMENT PROTEINS mimicked by substitution of charged amino acids at PKC Douglas D. Root', Jian-Ping Jin2; 'University of North Texas, PO phosphorylation sites. Box 305220, Denton, Texas 76203-5220, 2Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 389-Pos Board # B245 SKELETAL Troponin T plays a critical intermediary role between the troponin AND CARDIAC TROPONIN ISOFORMS complex and tropomyosin's regulation of actomyosin interactions. SHOW DIFFERENT REGULATORY PROPETIES Functional correlations between troponin Ts structure and its Robin Maytum', Barbara Westerdorf2, Kornelia Jaquet2, Michael interactions with other troponin subunits and tropomyosin are Andrew Geeves'; 'University of Kent at Canterbury, Canterbury, important for understanding the effects of troponin T mutations Kent CT2 7NY United Kingdom, 2Rhur-University of Bochum, that give rise to cardiomyopathy and heart failure. By investigating Universitiaestr. 150, Bochum, 44081 Germany the effects on intrinsic and extrinsic spectroscopic probes of Skeletal and cardiac muscle show considerable differences troponin T isoforms and mutants, we have mapped the origin of between their troponin isoforms, the most significant of which are specific fluorescent signals and their responses to protein that cardiac TnC has only one calcium binding site and the TnI interactions. A troponin T construct containing a single TRP236, contains phosphorylation sites which have been shown to modulate indicates that it is the major source of intrinsic fluorescence with a the sensitivity to calcium. Comparison of the regulatory properties peak at 348 nm and is strongly quenched by tropomyosin binding. of skeletal and cardiac troponins using kinetic measurements to This single TRP236 troponin T also exhibits a peak at 330 nm that determine the occupancy of the Blocked (B-) state (from the 3- appears to result from a tyrosinate rather than a tryptophan. The state model of McKillop and Geeves) has shown little difference tyrosinate was mapped to TYR187 based on sequence alignments between them with similar KB values for both. Using a sensitive SI of isoforms, sensitivity to pH, and FRET quenching analysis. This titration technique we show that S1 binding to regulated filaments tyrosinate fluorescence is quenched by tropomyosin binding. containing either skeletal or cardiac troponin show significant Fluorescein labeling of an isofonn containing a single CYS263 differences. In the presence of calcium both appear very similar quenches intrinsic tryptophan fluorescence, and the fluorescein's with virtually identical SI binding curves. However in the absence anisotropy is increased substantially upon reconstitution into the of calcium the binding curves differ considerably. Fitting of these troponin complex. curves to the 3-state model (Maytum et al 1999 Biochem. 38, 1102) shows that the difference between them can be accounted for 392-Pos Board # B248 purely by a different value for KT the closed-open (C to M state) ORIENTATION CHANGE OF TROPONIN C (TNC) equilibrium for the two types. While the skeletal system shows a 4 DOMAINS ON ACTIVATION OF SKELETAL MUSCLE. fold to in change (0.15 0.04) KT, the cardiac system shows no Roisean E. Yin-Biao Sun2, between the two states 0.2 for Ferguson', Andrew S. Brack2, John change (approx both). E.T. Comfie', David R. Trentham', Malcolm Irving2; 'National Institute for Medical Research, London, NW7 IAA United 390-Pos Board # B246 Kingdom, 2School of Biomedical Sciences, King's College FORCE GENERATION IN SINGLE RABBIT PSOAS London, London, SEI 1UL United Kingdom MYOFIBRILS AFTER REPLACEMENT OF NATIVE TNC WITH CARDIAC TNC. We are investigating the structural basis of regulation by TnC using polarized fluorescence from bifunctional rhodamine (BR) Nicoletta Piroddil, Earl Homsher2, Larry S Tobacman3, Chiara probes attached to pairs of cysteine residues (Corzie et al. Nature Tesi', Corrado Poggesl; 'Universita di Firenze, Firenze, 1-50134 400, 425, 1999). Expressed TnC mutants in which either Glul7 Italy, 2UCLA School of Medicine, Los Angeles, CA 90095, and Ala24 on the A helix or Glu56 and Glu63 on the C helix had 3University of Iowa, Iowa City, IA 52242 been replaced by cysteines were covalently modified with BR, and Single myofibrils and thin bundles of few myofibrils isolated from the 1:1 BR-TnC conjugates isolated at >95% purity. Native TnC rabbit psoas muscle were maximally activated (pCa 4.50, 150 C) was extracted from single skinned fibers of rabbit psoas muscle and fully relaxed (pCa 8.00) by rapidly (10 ms) translating the and replaced with BR-TnC. The orientation distribution of the BR interface between two flowing streams of solution across the dipole with respect to the fiber axis was modeled as a Gaussian preparation (Tesi et al., Biophys. J., 2000,78, 3081-3092). The rate distribution with peak angle 0. Ca2' activation at sarcomere length of force development following Ca2+activation (kca=7.63 ± 0.37 s 2.4 gm, 10°C produced only small changes in 0 for the A helix , n = 24) was the same as the rate of force redevelopment probe, but 0 for the C helix probe (0c) increased by about 350. A following a release restretch (k,r 7.74 ± 0.35 s '). Brief perfusion change in the angle between the A and C helices on Ca2e binding is with a low ionic strength TnC extracting solution decreased expected from NMR data (Gagneet al. Nature Struct. Biol. 2, 784, isometric force without affecting kc, or kt,r Highly extracted 1995). The Ca2-dependent increase in Oc was almost independent myofibrils (3 min perfusion reduced force below 10% of the of filament overlap. control value) were reconstituted with either skeletal (s) or cardiac Supported by MRC and BBSRC. The TnC expression vector was a (c) TnC. Reconstitution of the myofibril with sTnC restored force generous gift from Dr L.B. Smillie.

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393-Pos Board # B249 395-Pos Board # B251 STRUCTURE OF THE N-DOMAIN OF CARDIAC INTERACTION OF THE HEART SPECIFIC N-TERMINAL TROPONIN C IN COMPLEX WITH BEPRIDIL AND ARM OF BISPHOSPHORYLATED HUMAN CARDIAC CARDIAC TROPONIN47i.63. TROPONIN I Monica X Li, Xu Wang, Leo Spyracopoulos, Brian D. Sykes; AnJa Schmidtmannt, Karin Lohmann2, Kornelia Jaquet'; 1Ruhr- University of Alberta, Canadian Institute of Health Research University Bochum, Bochum, 44780 Germany, 2DLR Bonn Group in Protein Structure and Function, Edmonton, Alberta T6G Troponin, a regulatory protein of the thin filament, consists of 2H7 Canada three subunits: Troponin T, the tropomyosin-binding subunit; Cardiac troponin C (cTnC) is the Ca2+-dependent switch for TroponinI, the inhibitory subunit and Troponin C, the Ca2+ contraction in cardiac muscle and a potential target for drugs in the binding subunit. In comparison to skeletal Troponin I the cardiac therapy of congestive heart failure. Ca2e binding to the regulatory isoform contains a N-terminal extension of 32 amino acids. This domain of cTnC (cNTnC) induces little structural changes but sets N-terminal extension contains two adjacent serine residues at the stage for cTnI binding. A large 'closed' to 'open' positions 22 and 23 (human isoform) which can be phosphorylated conformational transition occurs in the regulatory domain upon by the cAMP-dependent protein kinase upon 8-adrenergic binding cTnl,47.163 as shown by us [Li et al. (1999) Biochemistry, stimulation. Phosphorylation of these two serine residues leads to a 38, 8289-8298] or the Ca2e sensitizer bepridil as shown by Cohen's conformational change of Troponin I and lowers the affinity of group [Li et al. (2000) PNAS, 97, 5140-5145]. This raises the Troponin I towards Troponin C and T. This probably induces a question of whether cTnIl,47.163 and bepridil compete for reduction in the affinity of Troponin C towards Ca2+. However, cNTnC*Ca2e. In this work, we examined the binding of bepridil to this mechanism is not known. It has been supposed that the N- cNTnC*Ca2+ in the absence and presence of cTnl,47-163, termninal extension is fixed to Troponin I itself, when respectively, and of cTnI147.163 tocNTnC-Ca2 in the absence and dephosphorylated.The bisphosphorylated N-terminal extension is presence of bepridil, respectively. The results show that although proposed to interact with Troponin C or Troponin T. both the drug and cTnI,47-163 peptide interacts with cNTnC*Ca, 3 1P-NMR-measurements revealed that Troponin C is probably not they do not displace each other, suggesting non-competitive the interaction partner of the bisphosphorylated extension of binding sites for the two ligands. We are in the process of TroponinI.Therefore, 3 1P-NMR-measurements of reconstituted determininf the NMR structure of the ternary complex bisphosphorylated Troponin I with Troponin C and deletion mutant cNTnC*Ca '*cTnI147.163*bepridil. This structure will provide proteins of Troponin T will be performed and the spectra will be insights into the features that may be important for the design of presented. cTnC-specific cardiac drugs. 396-Pos Board # B252 394-Pos Board # B250 3D ORGANIZATION OF TROPONIN ON RELAXED AND THE EFFECT OF FHC CAUSING MUTATIONS ON A ACTIVATED THIN FILAMENTS REVEALED BY EM AND HUMAN CARDIAC TROPONIN T PEPTIDE 3-D RECONSTRUCTION Thomas Palm, Sarah Graboski, Norma Greenfield, Sarah William Lehman', Michael Rosoll, Larry S Tobacman2, Roger Hitchcock-DeGregori; University of Medicine and Dentistry of Craig3; 'Boston University School Medicine, 80 East Concord New Jersey, Robert Wood Johnson Medical School, 675 Hoes Street, Boston, MA 02118, 2Univ. of Iowa Col. of Med., 200 Lane, Piscataway, NJ 08854 Hawkins Drive, Iowa City, IA 52242,3Univ. Massachusetts Med. Familial hypertrophic cardiomyopathy (FHC) is an inherited Sch., 55 Lake Avenue N., Worcester, MA 01655 disease that often leads to sudden cardiac death. Mutations in 3D reconstruction based on actin (Ac) helical symmetry has failed cardiac troponin T (hcTnT) account for about 15% of the cases. to reveal troponin (Tn) density along thin filaments because Tn Since whole TnT is sparingly soluble, FHC mutations were studied occurs only on every seventh Ac. We have partially overcome this in the recombinant fragment hcTnT7o.,70. We introduced the problem by studying filaments reconstituted with a short mutations 179N, R92Q, A104V, F101, A160E, and E163K into tropomyosin (Tm), interacting with 4 instead of 7 Ac monomers hcTnTio.170 and compared the stability and function of the FHC- (Landis et al., 1997). This increases the stoichiometry of Tm-Tn on peptides with the wild type peptide. The stability of the peptides Ac and the number of views displayed by Tn along filaments, was determined by CD melting studies at pH=6.5. CD creating a more favorable symmetry for 3-D analysis. In 3D spectroscopy was also used to estimate the ability of FHC-peptides reconstructions of negatively stained filaments, we detect clear, to form a ternary complex with N-, and C-terminal peptides of robust density that is distinct from Ac and Tm and therefore tropomyosin (TM). The ability of the peptides to bind to a-TM attributable to Tn. In the absence of Ca2+, Tn density emerges as a was assessed by affinity chromatography on a-TM-Sepharose at narrow stallc from Tm located on the Ac outer domain. The Tn pH=6.5. Their ability to promote TM binding to actin was extension widens to form a bulb-like cap that contacts a broad area measured by cosedimentation at pH=6.0, 300 mM NaCl, and 4 of the periphery of the Ac outer domain near Ac residues 1-4 and mM MgCl2. In all experiments, the properties of most FHC- 23-27. In the presence of Ca2, both Tm and Tn move away from peptides were similar to the wild type peptide with the exception of their low Ca2+ positions. Tn, while still attached to Tm, is released A104V and Fl10. A104V was less stable than wt and less able to and projects away from Ac as Tm moves towards the Ac inner promote a-TM binding to actin. P1101 was more stable than wt domain. These results are consistent with models of muscle and had little ability to form a ternary complex with TM-peptides. regulation in which Tn constrains Tm in an inhibitory position on Also, its binding to TM was much weaker than that of wt and its Ac, a constraint that is released on activation by Ca2. ability to promote a-TM binding to actin was impaired. These results show that the region around Fl 10 is very important for the 397-Pos Board # B253 stability and function of hcTnT and suggest that impaired CA2+-SENSING MECHANISMS IN CALMODULIN AND interaction with TM could cause FHC. Supported by NIH. TROPONIN-C Felicia Pitici; Wesleyan University, Middletown, CT 06459 Structural data (Houdusse et al., Structure 5:1695, 1997; Zhang et al., Nat. Struct. Biol. 2:758, 1995) have shown that single domains of calnodulin (CaM) and troponin-C (TnC) undergo similar changes from 'closed' to 'open' confonrmtions upon binding Can. In spite of the structural similarity of the domains, their transition

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mechanisms differ: e.g. the C-terminal domain of CaM (C-CaM) myosin and myosin associated proteins are re-expressed. It is has more than one intermediate that exchange between 'closed' and hypothesized that the acute quadriplegia is (1) begins with an 'open' forms (Evenas et al., J. Mol. Biol. 289:603, 1999), while the inexcitable sarcolemma, (2) followed by a block of myosin regulatory N-terminal domain of skeletal TnC (N-sTnC) has a synthesis at the transcriptional level, and finally (3) recovery of single 'closed' intermediate (Gagne et al., Biochem. 36:4386, protein synthesis and incorporation of myosin and myosin 1997). Vibrational analysis and molecular dynamics simulations associated proteins into the thick filaments. were used to identify the determinants for the observed different Ca2+-sensing mechanisms. Results show that intrinsic vibrations 400-Pos Board # B256 relate to the Ca2-induced transition, and that the preference for TARGETED ABLATION OF THE MOUSE CARDIAC transition is specific for Ca2+-occupancy: holo, but not apo N-sTnC MYOSIN BINDING PROTEIN C GENE. exhibits a strong propensity, while holo and apo C-CaM have limited propensities. A novel view of the Ca2-induced transition Samantha P. Harris', Christopher R. Bartley', Kerry S. in which the same structural elements that block the McDonald2, Patricia A. Powers', Richard L. Moss'; 'University of emerges, Wisconsin, Madison, WI 53706, of Missouri, access of functional targets of these proteins, also play an 2University autoinhibitory role in the transition related to Ca2e binding. Columbia, MO 65212 Mutations in the cardiac myosin binding protein C (MyBP-C) gene 398-Pos Board # B254 are a common inherited cause of familial hypertrophic MYOFILAMENT PROTEIN ALTERATIONS IN A SWINE cardiomyopathy (FHC). Although MyBP-C is likely to play ISCHEMIC-INDUCED HEART FAILURE MODEL structural and functional roles in proper sarcomere assembly and contraction, the mechanisms by which MyBP-C affects cardiac Irina Neverova, Jennifer E. Van Eyk; Queen's University, function are not well understood. To investigate deficits in MyBP- Botterell Hall, room 414, Kingston, Ontario K7L 3N6 Canada C we used homologous recombination to "knock-out" the MyBP-C In chronic disease, such as heart failure (HF), the complexity of gene in mouse heart. The targeting vector consisted of 5' and 3' adaptive changes to cardiac function is mirrored at the level of homology units corresponding to sequences flanking exons 3-10. cellular proteins. We developed an in vivo ischemic-induced HF Exons 3-10 were replaced with a gene for neomycin resistance. model in swine by occluding the left anterior descending coronary After electroporation into embryonic stem (ES) cells, clones artery (LAD). Six weeks post-surgery, all LAD-ligated animals resistant to G418 and ganciclovir were selected. Targeting was had reduced cardiac index and ejection fraction (39.3% and 24.6%, verified by Southern blot and clones were injected into blastocysts. respectively) when compared to sham-operated swine. Left Chimeric animals were bred for germline transmission of the ventricular tissue, remote from infarcted area, was analysed by mutant allele and heterozygous Fl progeny were mated. Mice SDS-PAGE, 2-DE, western blot analysis and mass spectrometry homozygous for the MyBP-C mutation were born in normal either as a whole tissue homogenate or myofilament fraction. The Mendelian inheritance ratios and were viable into adulthood. myofilament fraction was prepared using our newly developed These results suggest that MyBP-C is not essential for sarcomere selective sequential extraction method, based on differential assembly and function during embryogenesis and cardiac protein solubility at various pH. This technique allowed us to development. Analyses of cardiac phenotype in these mice will preserve protein integrity and the status of post-translational provide information regarding the role of MyBP-C in cardiac modifications (PTM). Compared to the sham-operated, LAD- function. [Supported by NIH47053]. ligated animals had an elevated level of MLC I and alpha-actinin up to 5 and 3 fold respectively, depending on the severity of HF. No in 401-Pos Board # B257 changes abundance of tropomyosin and troponin were found. CROSS-BRIDGE VERSUS THIN FILAMENT we did not Although detect changes in isoforms of TnT nor the CONTRIBUTIONS IN COOPERATIVE ACTIVATION OF level of TnI, different PTM products of these proteins were found in myofilament fraction of LAD animals, including multiple SKELETAL MUSCLE. phosphorylated and degraded forms of Tnl. Thus, the Michael Regnier, Scott B. Myrick; University of Washington, complement Box 357962, Seattle, and status of proteins within a myocytes of diseased heart were Washington 98195-7962 altered, both through gene-mediated and PTM processes. Ca2+ activation of striated muscle involves cooperative interactions of cross-bridges and thin filament (TF) regulatory 399-Pos Board # B255 proteins. To investigate TF contribution in cooperative activation, ACUTE QUADRIPLEGIC MYOPATHY AND LOSS OF we extracted native TnC from skinned rabbit psoas muscle fibers MYOSIN IN ICU PATIENTS. UNDERLYING CELLULAR and reconstituted Tn complexes with low ratios of purified AND MOLECULAR MECHANISMS. sTnC/TnC mutant with inactive N-terminal Ca2+ binding sites Raghu Kolluri&, Hakan Zackrisson2, Lars Larsson'; 'Pennsylvania (D27A, D63A; xxsTnC) to compromise regulatory protein State interactions. Cross-bridge contribution was studied by varying University, 117 Noll Lab, University Park, PA 16803, thin-thick filament lattice spacing with Dextran T-500 and/or by 2Karolinska Hospital, Sweden, Stockholm, Sweden replacing ATP with 2 deoxy-ATP (dATP). Prior to extraction of More than 20 years ago MacFarlane and Rosenthal (1977) reported native TnC, addition of 4% Dextran did not affect isometric force a case of acute quadriplegia in a 24 year old woman after treatment at pCa 4.0 (Fmax), but increased force by 20 +2 % at pCa 6.0 (n = with non-depolarizing neuromuscular blocking agents (NMBA) 7 fibers). Fmax with 4% Dextran was not altered by dATP (a and corticosteroids (CS). The aim of this study is to investigate the strong cross-bridge augmentor) but increased force at pCa 6.0 by cellular and molecular mechanisms underlying the acute 25%. Following complete sTnC extraction and reconstitution with quadriplegia in intensive care unit patients. We have employed an sTnC/xxsTnC, reconstituted Fmax (rFmax) was 49 +4% of control in vivo porcine model of AQM, where the animal was Fmax and pCa 6.0 force was only 20 ± 3% of rFmax compared mechanically ventilated and exposed to NMBA, CS, and sepsis. with 46 ± 2% of Fmax prior to extraction, indicating reduced Expression of myofibrillar proteins and mRNA, and regulation of calcium sensitivity of activation. Under these conditions, addition myofibrillar protein synthesis and degradation are being studied of 4% Dextran increased rFmax by 17 ± 2% and pCa 6.0 force by using Confocal Microscopy, PCR and Microarray techniques. 47 ± 3% and dATP increased both rFmax and pCa 6.0 force as Electro-physiological studies are also conducted. In the acute well. These results indicate that when the number of functioning stage, there is a partial or complete loss of thick filament proteins, regulatory units is reduced, increased strong cross-bridge binding a disorganization of the normal sarcomere structure and loss of the has a larger effect on activation of skeletal muscle TFs. HL52558. force generating capacity at the single cell level. During recovery,

89a 402-406.-- .-- POSTERS Sunday- 7

402-Pos Board # B258 Compared to HCRLC-WT reconstituted myofibrils, the E22K and MYOSIN SUBFRAGMENT 1 BINDING TO ACTIN AND R58Q mutations, that are located in the immediate extension of the ACTIN-TROPOMYOSIN STUDIED BY ISOTHERMAL helices flanking the Ca2+ binding site of RLC, dramatically TITRATION CALORIMETRY decreased the Ca2e sensitivity of the ATPase, with E22K also Tanya Freedman, Knut Langsetmo, Sherwin Samuel Lehrer; reducing inhibition (-Ca2+). These two mutants had greatly reduced Boston Biomedical Research Institute, 64 Grove Street, Ca2+ affinity compared to HCRLC-WT. The Kca for E22K was Watertown, MA 02472 decreased 17-fold and the R58Q mutant did not bind Ca2e. The other FHC mutants, A13T, F18L and P95A, located near the When bound to actin, tropomyosin (Tm) equilibrates between two phosphorylation site (Ser 15), significantly diminished the states (closed and open) producing cooperativity in the binding of maximal level of ATPase activity compared to HCRLC-WT. myosin subfragment I (SI) to the actin thin filament. To obtain These results suggest that the Ca2+ binding and phosphorylation thermodynamic parameters associated with effects of Tm on S1 sites play key roles in the regulation of cardiac muscle contraction. binding to actin, isothermal titration calorimetry (lTC) studies Supported by AHA 9808237Vand NIHAR45183. were begun (VP-ITC from MicroCal). 10 jl amounts of SI were injected into stirred solutions of degassed actin and actinTm at 3-5 ^M, in 50 mM NaCl, 5 mM 0.1 mM CaCl2, 5 'C. A small 405-Pos Board # B261 MgC12, ISOLATION OF THE GENE FOR MOUSE FAST quantity of apyrase was added to remove interfering ATP just before each run. Reliable binding isotherms of AH vs SI/actin SKELETAL MYOSIN REGULATORY LIGHT CHAIN, AND were obtained after initial unstable readings at low SI/actin ratios. CHARACTERIZATION OF THE PROMOTER Analysis at the higher SI/actin ratios where actinTm is in the open Fanny Morales, Danuta Szczesna, Todd E Miller, Fatima de state gave values of AH of 25 and 42 + 5 keal/mole for SI binding Freitas, James D Potter; University of Miami, 1600 N.W. 10th to actin and actinTm, respectively. Thus Tm in the open state on Ave., Miami, FL 33136 actin increases AH significantly. This can occur either by an We have isolated a clone from a mouse genomic library that indirect effect of Tm on each actin subunit or more probably by a contains the entire gene for the mouse fast skeletal myosin direct additional interaction of actin-bound SI with Tm. regulatory light chain (LC2). The identity of this clone has been (Supported by NIH HL 22461) confirmed by restriction mapping and sequencing. This gene consists of seven exons that span approximately 2.5 kb. One 403-Pos Board # B259 unusual feature of this gene is the presence of a Bi repetitive NEBULETTE ISOFORM EXPRESSION AND element located about 800 bp upstream of the start of transcription. INTERACTION WITH THIN FILAMENT PROTEINS. Another unusual feature is the presence of the gene coding for Ozgur Ogut, J.-P. Jin; Case Western Reserve University, 10900 diff6, the 3' end of which is situated about 180 bp away from the 3' Euclid Avenue SOM E527, Cleveland, Ohio 44106 end of the LC2 gene. This clone contained about 1.7 kb of DNA upstream of the start of transcription, so we have investigated the Nebulette and nebulin are highly homologous proteins expressed in promoter for the LC2 gene by cloning different 5' fragments in cardiac and skeletal Both are muscles, respectively. proteins front of a luciferase reporter (pGL3 basic), and transfecting mouse formed primarily by the repeating sequence of 35 anmino acid units, C2C12 cells. results of these which have been shown to The studies suggest the LC2 promoter bind F-actin. To further study the role is highly active in this skeletal muscle cell line, and of and we a putative nebulin nebulette, cloned 5-unit chicken nebulette regulatory elements have been identified, including those for cDNA reverse fragment by transcription PCR according to MyoD, and members of the MEF family. RT-PCR showed published sequence data (C.L. Moncman and K. Wang, Cell Motil. We defined transcripts from this gene were abundant in muscle, and trace Cytoskeleton (1995) 32:205-25). individual nebulette levels were found in other mouse tissues. Supported by NIH grants units based on exon the boundaries found from partial sequencing HL42325 and AR45183. of the mouse nebulin gene. The cloned nebulette protein was expressed in E.coli and purified to homogeneity. The 5-unit nebulette protein was fairly soluble (>4 mg/mL in H20) in contrast 406-Pos Board # B262 to human (J.-P. Jin and K. WangJ. Biol. Chem. (1991) 266:21215- A WORM-LIKE-CHAIN MODEL FOR COOPERATIVE 23) and mouse nebulin fragments of comparable sizes. The THIN-FILAMENT REGULATION OF STRIATED expressed protein was used as an immunogen to raise mouse MUSCLE. polyclonal antibodies against nebulette. Westem blots revealed up David Aitchison Smith', Michael A Geeves2; 'King's College to 2 nebulette isoforms expressed in chicken and bovine heart. London, Guy's Campus, London, SEI 1UL United Kingdom, Interestingly, the ratio of the two isoforms expressed in the bovine 2Univ. of Kent in Canterbury, UKC, Canterbury, Kent CT2 7NJ heart varied by chambers. Further experiments will be presented United Kingdom focusing on the in vitro interactions of the cloned nebulette Existing theories of cooperative thin-filament regulation of actin- fragment with muscle thin filament proteins. myosin interactions assume rigid weakly-interacting tropomyosin (Tm) monomers, implying that the size of the cooperative unit is 404-Pos Board # B260 the monomer length (n = 7 actin units); however, kinetic data for FAMILIAL HYPERTROPHIC CARDIOMYOPATHY A-Tm-troponin(Tn) yields n = 10-12 (Geeves and Lehrer. 1994. MUTATIONS OF HUMAN CARDIAC MYOSIN Biophys. J. 67:273), suggesting that tropomyosin acts as a flexible REGULATORY LIGHT CHAINS AFFECT ATPASE chain over the whole filament. A worm-like-chain model for ACTIVITY IN RECONSTITUTED MYOFIBRILS. tropomyosin is proposed where changes in its angular position ¢(s) Danuta Szczesna, Georgianna Guzman, Debalina Ghosh , James at distance s are controlled by bending force K4f'(s) and a restoring D. Potter; University of Miami School of Medicine, 1600 N.W. force at(s) where =0 includes the myosin-binding interface. The 10th Ave, Miami, FL 33136 set of thermally-excited Tm configurations in the absence of bound The wild-type (WT) and five mutants of human cardiac myosin myosin or TnI defines the 'closed' state. 'Open' or 'blocked' regulatory light chains (HCRLC): A13T, F18L, E22K, R58Q and regions of the filament are induced by bound myosin or Tnl, which P95A, that cause Familial Hypertrophic Cardiomyopathy (FHC) induce +ve or -ve kinks respectively in the Tm chain. The energies were reconstituted in RLC-depleted porcine cardiac myofibrils. of M-M, Tnl-TnI and M-Tnl interactions reflect the energy cost of Depletion of the endogenous RLC resulted in normal activation these kinks, and their range is set by the persistence length (+Ca2+), dramatically impaired inhibition (-Ca2+) and slightly {=(Iy1/4, which facilitates binding of additional myosins under a increased Ca2, sensitivity of myofibrillar ATPase activity.

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single +ve kink. Rapid attachment equilibrium of TnI at any Ca++ their interactions with actin, myosin and each other. As with level may also be required. bacterially expressed muscle tropomyosin, ala-ser N-terminal extensions of TPM1 and TPM2 were required for actin binding. 407-Pos Board # B263 Actin affinity was measured by co-sedimentation of 3H-labeled TROPOMYOSIN CONTAINING TRYPTOPHAN TPM2, and by competition between TPMI and 3H-TPM2 (with ANALOGS: PROBES OF SPECIFIC THIN FILAMENT their different lengths considered in the analysis). Unlike PROTEIN INTERACTIONS vertebrate muscle and non-muscle tropomyosins, neither TPM1 Aurea Denise de Sousa, Chuck Shaker Farah; Universidade de nor TPM2 bound much more tightly to actin-myosin Sl than to Sao Paulo, Av. Prof. Lineu Prestes 748, I.Q. /BI.0, S.12, Sao Paulo, bare actin. Correspondingly, all other tropomyosins increase SP 05508-900 Brazil myosin S1-ADP affinity for pyrene-actin several-fold, but yeast TPMs did not. Nevertheless, yeast TPMs modulated myosin Tropomyosin (Tm) is a coiled-coil protein that interacts with function in an isoform-specific manner. In in vitro motility troponin (Tn) to modulate muscle contraction via its Ca2+- experiments a lower myosin concentration was required to activate dependent repositioning on the actin filament. Analysis of Tm movement of TPM1- than TPM2-actin. Myosin S1-ADP bound isoform sequences indicated that residues 258-275 present a cooperatively to TPM1-actin, but non-cooperatively to TPM2- striated muscle-specific pattern atypical for a coiled-coil structure. actin. Curve-fitting suggests that tropomyosin binding to the actin Based on these observations, we designed 5 Tm mutants and inner domain invariably enhances myosin-actin binding compared expressed them under conditions in which tryptophan (Trp), 5- to the absence of tropomyosin, but to a degree that depends upon hydroxytryptophan or 7-azatryptophan was selectively tropomyosin isoform. This causes a TPM isoform-specific incorporated. The fluorescence of the Trp analogs can be modulation of myosin function. selectively excited and used as a tool to study interactions involving the C-terminal of Tm. All the proteins retained Tn and actin binding ability. We found that the probes' fluorescence 410-Pos Board # B266 at 261 and 263 are sensitive to actin and Tn PHOSPHORYLATION OF THE REGULATORY LIGHT intensity positions CHAIN OF MYOSIN FROM TARANTULA binding respectively. We are employing these mutants to STRIATED investigate the thermodynamics of Tm/Tn/actin interactions. MUSCLE. Position 269 is sensitive to both actin and Tn binding and to Carlos Hidalgol, R. Craig', M. Ikebe', R. Padr6n2; 'UMass Med. increasing ionic strength, which suggests that this probe is Schl., Worcester, MA, 2IVIC, Caracas, Venezuela reporting Tm polymerization. These mutants will assist us to gain a Contraction is modulated in many striated muscles by Ca2+-CaM structural and thermodynamic understanding of the protein-protein dependent phosphorylation of myosin regulatory light chain (RLC) interactions involved in the transmission of the calcium-binding by myosin light chain kinase. We have studied the biochemical signal to the actin-myosin binding interface. mechanism of RLC phosphorylation in tarantula striated muscle to SUPPORTED BY FAPESP better understand the basis of myosin-linked regulation. In an earlier study it was concluded that the RLC occurred as two 408-Pos Board # B264 species, both of which could be phosphorylated. We present here DIFFERENTIAL MOBILITY OF SKELETAL AND evidence that only a single species exists, and that this can be CARDIAC TROPOMYOSIN ON CA+2 OR MG+" ACTIN IN phosphorylated at one or two sites. We find substantial level of REGULATED THIN FILAMENTS PLUS SUBFRAGMENT basal phosphorylation at the first site in relaxed muscle. This is 1. augmented on activation followed by partial phosphorylation of the second site. We find also that [Ca?+2f has a dual effect on RLC Richard D. Ludescher, Iype K. Chandy, Alfredo A. Perez de phosphorylation depending on its concentration. At low Alejo Il; Rutgers University, 65 Dudley Road, New Brunswick, concentration (relaxation) there is only basal RLC New Jersey 08901 phosphorylation; at higher concentrations RLC phosphorylation is We have monitored the microsecond rotational dynamics of stimulated; while at still higher physiological concentrations it is erythrosin-labeled rabbit skeletal and cardiac tropomyosin using partially inhibited, suggesting an additional mechanism for fine- steady-state phosphorescence emission anisotropy (r). In partially tuning muscle contractile activity. (Supported by grants from NIH, reconstituted filaments without Tn, skeletal Tm is slightly more HHMI and CONICIT). mobile on Mg+2 than Ca*2 actin (r = 0.020 vs. 0.025, respectively) and less mobile on Mg+2 actin with saturating S1 (r = 0.059 vs. 411-Pos Board # B267 0.049); cardiac Tm, however, is less mobile on both Mg+2 actin (r MODULATION OF MECHANICAL PROPERTIES DUE TO = 0.018 vs. 0.010) and on Mg+2 actin with saturating SI (r = 0.067 REGULATORY LIGHT CHAIN PHOSPHORYLATION IN vs. 0.054). Under all filament conditions, skeletal and cardiac Tm MYOCARDIUM. exhibit differential mobility. In a fully reconstituted filaments with Tn, skeletal Tm is nearly immobile on both Mg+2 and Ca+2 actin in Melinda S. Huiting-Hollander1, Ju Chen2, Po-Hsien Chu2, the absence of Ca+2 (blocked state) (r = 0.074 vs. 0.067), becomes Richard L. Moss1; 'University of Wisconsin, 1300 University Ave. more mobile on both types of actin in the presence of Ca+2 (closed 115 SMI, Madison, WI 53706, 2University of California at San state) (r = 0.050 vs. 0.049), and is nearly immobilized by addition Diego, La Jolla, CA 92093-0613 of saturating S1 (open state) (r = 0.071 vs. 0.075). Cardiac Tm The roles of regulatory light chain (RLC) phosphorylation in exhibits similar mobility in the fully reconstituted complexes but myocardial contraction were studied in small, multicellular (1-3 has consistently higher anisotropy values than skeletal Tm. myocytes) preparations from control mice and knock-in mice Supported by American Heart Association Heritage Affiliate. expressing a non-phosphorylatable fonn of cardiac RLC (SISA). In control myocardium the resting level of RLC phosphorylation 409-Pos Board # B265 was 39%±2% of total RLC, as determined by isoelectric focusing. MODULATION OF MYOSIN FUNCTION BY Ca2 -sensitivity of force (pCa,o) and Ca2-dependence of the rate TROPOMYOSIN ISOFORMS. constant of force redevelopment (kt) after rapid slackening and James M. E. restretch of the preparation were measured to assess mechanical Strand', Nili2, Homsher2, L. S. Tobacman'; of was of Iowa, 2Te of California at Los performance. Ca2e-sensitivity force reduced in S1SA 'University University Angeles preparations compared to control: pCaso was 5.63±.01 in S15A Yeast tropomyosins are the shortest known tropomyosins, and 5 preparations compared to 5.69±.01 in control (p<0.05). Absolute when bound to actin span either monomers (TPM1) or only 4 values of k,4 during maximal activation did not differ between (TPM2). We expressed TPMI and TPM2 in bacteria and studied

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S1SA and control. In addition, no change was observed in relative after intermittent fatiguing stimulation. (Support: NHLBI-NIH HL ktr between SISA and control at any pCa value. Based on these 60304) results, we conclude that RLC phosphorylation increases Ca2+- sensitivity of force in myocardium but increasing the level of RLC 414-Pos Board # B270 phosphorylation from 0% to 40% does not influence the rate of INTRA-MOLECULAR INTERACTIONS IN force development. Additional experiments will be done to CALMODULIN-CALDESMON FUSION PROTEINS examine the effects of 100% RLC phosphorylation on kt. Hongqiu Guo, C.-L. Albert Wang; Boston Biomedical Research Supported by NIH HL47053 (RLM), HL61659 (JC), Institute, 64 Grove Street, Watertown, MA 02472 AHA9960031Y (JC). To investigate the interaction between calmodulin (CaM) and caldesmon (CaD), we have expressed recombinant CaM fused at 412-Pos Board # B268 its C-terminus with a CaD fragment that contains the CaM-binding MAPPING PROTEIN INTERFACES BETWEEEN sites (CCF2, from G666 to P771, and CCF3, from G666 to K710). NEBULIN AND CALMODULIN: THE APPLICATION OF A The purified fusion proteins exhibit calcium-dependent increases FLUOROGENIC CROSS-LINKER AND MASS in the Trp fluorescence, reflecting the interaction between the CaM SPECTROMETRY. and the CaD domains. This fluorescence change, however, Andrea Sinz, Kuan Wang; LPB/NLAMS/NIH, 9000 Rockville provides no information about the binding strength between the Pike, Bethesda, Maryland 20892 two protein moieties. To compare the intra-molecular binding Nebulin is a giant multifunctional protein that is thought to serve strength between the two constructs, we have explored the use of both as a length-regulating protein ruler, as well as calcium/CaM- differential scanning calorimetry. In the absence of calcium CCF2 mediated regulatory protein on the thin filaments of the skeletal undergoes a thermal transition at 59 C, as does CaM alone, muscle sarcomere. To define molecular interfaces between nebulin indicating that under this condition there is no interaction between and CaM, we thiolated lysines of CaM and ND66, a four-module the CaM- and the CaD-moieties, since neither CaD nor its fragment near the C-terminus of nebulin, with 2-iminothiolane and fragments are known to have clearly defined melting temperatures. cross-linked the complex with dibromobimane, which alkylates In the presence of calcium, both CCF2 and CCF3 exhibit two thiol pairs within -6 A of each other to form a fluorescent adduct. transitions: one at 101 C for CCF2 and 90 C for CCF3, and the Such a two-stage cross-linking generated mainly 1:1 complexes of other at 118-119 C for both. The transitions at lower temperatures ND66 and CaM, with limited extent of intramolecular cross- most likely represent the dissociation of the CaM- and the CaD- linking. In-gel chymotryptic digestion of the complexes yielded moieties, whereas the transitions at the higher temperature may peptides that were first screened by HPLC with fluorescence reflect unfolding of the dissociated CaM moiety. That the intra- detection and then scored for cross-linking with mass molecular complex collapses at a higher temperature for CCF2 spectrometry. Intermlecualr cross-linking of than for CCF3 is consistent with the idea that the longer CaD YKENMGKGTPLPVTPEM in ND66 to CaM indicate that the sequence favors a stronger interaction with the CaM-domain. nebulin/CaM interface is close to, and may overlap with, the Supported by NIH P01-41637. nebulin/actin interface. This proximity suggests a potential competition between CaM and actin for this nebulin interface. 415-Pos Board # B271 Intramolecular cross-linking suggests the interaction of two lobes DYNAMICS OF RHOA AND ROKA TRANSLOCATION IN across the central helix in CaM and hints an association of SINGLE LIVING CELLS noncontiguous nebulin modules in solution. Koji Miyazald, Mltsuo Ikebe; University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 413-Pos Board # B269 It has been suggested that small G-protein RhoA and one of its HYPOXIA AND FATIGUE-INDUCED MODIFICATION OF target kinases, ROKa translocate to plasma membrane fraction. FUNCTION AND PROTEINS IN INTACT AND SKINNED However, the relationship of translocation kinetics between these MURINE DIAPHRAGM MUSCLE. two molecules is still unknown. We clarified the molecular Marco de Paula Brotto, Sheila A van-Leyen, Leticia S Brotto, mechanisms of their translocation. We co-transfected COS-7 cells Christopher M Nosek, Thomas M Nosek; Case Westem Reserve with CFP-tagged RhoA and YFP-tagged ROKa, or their mutants, University, School of Medicine B-13, Cleveland, OH 44106 and monitored the localization of two different fluorescent tagged- In skeletal muscles, fatiguing stimulation renders muscles hypoxic. molecules in single living cells during EGF stimulation. Both Hypoxia is implicated in the production of reactive oxygen species RhoA (WT) and ROKa (WT) are translocated to raffling (ROS). We have previously demonstrated (Pflugers Arch (Eur. J. membrane with EGF stimulation. A ROKa mutant, in which the Physiol.) 440: 727-734, 2000) that fatiguing stimulation under kinase activity is desrupted, was also able to translocate to the hypoxic conditions disrupts both the excitation-contraction membrane with Rho (WT). On the other hand, the deletion of the coupling (ECC) process and the isometric contractile properties PH domain or the disruption of Rho-binding ability abolished the (ICP) in intact diaphragm muscle strips. ROS produced during translocation of ROKa to the membrane after EGF stimulation. muscle fatigue may be responsible for these effects by causing The results demonstrate that the binding of RhoA and the PH fragmentation/degradation of contractile proteins. We report for domain are essential for the translocation of ROKa, but the first time that hypoxic-fatigue decreased maximum calcium- activation of enzymatic activity is not directly related to the activated force and calcium sensitivity of the contractile apparatus translocation mechanism. (Supported by NIH grants HL60831 and of the mouse diaphragm; this was associated with the degradation HL61426) of TnI and TnC. We also found that although actin and TnT were not degraded, they were oxidized under hypoxia, control-fatigue 416-Pos Board # B272 and hypoxic-fatigue conditions. Reconstitution of a recombinant STRUCTURE OF CALMODULIN IN THE TRANSITION complex of troponins into the skinned muscle fibers from the STATE. hypoxic-fatigue group significantly recovered Fmax. Because troponins are involved in regulating the interaction between actin Zenon Grabarek; Boston Biomedical Research Institute, 64 and myosin during the cross-bridge cycle, the degradation of Tnl Grove St., Watertown, MA 02472 and TnC might explain the effects of hypoxia-fatigue on the ICP. We report a 2.4 A resolution structure of a calmodulin mutant The need for re-synthesis and repair of contractile proteins may (CaM41/75) in which the N-terminal domain is locked in the explain the slow recovery (24-72 hrs) of fatigue known to occur

92a S ouiiual PCVSTPPPO,RTFR.X %..# %.j X IIS ArL41 %E-T.A9A6-20

Ca2+-free conformation by a disulfide bond between Cys residues reconstituted myocardium. The results were analyzed by the substituted for Q41 and K75. The disulfide bond decreases the following cross-bridge scheme, where A=actin, M=myosin, S=MgATP and D=MgADP. In control myocardium, K, was 9.1 affinity for Ca2+ and reversibly inactivates the protein. In addition mM I, K2 was 2.6, K4 was 0.76, and K5 was 0.14 mM-. In actin Asn was substituted for Asp64 - a mutation that further decreases D , AMS - the affinity for Ca2+. CaM41/75 forms tetragonal crystals (space k2 AMDP) k kP AMD AM *7-+ AM'S t= AM-uP 4AWD group P43212) at 4 °C in the presence of 5 mM CaC12, 32% 2- K0 S k2 MS * MDp k4 K5 methyl-2,4-penthanediol (MPD), 10% t-butanol and 20 mM filament-reconstituted myocardium without regulatory proteins, K, cacodylate buffer pH 5.2. The structure was solved by MAD decreased to decreased to phasing of a 0.18x, K2 0.18x, K4 increased to 7.0x, seleno-Met substituted protein and refined to and Ks to 2.8x. CaM41/75 has an increased Those kinetic constants regained original Rfree=0.24. extended conformation in the values when further reconstituted with Tm and Tn. These results crystal, similar to that of the wild type protein. The N-terminal demonstrate that presence of regulatory proteins promotes cross- domain is closed with the four undistorted helices A-D making bridge detachment. contacts with each other. Ca2+ ions are bound at both N-terminal sites, however Glu at position 12 of the loop, which normally 419-Pos Board # B275 provides a bidentate ligand for Ca2+ does not reach the Ca2+ and INCREASED SUSCEPTIBILITY TO FATIGUE OF SLOW the corresponding coordination positions are occupied by H20 in AND FAST TWITCH MUSCLES FROM MICE LACKING site I and by the hydroxyl groups of MPD in site II. The structure THE MG29 GENE represents a transition state which occurs after the initial Ca2+ Ramki Yelleshpur Nagarajl, Christopher Nosekl, Marco Brotto', binding and preceds the movement of the helices and the opening Miyuki Nishi2, Hiroshi Takeshima2, Thomas Nosek', Jianjie Ma'; of the domain. 'Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44016, 2Kurume University, Fukuoka 417-Pos Board # B273 Mitsugumin 29 (MG29), a triad junctional protein in skeletal has been identified to A SKELETAL MUSCLE TISSUE RESTRICTED FKBP12- muscle, play roles in the formation ofprecise DEFICIENT MOUSE MODEL junctional membrane structures important for efficient signal conversion in excitation-contraction coupling. We carried out WEI TANG, Ximena Sanchez, Julianne S Clancy, Michael B experiments to not only study the role of MG29 in normal muscle Reid, Martin M. Matzuk, Susan L Hamilton; Baylor College of contraction but also to determine its role in muscle fatigue. We Medicine, One Baylor Plaza, Houston, TX 77030 compared the contractile properties of extensor digitorum longus FKBP12 (FK506 Binding Protein, 12KD) is a ubiquitously (EDL), soleus (SOL), and diaphragm (DPH) isolated from mice expressed cytoplasmic protein. It physically interacts with and lacking the MG29 gene and wild-type mice prior to and after modulates multiple intracellular calcium release channels including fatigue. Our results indicate that the mutant EDL and SOL muscles the tetrameric skeletal muscle calcium release channel (RyRI). In are more susceptible to fatigue than the wild-type muscles. The FKBP12-deficient mice, both skeletal RyRl and cardiac RyR2 mutant muscles not only fatigued to a greater extent, but also have altered single-channel properties. The mice have apparently recovered significantly less than the wild-type muscles. Following normal skeletal muscle development through embryogenesis, but fatigue, the mutant EDL and SOL muscles produced a much lower they have severe cardiac defects. Because of early lethality in the twitch force than the wild-type mice, and also fatiguing produced a deficient mice, the role of FKBP12 in skeletal muscle is not well much greater downward shift in the force-frequency relationship in understood. We have generated skeletal muscle restricted the mutant mice when compared to the wild-type controls. Our FKBPI 2-deficient mice using the Cre-loxP system. Post-natal mice results indicate that fatiguing affects the contractile properties of exhibit a skeletal muscle specific decrease in the FKBP12 protein, the mutant EDL and SOL muscles, and the effect of fatigue in but display no gross abnormalities in ambulation or other motor these mutant muscles could be mainly due to an alteration of the tasks associated with unrestrained behavior, nor do they differ in intracellular Ca homeostasis. their body weights. Contractile properties of excised diaphragm fiber bundles are, however, abnormal; less force is developed Actin & Actin-Binding Proteins during twitch and low-frequency (10-100Hz) tetanic contractions, shifting the force-frequency relationship rightward. Passive tension is also elevated at optimal length and the passive length-tension is 420-Pos Board # B276 shifted to the left. These findings suggest that FKBP12 is MEASUREMENT OF MYOFIBRIL THIN FILAMENT important in either controlling calcium release from SR during rest LENGTHS BY DISTRIBUTED DECONVOLUTION and contraction, or maintaining proper myofiber structure in Ryan Littlefield, Velia M. Fowler; The Scripps Research Institute, skeletal muscle. 10550 N. Torrey Pines Rd., La Jolla, CA 92037 The lengths of the thin filaments in isolated chicken myofibrils 418-Pos Board # B274 were determined by distributed deconvolution, a new method that ELEMENTARY STEPS OF ACTIN FILAMENT- quantitatively determines the location of probes in myofibrils from RECONSTITUTED BOVINE MYOCARDIUM DEDUCED conventional fluorescence microscope images. Thin filament BY SINUSOIDAL ANALYSIS. lengths were determined using two fluorescent probes: bodipy- Hideaki Fujita, Masataka Kawai, ; Dept. of Anatomy and Cell phallacidin to stain along the thin filament and antibodies against Biology, Iowa City, IA 52242 tropomodulin (Tmod), the pointed-end capping protein, to stain thin filament pointed ends. The thin filament lengths in pectoralis We investigated the role of regulatory proteins in elementary steps major (PM) myofibrils were 1.003 gim, in agreement with previous of the cross-bridge cycle using bovine myocardium in which the measurements. Using Z-axis series of PM myofibrils, we actin filamet was reconstituted in the absence of tropomyosin (Tm) determined that the thin filament lengths were extremely and troponin (Tn) as reported previously (Fujita et al., 1996). reproducible over a large range of focus, allowing the comparison Elementary steps of cross-bridge cycle was deduced from of values determined from different images. Statistical analysis sinusoidal analysis. The effects of MgATP and inorganic using jack-knife tests indicated that the precision of the phosphate (Pi) on the rate constants of exponential processes were length studied in measurements were less than 20 nm. We used this method to control, actin filament-reconstituted, and thin filament- determine for the first time that the thin filament lengths in

93a zt9A-d95 420-425 POST'ERSPOSERSv.. _ , J Sundav

posterior latissimus dorsai (PLD) myofibrils were 1.110 1sm. For 423-Pos Board # B279 both PM and PLD myofibrils, both probes were less than 20 nm LIMULUS ACROSOMAL BUNDLE BEYOND 20A different, indicating that both phallacidin and Tmod fluorescence RESOLUTION were accurate probes for measurement of thin filament lengths. Michael F. Schmid', Michael B. Sherman2, Paul Matsudaira3, Thus, the application of distributed deconvolution to isolated Guillermina Wailer 3, Wah Chiul; 'Baylor College of Medicine, myofibrils is a simple way to determine thin filament lengths with One Baylor Plaza, Houston, TX 77030, 2Purdue University, West nanometer precision. Lafayette, IN 47907, 3Mass. Inst. of Tech., Boston, MA 02142 Limulus sperm contains a dynamic macromolecular structure that 421-Pos Board # B277 rapidly extends a 50-lsm process called the true discharge. At its MICROSCOPIC ANALYSIS OF POLYMERIZATION core is a bundle of filaments with crystalline order composed of a PROCESS OF SINGLE ACTIN FILAMENTS. complex of actin, scruin and calmodulin. We have used electron Iluko Fujiwara, Shin Takahashi, Hisashi Tadakuma, Takashi crystallographic analysis of single images of bundles preserved in Funatsu, Shin'ichi Ishiwata; Waseda Univ., 3-4-1 Okubo, vitreous ice to reconstruct their structure beyond 20 A resolution. Shinjyuku-ku, Tokyo 169-8555 Japan No helical or crystallographic symmetry (other than P1) was used We have studied the polymerization-depolymerization dynamics of in the reconstruction procedure The axial positions and azimuthal individual actin filaments under a fluorescence microscope. orientations of each of the 28 actin-scruin subunits in a unit cell Fluorescent dye (10% TMR-5-MA)-labeled actin molecules were were computed by cross-correlation with an actin-scruin subunit used for image-processing of polymerized filaments under isolated from our previous reconstruction of a single filament. The evanescent field illumination at room temperature. The time locations were then used to calculate an averaged actin-scruin course of polymerization after the addition of salts (30mM KCI, subunit and then to generate an averaged filament with a better 2mM MgC12, 4mM ATP, pH 7.0) was composed of two phases, signal to noise ratio. This averaged map clearly resolves the two i.e., the polymerization phase and the following steady phase. The domain features of the scruin molecule, which wraps around the f- average polymerization and depolymerization rates estimated from actin. Our 3-D reconstruction reveals an actin-scruin helix that is the polymerization phase were, respectively, 1.0x107/M/s and 0.6/s azimuthally modulated by the influence of the interactions of a for Mg-actin, 6.1x106/M/s and 0.86/s for Ca-actin, consistent with filament with its neighbors. There are a variety of density those obtained in solution experiments. The analysis of time connections with neighboring filaments involving scruin. Our correlation of length fluctuation at the steady phase showed that structure indicates that promiscuous scruin-scruin contacts are the polymerization and depolymerization that occur at both ends of the major determinants of bundle stability in the true discharge. It also filaments can be explained by the stochastic (diffusion) process. suggests that rearrangements would be permitted, which can Based on the diffusion model (the size of actin subunits, 2.7 nm), facilitate the transition from the coiled to the true discharge form. we estimated the polymerization and depolymerization rates as 1.2xI08/M/s and 7.6/s for Mg-actin, 7.9x107/M/s and Il/s for Ca- 424-Pos Board # B280 actin, respectively. These values were 12-13 times as large as STRUCTURE OF DISULFIDE CROSS-LINKED MUTANT those obtained from the polymerization phase. The reason for this YEAST ACTIN apparent discrepancy is discussed. Albina A. Orlova', Vitold E Galkin2, Eldar Kim3, Alexandr Shvetsov3, Emil Reisler3, Edward H. Egelman'; 'University of 422-Pos Board # B278 Virginia, Charlottesville, VA 22908, 2lnstitute of Cytology, RAS, THE 3-D STRUCTURE OF SMOOTH MUSCLE ac-ACTININ Saint-Petersburg, Russian Federation, 3University of California, BY CRYOEM REVEALS TWO MODELS OF ACTIN Los-Angeles, CA 90095 BINDING An intermolecular disulfide bond between residues 41 and 374 was Jun Liu, Dianne W Taylor, Kenneth A Taylor; Florida State introduced into F-actin by oxidation of the Gln4lCys yeast actin University, Tallahassee, Florida 32306 mutant filaments (Kim et al, JMB, 2999,421,2000). This disulfide 2-D arrays of smooth muscle a-actinin were formed on positively bridge can be used both to test the existing models of F-actin charged lipid monolayer and preserved frozen hydrated for structure and probe the flexibility of the C-terminus and the cryoEM. The 3-D density map at 2.0 nm resolution was generated DNAasel-binding loop. We have used EM and image analysis to using conventional methods. Atomic structures, homologous to the study these filaments. We show that the formation of angle-layer 3 domains of a-actinin, i.e. actin binding, calmodulin-like and 3- aggregates of crosslinked actin under phisiological conditions may helix motif, were docked manually into the molecular envelope be due to a redistribution of charges on the actin surface, since and refined using real space protocol to produce a pseudo atomic similar structures are only observed with wild type protein at acidic model. The central domain contains 4 paired, antiparallel 3-helix pH or in the presence of high concentrations of divalent cations. motifs with an overall 750 left-handed twist and slightly bend Using a novel method of image analysis for helical filaments which restricts the placement of the rod atomic model to one (Egelman, Ultramicroscopy, 2000) we find that the distributions of orientation. Locally, the central domain has 2-fold symmetry. The filament twist in disulfide cross-linked actin and in control Mg- end domains, consisting of the N-terninal actin binding and the C- actin are different. Three-dimensional reconstructions of S-S actin terminal calmodulin-like have two different structures that are reveal a change of electron density between two strands of actin oriented -90° to each other. The end domains are not related by that can be interpreted in terms of subdomain shifts. any local or crystallographic 2-fold rotation axis. The end domain structures can be used to generate 2 different conformations of a- 425-Pos Board # B281 actinin by placing one end domain structure at each end of the ENERGY OF AN ACTIN SPRING central domain. One of these has a conformation suitable for Jennifer H Shin', Jason Sutin2, Peter T So3, Paul Matsudairae, L crosslinking parallel actin filaments into a polar bundle; the other Mahadevan3; 'Massachusetts Institute of Technology, Department for crosslinking antiparallel actin filaments into a bipolar bundle. of Mechanical Engineering, Cambridge, MA 02139, 2University of Supported by NIH. Illinois at Urbana-Champaign, Urbana, IL 61801, 3Massachusetts Institute of Technology, 4Massacusetts Institute of Technology, Department of Biology, Cambridge, MA 02139 We study the dynamics of the acrosomal process in horseshoe crab (Limulus polyphemus) sperm. In its native state, the acrosomal bundle consists of a 60pm para-crystalline helical coil of bent,

94a SuAdAy POSTES Sundav POSTERS- 4 2 425-429A2

twisted actin filaments. In the presence of Ca > 2+, the actin surrounding polymers into account. Furthermore, theoretical binding protein, scruin, undergoes a conformation change, causing considerations within the frame of the same model help us to the individual actin filaments to untwist. This leads to the explain the observed changes in mechanical properties upon straightening of the 601im long bundle propelled through a nuclear addition of Cortexillin I, an actin-bundling protein from channel at a mean velocity of 15pm/s at room temperature. This Dictyostelium. Adding this protein in different concentrations, we unique process of actin-based motility involves only have investigated the changes in mechanical properties, performing conformational changes in the proteins in the absence of ATP or dynamic light scattering measurements at various wave- vectors. actin polymerization. Its velocity is constant throughout the entire Rheological measurements with an oscillating disc rheometer and extension, suggesting that the uncoiling of the bundle is a localized electron microscopy gave us additional information about the event that propagates in a zipper-like fashion. The average velocity interesting properties of these biologically important systems. of the acrosomal process increases as the temperature is raised. The bending stiffness, El, of the actin bundle was measured using 428-Pos Board # B284 two methods. One involves the analysis of the bending shape at PROPULSION AND COMET-TAIL FORMATION BY equilibrium in a steady flow and the other uses the relationship ACTIN POLYMERIZATION between the deflection of the bundle and the concentrated load of the force to the bead attached at the of the bundle. An Jonathan Isaac Katz, Anders E. Carlsson; Washington U., 1 magnetic tip Brookings Dr., St. Louis, Mo. 63130 estimate for the initially stored energy was then obtained to be of the order of 7x104 ergs. We have developed and numerically simulated a model of propulsion by actin polymerization and formation of a "comet tail" 426-Pos Board # B282 of F-actin. We consider a rigid sphere or disc uniformly covered THERMODYNAMICS OF THE POLYMERIZATION OF with sites (ActA) at which F-actin is bound and may polymerize. ACTIN Intercalation of G-actin displaces the sphere and polymer along the polymer's axis, in opposite directions, by amounts inversely Jeffrey G. Forbes, Priya Shantharaj, Sandra C. Greer; University proportional to their viscous drags. As the is and of sphere displaced Maryland, College Park, MD 20740-2111 rotates all the polymers are subject to drag in the surrounding flow Actin is ubiquitous in eukaryotic cells and it is crucial for field. We consider two simple boundary conditions: 1. The maintaining cellular structure and effecting motility. In fulfilling polymers remain perpendicular to the surface of the sphere, but are its various roles in the cytoskeleton, actin cycles between the free to slide along it; 2. The polymers are hinged at fixed points of monomeric G-actin form and the polymeric F-actin form. The attachment. In each case small random deviations from symmetry polymerization of actin is an entropy-driven process, the extent of grow exponentially as motion in one direction sweeps the which increases with increasing temperature. Likewise, a reduction polymers back, so further intercalation augments the directed in temperature will result in the loss of actin monomers from the motion. Numerical simulation, intercalating monomers randomly polymers. As the polymerization process is dependent, in part, on at the assumed polymerization sites, shows that the second the removal of water molecules surrounding a hydrophobic boundary condition leads to runaway rotation and little translation, surface, deuterium oxide should have a noticeable effect on the but that the first boundary condition leads to formation of a polymerization. In order to understand the equilibrium coherent comet tail and effective propulsion of the sphere. polymerization of actin, we need to understand the Propulsion is not effective until the polymers have grown, giving a thermodynamics of the process. To further our understanding of significant latency time, consistent with experiment. the thermodynamics of actin polymerization, we have used pyrene- labeled actin to study how the extent of polymerization of actin 429-Pos Board # B285 changes with temperature at low potassium concentrations. We ATOMIC MODEL OF COFILIN/F-ACTIN CONSTRUCTED have studied the polymerization process from 4 to 40°C in both FROM A TRUSSED NETWORK OF RESOLUTION- H20 and D20, and as expected, the extent of polymerization INVARIANT LANDMARKS increases with temperature. An feature of the extent of unexpected Willy Wriggers', Vitold Galkin2, Albina Orlova2, Edward H. polymerization is that it shows a maximum at higher temperatures. This has been seen with neutron under Egelman2; 1The Scripps Research Institute, 10550 N. Torrey Pines previously scattering Rd., La Jolla, CA 92037, 2University of Box similar conditions. We suggest that the polymerization of actin can Virginia, 800733, be described by the same statistical mechanical models used for Charlottesville, VA 22908 other second order phase transitions and other polymerizations. Molecular modeling and information processing techniques were combined to uniquely define a molecular model of cofilin/F-actin, 427-Pos Board # B283 based solely on the monomeric crystal structures and on an MECHANICAL PROPERTIES OF ENTANGLED AND electron microscopy (EM) reconstruction of actin filaments CROSSLINKED ACTIN SOLUTIONS decorated with plant-ADF. The fitting relied on resolution- Alois K invariant landmark skeletons (to be distributed with the Situs Popp', Erich Sackmann2, Guenther Gerisch3, Erwin Frey1; docking package) that enable the registration of individual 'Harvard University, 9 Oxford St., Cambridge, MA 02139, actin 2Technische structures with the EM map. The resulting F-actin model is Universitaet Muenchen, James- Franck- Strasse, practically identical (2.7A rmsd) to the (symmetry corrected) Garching, 85747 Germany, 3Max-Planck-Institut fuer Biochemie, model Am 18 Holmes of F-actin. Subtracting the effective actin density, Klopferspitz a, Martinsried, 82152 Germany we obtained individual plant-ADF densitites that yielded a unique Applying dynamic light scattering and bulk rheology, we have fit of cofilin's atomic structure relative to F-actin. The resulting investigated mechanical properties of semi-dilute actin solutions, position of cofilin is very similar to the G-actin binding mode and the influence of a crosslinking protein copolymerized with predicted with Tang & Janmey (Wriggers et al., JMB 282:921, actin in different concentrations. With the light scattering data, we 1998). The main difference between cofilin's predicted F-actin have performed a linear mode analysis according to the theory of binding mode relative to the G-actin mode is a reduced number of Kroy&Frey (e.g. Phys.Rev.E 55, 3092-3101,1997) to obtain peripheral salt-bridges that allow cofilin to swivel by -10°about estimates of mechanical properties of entangled actin gels. We the main contacts at res. 96 and 123, thereby avoiding a steric clash have determnined several quantities like persistence length, with the adjacent actin monomer in the filament. Our model is entanglement length and mesh size, using a model that takes the fully consistent with actin/cofilin interface data from mutagenesis tube- like confinement of a single entangled actin chain by the experiments.

95a A'IflAA A,tMLAPV-I,jTFP i -dLu POSTERS SiindavLi 41nd2v4

430-Pos Board # B286 polymerization. Profilin increased the F-actin depolymerization COFILIN-INDUCED STRUCTURAL CHANGES AT THE rate in buffers containing either ATP or ADP and 20 mM INTERMONOMER INTERFACES IN F-ACTIN. phosphate. The polymerization rate constant for 1 1iM MgATP- actin was decreased to half-maximal value by about 20 yM Andrey A. Bobkovl, Sergey Vorobiev2, Steven C. Almo2, Andras profilin; in contrast, profilin at a concentration equimolar with the Muhlrad', Emil Reisler'; UCLA, Los Angeles, CA 90095, 2Albert actin has little effect on the observed polymerization rate constant. Einstein College of Medicine, New York, NY 10461 The data indicate that profilin interaction with the actin filament Cofilin has been shown to change the twist (McGough et al., J. barbed end blocks association of monomeric actin and increases Cell Biol. 74:764) and to weaken lateral contacts (McGough and the dissociation rate of the terminal actin subunit. Elongation by Chiu, J. Mol. Biol. 291:513) in actin filaments. We studied these the profilin-MgADP-actin complex indicates that ATP hydrolysis effects in solution by investigating the interaction of yeast cofilin is not required. We suggest that the binding of profilin-actin to the with Q41C and Q41C/C374S mutant yeast F-actins and ANP- actin filament barbed end induces an actin conformational change labeled and cross-linked rabbit skeletal F-actin. Cofilin resulting in weakening of profilin affinity This work supported by significantly decreased the excimer fluorescence produced by the the Department of Veterans Affairs. interaction of two pyrenes attached to C41 and C374 on the Q41C mutant and the energy transfer (FRET) from tryptophans to 433-Pos Board # B289 AEDANS bound to C41 on the Q41/C374 mutant. FRET was also THE EFFECT OF PROFILIN ON THE INTERACTION dramatically decreased between tryptophans and ANP attached to BETWEEN ACTIN AND CYTOCHALASIN D Q41 of rabbit skeletal actin. However, FRET was much less affected when F-actin was intrastrand cross-linked by ANP Lynn A Selden, Henry J Kinosian, James E Estes, Lewis C between Q41 and C374. Cofilin caused also dissociation of Gershman; Veterans Affairs Medical Center, 113 Holland Ave, rhodamine-phalloidin from F-actin. Since the FRET changes are Albany, NY 12208 due to the changes at the intramonomer interface along the same We have investigated the effects of profilin on the interaction strand of the filament while the dissociation of phalloidin is related between cytochalasin D (CD) and vertebrate non-muscle actin. to altered lateral contacts our results indicate that cofilin affects The interaction between CD and Mg-ATP-actin was monitored by both the longitudinal and lateral contacts between protomers in F- measuring the CD-induced Mg-ATP-actin ATPase. The data can actin. be fit by a model in which CD and profilin bind competitively to Mg-ATP-actin with Kd's of 0.06 jiM and 0.15 pM, respectively, 431-Pos Board # B287 with CD binding resulting in an ATPase with a rate constant, kh = ACTIN CAN BIND TWO ADF MOLECULES 0.15 s'. The effect of profilin on this CD-induced ATPase reaction is seemingly ambiguous. At low profilin:CD ratios there Vitold E. GaUdn', Albina Orlova2, Natalya A. Lukoyanova3, is in an increase in the observed steady state ATPase rate, but as Edward H. Egelman2; 'Institute of Cytology, RAS, Saint- the profilin:CD ratio is increased the ATPase reaction is inhibited. Petersburg, Russian Federation, 2University of Virginia, Our model explains these results; at low profilin:CD ratios, Charlottesville, VA 22908, 3ITEB, RAS, Pushchino, Moscow profilin acts to increase the ATPase rate by increasing the rate region, Russian Federation limiting exchange of ATP for actin-bound ADP during the Proteins in the ADF/cofilin family have been shown to change the reaction. As the profilin:CD ratio is increased, profilin twist of actin by -5 degrees per subunit, and it has been suggested competitively inhibits CD binding resulting in inhibition of the that inducing this untwisted state is a part of the mechanism of CD-induced ATPase. These experiments highlight the high filament severing and depolymerization (McCough et al., 1997). affinity of profilin for non-muscle actin and its potent effect on the Using EM and a novel method of image analysis for helical regeneration of ATP-actin from ADP-actin. Inhibition of the CD- filaments we show that segments of actin filaments can be found in induced ATPase by profilin may be useful in furthering our a similar highly untwisted state in the absence ADF. This suggests understanding the interaction between CD and actin. Supported by that ADF may shift the mean twist of actin population to an the Department of Veterans Affairs. already exiting state, rather than inducing a new conformation. We can find segments of actin filaments fully decorated with ADF that 434-Pos Board # B290 have a less untwisted state -3 degrees per subunit, suggesting that DIFFERENCES the full change of twist is not essential for binding as has been CONFORMATIONAL AND DYNAMIC proposed. We see a second molecule of human ADF bound to a BETWEEN ACTIN FILAMENTS POLYMERIZED FROM site opposite from that of the primary ADF binding site. This ATP- OR ADP-ACTIN MONOMERS explains sites on actin implicated in binding ADF that cannot be Bdla Somogyi, EmOke B6dis, Gabor Hild, Krisztina Szarka, accounted for by earlier models. Kinetic observations suggested Mikl6s Nyitrai; University of Pecs, Faculty of Medicine, Szigeti that human ADF more efficiently depolymerizes rabbit muscle str. 12., Pecs, Hungary H-7624 Hungary actin in comparison with plant ADF. Since plant and human ADF Conformational and dynamic properties of actin filaments shift actin to the same untwisted state, the ability of human ADF to polymerized from ATP- or ADP-actin monomers were compared bind two molecules per one actin subunit might explain the by using fluorescence spectroscopic methods. The fluorescence difference in the activity of these proteins. intensity of 1AEDANS attached to the Cys374 residue of actin was smaller in filaments from ADP-actin than in filaments from ATP- 432-Pos Board # B288 actin monomers, which reflected a nucleotide-induced POLYMERIZATION OF VERTEBRATE NON-MUSCLE conformational difference in subdomain 1 of the monomer. Radial ACTIN IN THE PRESENCE OF PROFILIN. coordinate calculations revealed that this conformational difference Henry J. Kinosian, Lynn A. Selden, Lewis C. Gershman, James did not modify the distance of Cys374 from the longitudinal E. VA Medical Center, 113 Holland Avenue, filament axis. Temperature-dependent fluorescence resonance Estes; Stratton energy transfer measurements between donor and acceptor Albany, NY 12208 molecules on Cys374 of neighboring actin protomers revealed that The polymerization and depolymerization time courses for the inter-monomer flexibility of filaments assembled from ADP- vertebrate non-muscle actin nucleated with spectrin seeds were actin monomers were substantially greater than the one of followed by light scattering. Profilin was found to decrease both filaments from ATP-actin monomers. Flexibility was reduced by the rate and extent of MgATP-actin and MgADP-actin phalloidin in both types of filaments. polymerization. Moreover, profilin is a much more potent inhibitor of non-muscle actin polymerization than muscle actin

96a SundavLi "LA%444 T POSTERSA. %-, 0-0 JL - - %.-, 435-439I ., -.1 I .., .,

435-Pos Board # B291 employed to the full length molecule as well as large fragments FURTHER EXAMINATION OF THE EFFECT OF which have been expressed by use of an improved E.coli FESSELIN ON ACTIN POLYMERIZATION expression system for E-type Tmod. The C-terminal half represents a compact globular cooperatively melting domain Brent M Beall, Joseph M Chalovich; East Carolina University whereas the N-terminal half is elongated and has no definite School of Medicine, 600 Moye Blvd, Greenville, NC 27858 tertiary structure in solution. Upon binding to tropomyosin, Fesselin is a proline rich actin-binding protein found in smooth structure of this region may be altered. We propose a model for muscle (Leinweber et al., J. Musc. Res. Cell Motil., 1999) that has Tmod-tropomyosin-actin complex; the elongated N-terminal half some sequence similarity to another actin-binding protein of Tmod binds side by side to the N-terminal region of synaptopodin (Mundel et al., J. Cell Biol., 1997). Fesselin is tropomyosin and that the C-terminal half is protruded from the P- isolated as a pair of polypeptides of molecular weight 103 kDa and end being slightly bent towards (and may also interact with) the P- 79 kDa. We reported earlier that at low ionc strength (2 mM end actin, thereby blocks the P-end. Since the P-end capping may MgCI2, 5 mM Tris-HCI pH 7.8, 0.2 mM ATP) the mixture of be soft and dynamic in nature [Littlefield & Fowler (1998) Annu fesselin polypeptides accelerated smooth and skeletal muscle actin Rev Cell Dev Biol, 14: 487-525], the two pairs of interactions may polymerization, eliminated the lag phase of polymerization and cross-talk to each other. reduced the critical concentration of smooth muscle actin (Beall & Chalovich, Biophys. J. 238A, 2000). We have now extended these was 438-Pos Board # B294 observations. At the original conditions, actin polymerization STRUCTURE AND FUNCTION OF induced by adding sonicated F-actin "seeds". Actin HEADPIECE polymerization occurred rapidly, without a lag phase, just as it had DOMAINS done in the presence of fesselin. This suggests the possibility that Didem Vardar, Benjamin S. Frank, C. James McKnight; Boston fesselin accelerates actin nucleation. The addition of both fesselin University Sch. of Medicine, 700 Albany Street W427, Boston, and actin "seeds" resulted in a further increase in the MA 02118 polymerization rate. This is evidence that fesselin accelerates the Headpiece (HP) is a unique, modular F-actin recognition motif elongation step of actin polymerization. The two fesselin found at the C-terminus of larger "core" domains in a variety of polypeptide chains were separated by reverse phase HPLC. Both cytoskeletal proteins. There are over 25 HP-containing proteins of the resulting fesselin polypeptides accelerated actin identified in plants and animals. Despite their high sequence polymerization. The relationship between these two fesselin homology, not all HP domains bind F-actin. Here we report the polypeptides remains unclear. Finally, fesselin was observed to binding affinities for ten different HP constructs, studied by in retain its activity in the presence of 100 mM KCI. vitro actin sedimentation assays. Their binding constants can be grouped into low-micromolar, high-micromolar, and nonspecific 436-Pos Board # B292 categories. Circular dichroism, gel filtration chromatography, and REGULATION OF F-ACTIN ARCHITECTURE USING NMR studies indicate similar structures for these domains. SIMPLE MULTIVALENT IONS. Mapping residues identified in actin-binding on the high-affinity villin HP NMR structure, reveals a "crown" of alternating charges Gerard C. L. Wong', Alison Lin2, Jay X. Tang3, Youli Li2, Paul that separates a hydrophobic "cap", and a patch of positive charges A. Janmey4, Cyrus R. Safinya2; 'University of Illinois at Urbana- as the F-actin binding face. Computer modeling of other HP Champaign, Urbana, IL 61801, 2University of California at Santa of domains based on the HP67 structure, suggests significant changes Barbara, 3University of Indiana at Bloomington, 4University on the actin binding face for low-affinity and nonspecifically Pennsylvannia binding HP domains. It is proposed that the differences in the Intuitively, two like-charged macromolecules are expected to repel binding affinity are due to critical alterations on the surface charge one another. It is not surprising that the widely used Poisson- distribution of the identified binding face, rather than major Boltzmann mean-field theory predicts that two-like charged structural changes in the unique HP fold. These findings make the cylinders will always repel one another. In the presence of HP domain ideal for spatially and temporally regulated multivalent cations, however, many biopolymers actually attract cytoskeletal recognition. one another and condense into a compact state. F-actin is a rigid helical polyampholyte with a charge density -4e/nm, and a persistence length of -10 microns. We show that the global 439-Pos Board # B295 organization of F-actin filaments can be reversibly modulated PHOSPHORYLATION OF DEMATIN HEADPIECE between different architectures by using different concentrations of RESULTS IN A CONFORMATIONAL CHANGE IN A simple multivalent ions, without employing any architecture- DISTAL DYNAMIC LOOP specific actin-crosslinking proteins. We unambiguously Benjamin S Franke, Didem Vardar', Athar H Chishti2, C. James demonstrate the existence of two distinct phases: a liquid McKnight'; 'Boston University School of Medicine, 700 Albany crystalline network phase and a parallel bundled phase. Street, Boston, MA 02118, 2St. Elizabet's Medical Center, 736 Cambridge Street, Boston, MA 02135-2997 437-Pos Board # B293 Headpiece domains are modular, -75 amino-acid F-actin binding TROPOMODULIN: DISTINCT PROPERTIES OF N- AND motifs found at the C-termini of several families of actin- C-TERMINAL HALVES associated proteins. The NMR structure of human dematin Inna Krieger', Elizaveta headpiece (DHP) is similar to that of the previously determined for Alla Kostyukova', Tetsuro Fujisawa', villin headpiece and retains the hydrophobic "cap", alternating Tiktopulo2, Kayo Maeda', Yuichiro Maeda'; 'RIKEN Harima on the actin-binding Institute at SPring-8, 1-1-1 Kouto, Mikazuki, Sayo, Hyogo 679- charge "crown" and positive "patch" features 5148 Japan, 2Institute of Protein Research, RAS, Pushchino, face, as well a buried histidine and salt bridge. However, unlike Moscow Region 142292 Russian FederationTropomodulin (Tmod) villin or any other headpiece domain, DHP can be phosphorylated is the unique P-end capping protein of actin-tropomyosin filament. at a serine (S74) in vivo. This phosphorylation results in a loss of N- and C-terminal halves have distinct functional roles; the N- actin bundling, but not binding, by full length dematin in vitro. terminal half interacts with the N-terminal region of tropomyosin NMR chemical shift changes in phosphorylated DHP indicate that whereas the C-terminal half is required for capping activity. For there is little change in structure at the site of phosphorylation but systematic structural characterization of each half, limited large changes in the conformation occur in the variable length loop proteolysis, circular dichroism, differential scanning (V-loop, residues 20-28). The V-loop is far from the site of and small angle X-ray scattering have been phosphorylation in sequence, but close in space in the DHP NMR microcalorimetry structure. The structure of the V-loop in unphosphorylated DHP is

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not well defined and 15N-relaxation experiments demonstrate that 442-Pos Board # B298 it is dynamic and undergoing significant motions. The basic DETECTION OF THIOL NITROSYLATED PROTEINS IN residues within the DHP V-loop may form a salt bridge with the SMOOTH MUSCLE phosphate group on S74, structuring and ordering the dynamic V- Barbara D. Leinweber; University of Nevada, Reno, NV 89557 loop. Recent studies have identified a guanosine 3',5'-cyclic monophosphate (cGMP) -independent component of smooth 440-Pos Board # B296 muscle relaxation in response to nitric oxide donors. The INTERACTIONS OF CAPPING PROTEIN AND ARP2/3 mechanism of cGMP-independent relaxation may involve thiol COMPLEX WITH ACTIN FILAMENTS IN LIVING CELLS nitrosylation of one or more cellular constituents. An assay was STUDIED BY FLUORESCENCE CORRELATION developed to detect thiol nitrosylation of proteins in SPECTROSCOPY polyacrylamide gels. Nitric oxide released from protein thiols by Saveez Saffarian, Tetsuro Wakatsuki, Dorothy A. Schafer, Elliot 365 nm UV irradiation under aerobic conditions is capable of L. Elson; Washington University, I Brooksings Drive, CB 1105, oxidizing the non-fluorescent compound dihydrorhodamine to St. Louis, MO 63130 rhodamine. This reaction is selective for thiol nitrosylation. Capping Protein and the Arp2/3 complex are essential for Smooth muscle myosin subfragment I and actin incubated with the regulating the polymerization of actin filaments that drives nitric oxide donor nitrosoglutathione exhibited a fifty fold increase membrane protrusion during cell spreading and locomotion. in rhodamine fluorescence compared to control proteins treated Current models suggest that the Arp2/3 complex nucleates actin with glutathione. Significant increases in thiol nitrosylation were polymerization while the Capping Protein blocks polymerization also seen in strips of canine colon treated with 2.5 mM of filaments that are not properly disposed to drive membrane nitrosoglutathione. Myosin and actin, or proteins of the same protrusion. Our goal is to characterize the interactions of these molecular weight, were the predominate thiol nitrosylated proteins proteins with the actin cytoskeleton in different regions of active in smooth muscle. A protein which migrates with a-actinin is also cells, e.g., in the protruding lamellipodium and in the cell body, thiol nitrosylated. This study demonstrates thiol nitrosylation of using Fluorescence Correlation Spectroscopy (FCS). Using an specific proteins in smooth muscle. instrument that combines FCS measurements with scanning confocal microscopy (Zeiss Confocor II), we can perform FCS 443-Pos Board # B299 measurements at locations of interest identified in high-resolution DISSECTING PIP2-SENSITIVITY OF GELSOLIN confocal images of the cells. We have observed that capping Wujing Xian, Paul A. Janmey; University of Pennsylvania, 1080 protein and Arp2/3 complex, each fused with green fluorescent Vagelos Labs., Philadelphia, PA 19104-6383 and that the protein, undergo both fast and slow dynamic processes Gelsolin is an actin-binding protein that binds, caps and severs relative proportions of molecules participating in these processes actin filaments. Subsequent to severing, gelsolin binds actin varies substantially in different regions of a cell. Our working tightly (Kd-pM), yet phosphatidylinositol 4,5-bisphosphate (PIP2) hypothesis is that the slow components reflect interactions of the is capable of dissociating the actin-gelsolin complex, even though capping protein and Arp2/3 with actin filaments. the Kd for gelsolin-PLP2 is -pM. Our hypothesis is that PIP2 causes conformational changes at the PLP2-binding site(s), which in turn 441-Pos Board # B297 disrupt the actin-binding sites. Molecular dynamics calculations NEW FLUORESCENT PHALLOTOXIN CONJUGATES are performed to force a conformational change at one of the PIP2- WITH ABSORPTION AND EMISSION WAVELENGTHS binding sites, G(150-169), and the results show disruption of the BEYOND 630 NM. actin-binding sites, in agreement with the hypothesis. Next, the N- Hee Chol Kang, Walter K Metcalfe, Jason Kilgore, Richard P terminal PIP2-binding site G(150-169) is engineered onto the Haugland; Molecular Probes, Inc., P.O. Box 22010, Eugene, OR corresponding site in the homologous but normally PIP2- 97402-0469 insensitive C-terminal half. Conversely, this C-terminal site is Fluorescent phallotoxin conjugates have been widely used for engineered onto the site of G(150-169) in the N-terminal half. Both labeling F-actin in fixed and permeabilized cells due to their high the C- and N-terminal half mutants bind actin monomers. The C- actin specificity and low molecular weight. In fluorescence term mutant also binds PIP2, and its thermal stability is affected by microscopy of biological specimens, however, tissue PIP2 as well. It is possible that PIP2 induces partial unfolding of autofluorescence has often limited the usefulness of visible gelsolin, therefore disrupting the actin-binding sites. These results wavelength dyes. To allow improved signal detection, we have help define how gelsolin is regulated, and are encouraging first developed several new fluorescent phallotoxin conjugates with steps for engineering novel PIP2-sensitive proteins. dyes that absorb and emit at longer wavelengths where autofluorescence is not common. Alexa Fluor 633, 660 and 680 Microtubules & Microtubules-binding Proteins dyes have been used to prepare fluorescent phalloidin conjugates. The absorption maxima of Alexa Fluor 633, 660 and 680 444-Pos Board # B300 phalloidin conjugates are 632 nm, 663 nm and 679 nm, MICROTUBULE TREADMILLING IN VITRO respectively, and their emission maxima are 647 nm, 690 nm and INVESTIGATED BY FLUORESCENCE SPECKLE AND 702 nm, respectively. Due to the close match of the absorption CONFOCAL MICROSCOPY maximum of the Alexa Fluor 633 phalloidin to the 633 nm line of the He-Ne laser, this probe should be very useful for confocal laser Sonia Grego, Edward D Salmon; Univ North Carolina Chapel scanning miicroscopy. Preliminary studies indicate that these Hill, 607 Fordham Hall, CB#3280, Chapel Hill, NC 27599 fluorescent phalloidin conjugates label F-actin with high In vivo, mitotic spindle microtubules have been shown to exhibit specificity and strong fluorescence. The photostability of these treadmilling (termed flux) poleward, growing at plus and conjugates appears to be excellent, which is an important feature of disassembling at minus end; plus to minus treadmilling has also dyes used in confocal microscopy. The Alexa Fluor 633 phalloidin been observed during interphase (reviewed in 1997, Current Biol. produces a bright F-actin staining pattern that is easily 7:R369). Recently, Panda et al. (1999, P.N.A.S. 96:12459) using a distinguished from most other fluorophores. These far red emitting biochemical assays have reported treadmilling at a rate of 0.2 fluorescent phallotoxin conjugates are particularly suitable for Sum/min for pure tubulin MTs in 2% glycerol stabilizing buffer in multiple color labeling experiments. vitro and proposed that treadmilling is an intrinsic property of pure tubulin microtubules. To test this conclusion, we directly viewed the polymerization dynamics of individual microtubules assembled

98a R lv .uinLUAUII tn PCTv A A P('.TR.R.Ra W.Ji. .5. .LJA.i 54 AAA -AAG in vitro from porcine brain tubulin, using a 2% glycerol buffer to sites for MTs are arrayed within sleeves. The average penetration suppress dynamic instability. The fluorescence speckled of a MT into a sleeve is a function of load on the MT; larger microtubules were bound to the coverglass by kinesin motors and tensions shift the steady state position of the MT tip outwards the assembly dynamics of plus and minus ends were recorded with increasing the detachment probability. New MT tips enter the a spinning disc confocal fluorescence microscopy system. At sleeves by polymerization. When the tip of a polymerizing or steady state assembly some microtubules achieved treadmilling in depolymerizing MT lies within a sleeve, the kinetochore a plus to minus direction, others in a minus to plus direction due to experiences a force in the direction of MT tip movement. stochastic differences in dynamic instability between plus and Magnitude of polar ejection force on a chromosome depends on minus ends. The population as a whole exhibited very little net the spindle MT density, thus on the position of the chromosome growth in both directions (on average 0.01 lim/min). Supported by relative to the two poles. This force distribution biases the NIH GM24364 and GM60678. direction of movement of the kinetochores. At metaphase plate, chromosome oscillations are unbiased, the polar ejection forces 445-Pos Board # B301 being roughly equal. The model explains mitotic chromosome MERCURY AND CADMIUM EFFECTS ON movements without invoking elaborate sensing mechanisms (e.g. MICROTUBULE POLYMERISATION AND "smart kinetochore"). DEPOLYMERISATION 1 - Hill, T. (1985) Proc. Natl. Acad. Sci. USA, 82,44404-4408. Vlado Bujan, Stewart Yeung, Sanya Rushdi, James Delikatny, Supported by the Burroughs Wellcome Fund. Brett Hambly; University of Sydney, NSW 2006 Australia Mercury and cadmium are well known cytoskeletal toxic agents. 448-Pos Board # B304 However, the mechanism of toxicity is poorly understood. A major MICROTUBULES STATUS HAS NO EFFECT ON mechanism of mercury and cadmium toxicity may be due to their SHORTENING VELOCITY IN SINGLE PERMEABELIZED affinity for binding sulfydryls (Christie and Costa, 1984). Since VASCULAR SMOOTH MUSCLE. tubulin purified from mammalian brain has 12 and 8 cysteine R a Darl Swartz', Dahua Zhang', Jennifer Sherwood2; 'Indiana residues in its a and subunits, respectively, then quite complex University School of Medicine, 635 Barnhill Drive, Indianapolis, mechanisms of toxicity are expected. We have studied mercury IN 46202, 2Marquette University and cadmium effects on microtubule polymerisation and Microtubules have been shown to be involved in smooth muscle depolymerisation as a function of mercury and cadmium chloride contraction, possibly by serving as an intemal load which oppose concentration, protein concentration, the relative length of pre- contraction. To test this hypothesis, microtubule distribution and incubation time with GTP, mercury and cadmium chloride and the shortening velocity in freshly isolated single vascular smooth taxol using light scattering (turbidity) and transmission electron muscle cells (VSMCs) treated with microtubule modulating drugs microscopy. Our preliminary data show that microtubule were investigated. Inmuonocytochemical studies demonstrated polymerisation and depolymerisation is dependent on the that microtubules run mainly longitudinally in relaxed VSMCs. concentrations of protein and heavy metals. Effects of mercury They are oriented more obliquely, almost transversely to the long chloride are very sensitive to the relative length of pre-incubation axis of the cells after contraction, suggesting thatmicrotubules are time with GTP. It may indicate that toxicity of mercury chloride is compressed during shortening, and thus might impart a passive mediated via sulflhydryl group/s located in GTP binding pocket. internal load. Quantitative immunocytochemical analysis revealed Taxol recovers microtubule polymerisation significantly less than that, relative to the control group, colchicine (15 IAM) and GTP. The taxol-recovering effect does not depend on the nocodazole (15 ;LM) decreased the microtubule density by 40-60% taxol/mercury concentration ratio. while taxol (10 AM) increased the microtubule density by 46%. However, alteration of microtubule polymerization status by these 446-Pos Board # B302 microtubule-modulating drugs did not have a significant effect on ELECTROSTATIC CONTRIBUTIONS TO THE unloaded shortening velocity in permeabilized VSMCs. These STRUCTURE AND DYNAMICS OFMICROTUBULES results suggest that microtubules do not present an appreciable David Sept', Nathan Baker', Michael Holst2, J. Andrew internal load to dampen single VSMCs shortening in the present McCammon'; 'UC San Diego, 9500 Gilman Dr., La Jolla, CA experimental system. This work was done during the tenure of a 92093-0365, 2UCSD, 9500Gilman Dr., La Jolla, CA 92093 Fellowship from the American Heart Association, Midwest Microtubules are key components of the cytoskeleton and have Affiliate, Inc (#9910171Z). been the subject of intensive investigation for several decades. Two of the more interesting properties of microtubules are their 449-Pos Board # B305 helical, tube-like structure and their polymerization behavior EVIDENCE FOR INVOLVEMENT OF THE PKC-ALPHA (different rates at the + and - ends and dynamic instability). We AND RHOA/ROK PATHWAY IN STRETCH-INDUCED will present preliminary results which are aimed at elucidating the MYOGENIC TONE OF THE RABBIT BASILAR ARTERY underlying reasons for these different properties. Using very large- Kathleen G Morgan', Young-Ho Lee2; 'Boston Biomedical scale electrostatic calculations, we will show how electrostatic Research Institute, Yonsei University College of Medicine, C.P.O. interactions play a large role in determining microtubule structure Box 8044, Seoul, 120-752 and also affect polymerization at the two ends of the microtubule. We investigated the role of PKC- or RhoA proteins-induced Ca2+ sensitization in myogenic tone of the rabbit basilar artery by 447-Pos Board # B303 measuring Fura-2 signals, contractile responses, PKC A MODEL FOR DIRECTIONAL INSTABILITY DURING immunoblots, translocation of the PKC and RhoA proteins, and MITOTIC CHROMOSOME MOVEMENT phosphorylation of MLC20. Stretch evoked myogenic tone with Ajit Joglekar, Alan J. Hunt; University of Michigan, 300, N. increase in [Ca2+Ji only in the presence of extracellular Ca2+. Ingalls #952, Ann Arbor, MI 48109 Stretch-induced increase in (Ca2+]i & tensions were inhibited by We propose a stochastic model that predicts prometaphase treatment of tissue with nifedipine, but not ingadolinium. PKC chromosome movements resulting from motors coupled to inhibitors, H-7 & calphostin C, and Rho-kinase inhibitor, Y-27632, microtubule (MT) depolymerization at the kinetochores and inhibited a stretch-induced myogenic tone without changes in opposed polar ejection forces on the chromosome arms. The [Ca2+]i.Imunoblotting showed the presence of PKC-alpha and mechanism of force generation at kinetochores is an extension of a PKC-ipsilon in the basilar artery. PKC-alpha, but not PKC-ipsilon, thermodynamic model described by Hill (1). Kinetochore binding and RhoA proteins were translocated from cytosol to the cell

99a 449-454 POSTERS Sunday

membrane by stretch. Phosphorylation of MLC20 was increased 452-Pos Board # B308 by stretch and these increases were blocked by treatment of tissue NONLINEAR PREDICTION OF CORTICAL SYNAPTIC with H-7 & Y-27632. These results suggest a link between the RESPONSES TO COMPLEX STIMULI Ca2+ sensitization that occurs during the myogenic contraction Ingo C. Kleppe, Hugh P.C. Robinson; University of Cambridge, and activation of the PKC-alpha and RhoAlROK pathways. Downing St., Cambridge, CB2 3EG United Kingdom Support: HMP-00-B-21400-0057 (2000) Good Health R&D Central mammalian synapses show a large degree of response Project, Ministry of Health & Welfare, Korea. variability, but fluctuations at individual synapses are highly correlated to the short-term history of release. Therefore, the usual Neuronal Systems & Modeling approach of modelling synaptic function, by describing the trajectory of the ensemble average response, discards some of the 450-Pos Board # B306 dynamics and predictability of individual responses. Here we BRAIN CHIP: TRANSISTOR ARRAY FOR develop a general method, based on delay embedding of the EXTRACELLULAR RECORDING OF HIPPOCAMPAL sequences of both input spike intervals and output response SLICES amplitudes, which can be applied to construct predictive phase- Brigitte Juliane Besi, Peter Fromherz; Max-Planck-Institute for space models of the synaptic transfer function, for arbitrary timing Biochemistry, Am Klopferspitz 18a, Martinsried, 82152 of synaptic input. The method was characterized using surrogate data generated by a model of cortical short-term plasticity Until now field-effect transistors have been used to record action (Tsodyks & Markram, PNAS 94:719), with correlated or potentials from single nerve cells attached to the gate. For the first uncorrelated fluctuations. We show that this method measures the time it was now possible to measure the evoked activity in cultured correct number of underlying dynamical variables of the synaptic brain slices with transistors. model, provides optimal prediction of individual responses when We established the Gahwiler technique of rat brain slices on silicon fluctuations are correlated, that it quantifies the nonlinearity of the chips and cultured hippocampal slices of 5-7 day old Wistar rats on synapse, and that it identifies input patterns which produce reliable three different kinds of chips: responses. We demonstrate the application of this technique to data 1) A 4x4 array of 16 field-effect transistors (100 lsm spacing from rat cortical pyramidal neurons with synaptic inputs driven by between the gates) with a gate size of 10 itm x 80 ism (width x Poisson and other irregular processes. Supported by the BBSRC length), and opposite of the transistors 16 stimulation spots of 10 to (UK). 50 Am in diameter. 2) A linear array of 96 field-effect transistors with a gate size of 20 453-Pos Board # B309 jtm x 2 iLm and a distance between the gates of 22 Am. BURSTING IN CEREBELLAR GRANULE CELLS: 3) A linear array of 96 field-effect transistors with a gate size of EXPERIMENTS AND MODELING 2.6 lim x 1.8 ktm and a distance between tIh gates of 4.6 Am. Egidio D'Angelo, Thierry Nieus, Arianna Maffei, Simona Armano, By stimulating the Schaffer collaterals of the hippocampal culture Paola Rossi, Vanni Taglietti, Andrea Fontana, Giovanni Naldi; with an external tungsten electrode or with a stimulation spot University of Pavia (current pulses of 30 1tA to 60 AA, 100 As duration) we measured Neurons process information in a non-linear fashion generating local field potentials with all three chip types. The signals recorded oscillations, bursting and resonance enhancing responsiveness at had an amplitude in the range of 400 ltV to 3 mV. preferential frequencies. It has been proposed that slow The Transistor Arrays give the possibility to study the activity in repolarizing currents could be responsible both for oscillation/burst complete neuronal networks with high spatiotemporal resolution. termination, and for high-pass filtering causing resonance. However, different mechanisms including electrotonic effects, the 451-Pos Board # B307 expression of resurgent currents, and network feedback may also FM AND gS RESOLUTION IN MEMBRANE POTENTIAL be important. In this paper we have performed whole-cell patch- IMAGING REVEALS SEAL PROPERTIES OF CELLS AND clamp recordings from granule cells in acute rat cerebellar slices TISSUE IN 3D showing (1) theta-frequency (3-12 Hz) bursting and resonance and (2) a previously unidentified slow repolarizing K+ current (IK.ulOW) Dieter Braun, Peter Fromherz; Max-Planck Institute for A mathematical model based on Hodgkin- Huxley like equations Biochemistry, Am Klopferspitz 18A, Martinsried, D-82152 and implemented in the NEURON simulator (Hines) indicated that Voltage-sensitive fluorescence of passive membrane potentials of IK-SlOW was necessary for both bursting and resonance. A persistent cells which are stimulated from isolated planar electrodes has been (and potentially a resurgent) Na current exerted complex demonstrated recently by the authors with sub-jm and sub-Its amplifying actions on bursting and resonance, whereas electrotonic resolution by the use of either lock-in or transients averaging effects were excluded by the compact structure of the granule cell. techniques. The measurements on single cells match precisely the Theta-frequency bursting and resonance in granule cells may play predictions from a spacially resolved area contact model [1] and an important role in determining synchronization, rhythmicity, and are used to measure the seal resistance between cell and chip. As learning in the cerebellum. the thickness of this cleft is known from FLIC measurements [2], we can deduce its resistivity. We find that it is 4-fold enhanced for 454-Pos Board # B310 many mammalian cells as compared to the resistivity of the NS1619 MODULATES ACTION POTENTIAL FIRING IN medium. Transients of the membrane voltage are consistent with AFFERENT NEURONS FROM THE RAT DORSAL ROOT the lock-in measurements with time constants of 1-3,ss. A z-profile GANGLION. along the cell-cell contact within a group of cells proves a linear voltage drop along the z direction. In cell monolayers of HEK and Xu-Feng Zhang, Jianlin Feng, Rachel Davis-Taber, Victoria E. MDCK cells, both phase and transient signals show a seal Scott, Michael Coghlan, James P. Sullivan, Murali resistance of the cell-cell contact which is greatly enhanced for Gopalakirshnan, Char-Chang Shieh; Abbott Laboraotories MDCK cells [3] as expected from their tight junctions. The data NS1619 is a large-conductance Ca2+-activated K+ channel (BKca) results in a refined equivalent circuit for cell monolayers. opener with inhibitory effects on L-type Ca2' and voltage-gated K+ [1] Weis, Fromherz (1997) Phys.Rev.E 55, 877 channels. NS1619 has been shown to relax vascular and urinary bladder smooth muscles, however its effects on afferent neurons [2] Braun, Fromherz (1998) Phys.Rev.Lett. 81, 5241 remain unknown. To examine the effects of NS1619 in [3] Lo, Keese, Giaever (1995) Biophys.J. 69, 2800 modulating functional responses of afferent neurons, DRG neurons

lOOa Sundav POSTERS-POSTERSAL -4.445- 458

(L6-S 1 regions) from adult rats were enzymatically dissociated. Numerical simulations were performed following two scenarios: Membrane currents and action potentials were measured under one in which Ca_.er responds slowly to changes in Ca_cyt, and one normal physiological solutions using whole-cell voltage- and in which the Ca_er response is rapid. A current tail was predicted current-clamp, respectively. NS1619 (10 ,uM) increased whole-cell only in the case of the faster ER. Also, if the ER is emptied by a current amplitude by -20 % at 40 mV. These responses were SERCA pump blocker, the model predicts that Ca and current tails inhibited by 500 nM paxilline, indicating the activation of BKca should not be observed. These and additional numerical channels. In neurons with spontaneous firing activity, NS1619 simulations suggest the following: (1) the presence of Ca- and significantly suppressed (-80 %) the frequency of action potential current-tails following the pulse train indicate that the ER Ca firing by prolonging the refractory period. On the other hand, in concentration changes rapidly, (2) no tails are produced when the neurons where repetitive action potentials were elicited by ER is drained of Ca, and (3) the current tail is uncorrelated with the intracellular current injection, NS1619 reduced the firing fast bursting often seen in unclamped beta-cells. The last two frequency by -40%. These responses can be attributed to prolong predictions are supported by experimental data. the interspike interval by increasing the latency to action potential firing. These studies demonstrate that BKca channels play an 457-Pos Board # B313 important role in the modulation of activity in DRG neurons. A NOVEL METHOD FOR CORRECTING SPACE-CLAMP DISTORTED VOLTAGE-CLAMP RECORDINGS 455-Pos Board # B311 Andreas T. Schaefer, Bert Sakmann, Alon Kormgreen; MPI for INTERNAL ELECTRIC FIELD EFFECTS ON OUTER medical research, Jahnstr.29, Heidelberg, 69121 HAIR CELL ELECTROMOTILITY Incomplete space-clamp in voltage-clamp experiments leads to Robert M. Raphael', Aleksander S. Popell, William E. Brownell2; distortions of membrane currents rendering quantitative analysis 'Johns Hopkins University, 720 Rutland Ave., Baltimore, MD impossible. Here we present a simple numerical algorithm that 21205, 2Baylor College of Medicine, Houston, TX 77030 allows to correct these distortions. The method is based on A complete model of electromechanical coupling in biological stepwise estimating the underlying conductance by fitting membranes must consider not only the applied electric field but simulated to the experimentally recorded space-clamp distorted also the electric field within the membrane that results from clamp currents. We examine its potential for correcting space- intrinsic membrane dipoles. We have developed a new model of clamp-related errors using simulated potassium conductances that outer hair cell electromotility that explicitly considers the internal were inserted in either a long cable or in a detailed computer model electric field and calculates this field by the Lorentz local field of a neocortical pyramidal neuron. We show that the method relationship. The effect of the internal field manifests itself in enables accurate estimation of local densities and kinetics for all several experimental phenomena related to electromotility. channels tested not only in somatic but also in dendritic recordings. Application of tension to the membrane by increasing intracellular In addition, conductance gradients and conductance steps could be pressure shifts the voltage dependence of the nonlinear capacitance correctly retrieved to an accuracy of approx. 10% of the passive and reduces its magnitude: this effect is accounted for by a length constant without a priori assumptions about the channel reduction in the coupling between the applied and the internal distribution. Neither noise nor incomplete knowledge of passive field. Adsorption of salicylate to the membrane alters the intemal parameters significantly reduces the performance of the correction field and reduces the "effective" dipole moment of the voltage algorithm. The generality and robustness ofthe algorithm make it a sensor, thereby reducing electromotility. The voltage- and tension- useful tool for voltage-clamp analysis of most types of voltage- dependent diffusion of fluorescent probes in the membrane reflects gated currents independent of the recording location and the cell the altered internal electrical environment seen by the probe. morphology. Hence, consideration of the internal field unifies apparently unrelated experimental phenomena, and indicates that electromotility involves a voltage-induced change in internal Ligand-gated Channels membrane order that may be coupled to nanoscale membrane curvature. 458-Pos Board # B314 THE ROLE OF CA2*-ACTIVATED K CHANNELS IN THE 456-Pos Board # B312 PROLIFERATION OF PANCREATIC CARCINOMA UNMASKING ENDOPLASMIC RETICULUM CALCIUM CELLS. DYNAMICS IN PANCREATIC BETA-CELLS Katharina Ruff, Klaudia Giehl, Stephan Grissmer; University R. Bertram', A. Sherman2, P. B. Goforth3, M. Zhang3, L. S. Ulm, Albert-Einstein-Allee 11, Ulm, 89081 Satin3; 'Florida State Univ., Tallahassee, Florida 32306, 2NIH, Germany Bethesda, MD 20892, 3Medical Coll. of Virginia, Richmond, VA To find out whether ion channels might be important for the 23298 proliferation of pancreatic carcinoma cells we used the whole-cell recording mode of the patch-clamp technique in order to identify - We have used a voltage-clamp protocol with intracellular Ca in a first step- the functionally expressed ion channels in these measurements in mouse pancreatic beta-cells to investigate K(Ca) cells. In the BxPC-3 and MLAPaCa-2 pancreatic carcinoma cells, current and the influence on this current of Ca in the ER. The we could identify -500 functional K+ channels per cell. These protocol consists of depolarization to a plateau from which a train channels were observed using pipette solution buffering [Ca2+]i to of action potential-like depolarizations are made. Current rises I during the train and falls slowly after the train. Ca measurements pM, were voltage-independent, were blocked by CTX and indicate that the rise and fall of current correlate with Ca, blocked by Ba2 in a voltage-dependent manner indicating that suggesting that a K(Ca) current activates during the train and these channels belong to the clotrimazole sensitive Ca2+-activated slowly deactivates following the train, producing a current tail.

101a V111-IA -T..rN-AI1. Ir /. A %./U A A.IAfbl.3 3juiuic x

IKCa channels. The high expression of these IKCa channels desensitization. Our initial findings have shown some differences suggested a possible functional role for these channels in in VRl's properties when measured in this system versus what has proliferation. To test whether the proliferation of the carcinoma been reported in other preparations. cells depends on functional IKCa channels we measured the proliferation rate in MIAPaCa-2 cells in the absence (control) and 461-Pos Board # B317 presence of I FM clotrimazole. Cell number doubled about every CYCLIC-GMP-DEPENDENT ACTIVATION OF 24 h under control conditions. In the presence of 1 AM AQUAPORIN-1 ION CHANNELS. clotrimazole this doubling rate was reduced by more than 50 % Andrea J. Yool, Daniela Boassa; Depts. of Physiology and indicating that at least in MIAPaCa-2 cells functional IKCa Pharmacology and the Program in Neuroscience, University of channels are required for cell proliferation. Supported by grants Arizona College of Medicine, PO Box 24-5051, Tucson, AZ 85724 from the DFG (Gr848/8-1) and the BMBF (iZKF Ulm, B7). Aquaporins are mammalian water and solute channels with structural homology to potassium and cyclic-nucleotide-gated 459-Pos Board # B315 (CNG) channels. Sequence similarities exist between the carboxy CAPILLARY ELECTROPHORESIS AND PATCH CLAMP tail domains of Aquaporin-l (AQPI) and CNG channels (a-C DETECTION COUPLED TO PULSED FLOW region). Inside-out patches from oocytes expressing AQP1 SUPERFUSION showed cGMP-dependent activation of a large conductance non- Cecilia Farre, Andreas Sjdberg, Owe Orwar; Analytical selective cation channel (150 pS) that was not activated by cAMP, Chemistry, Kemivagen 10, Goteborg, SE-412 96 Sweden and not present in control oocytes. Two-electrode voltage clamp Techniques for identification of endogenous and synthetic ligands demonstrated dose-dependent activation with membrane-permeant interacting with ion channels is of central importance in cGMP agonists (8Br-cGMP or 8-pCPT-cGMP). The AQP1 understanding neuronal communication, as well as for screening conductance was inhibited by forskolin (10 jsM), suggesting an drug libraries. We present a pulsed-flow perfusion, patch-clamp antagonistic effect of cAMP. Ion substitution showed that the = > A detection scheme in capillary channel selectivity was K+ Cs' Na >> TEA' without ~-~ -'lelectrophoresis~ - - (CE-PC) where the appreciable anionic permeabiLity. Activation by cGMP was not nicotinic acetylcholine receptor inhibited by pretreatment of the oocytes with the kinase inhibitor agonists acetylcholine, carbachol and H7, suggesting that the channels were directly regulated by ligand nicotine were fractionated and binding rather than indirectly through kinase activity. An detected by a patch-clamped expanding role for aquaporins as ion channels, in addition to their 100 pA phaeochromcytoma (PC12) detector known function as water channels, has interesting implications for cell. Receptor ligands were detected the regulation of water and salt fluxes and potential transmembrane _ at physiologically relevant signaling in membranes expressing aquaporin channels. Supported W concentrations with high selectivity, by NIH ROI 59986. 202'5 ';5 using molecular recognition, and T~lonl high sensitivity from biological 462-Pos Board # B318 A & B: CCh 5 mM, Nic 0.1 mM amplification pathways. C: CCh 0.5 mM. Nic 0.01 mM ANION PERMEATION AND M2-DEPENDENT Desensitisation of ion channel SELECTIVITY OF MOD-1, A C. ELEGANS SEROTONIN- receptor responses is an abundant phenomenon, occurring at many GATED CHLORIDE CHANNEL ionotropic receptors with time scales ranging from ms to hours. To Claudine Menard1, Rajesh Ranganathan2, H. Robert Horvitz2, regain the high-conductance state of the acetylcholine receptors Stephen C Cannon'; 'Harvard Medical School, Mass Gen Hospital, during CE-PC detection, they were periodically resensitised by a Boston, MA 02114, 2MIT, Cambridge, MA 02139 pulsed flow of buffer, yielding a Gaussian-distributed ensemble of MOD-1 is a serotonin-gated channel recently cloned from c. peak-currents (fig, PC-trace B). Furthermore the pulsed-flow All known allowed for the resolution of carbachol and elegans. previously 5-HT-gated ionotropic receptors perfusion procedure (- (5-HT3aA,) are non-selective cation channels. MOD-1 is unique )-nicotine (fig, PC-trace B, C), which is impossible in traditional because it is highly permeable to chloride and impermeant to CE-PC agonist detection (fig, PC-trace A) due to the cations (Ranganathan et al. Nature 2000). We have further desensitisation of the receptor response. Also, acetylcholine was characterized the pore of MOD-1 by determining the selectivity detected in a crude biological sample of -200 lysed PC12 cells. sequence for anions and exploring the consequences of mutations in the M2 segment. MOD-1 was expressed in Xenopus oocytes 460-Pos Board # B316 and currents activated by 5-HT (1 pM) were measured by two- CHARACTERIZATION OF THE CLONED VANILLOID electrode voltage clamp. Shifts in the reversal potential caused by RECEPTOR, VRI, IN EXCISED PATCHES FROM a complete replacement of the extracellular anion revealed a XENOPUS OOCYTES permeability sequence of: SCN > Br' r > Cl >> F > Asp'>> Michael James Richards, Sharona E Gordon; University of Gluc'. This rank order is identical to that of GABAA and Gly Washington, 1959 NE Pacific Street, Box 356485, Seattle, channels and suggests a weak electrostatic interaction with binding Washington 98195-6485 sites on the pore. Three pore-lining rings of negative charge The cloned vanilloid receptor subtype 1 (VRI) is a nonselective contribute to cation permeability in nAChR and 5-HT3R channels. cation channel that is activated by the binding of capsaicin and MOD-1, like GABAAR and GlyR, has a positive charge at the other vanilloid-containing compounds. With the accumulating external ring and an Ala at the intermediate ring. Mutating this evidence suggesting that VRI is the integrator of multiple noxious Ala270 in MOD-1 to Glu (combined with APro269 and stimuli in the nociceptive pathway, a great deal of attention has Thr283Val) abolished the selectivity for Cl' over Gluc' (Pci =Pgiuc) been focused on this channel. However, little information and increased the permeability to K+ but not Na+. The effects of regarding VR1's basic properties when isolated of other cellular substituting additional negative charge at the external and internal components is available. Thus, we have studied the functional rings (both Lys.-4Asp) will be presented as well. These studies properties of VR1 in a reduced system - excised patches from indicate the M2 region of MOD-1 shares determinants for Xenopus oocytes. We examined such properties as the dose- anion/cation selectivity in common with other ligand-gated ion response relation for various ligands, voltage dependence, and channels.

102a Sundav- I POSTERS 463-4671

463-Pos Board # B319 Transient kinetic measurements, using the laser-pulse photolysis CHARACTERIZATION OF THE OUTER PORE REGION technique ', are consistent with a mechanism in which inhibitors OF THE APAMIN-SENSITIVE CA2+-ACTIVATED (MK-801 and cocaine) bind to an allosteric site with higher affinity K+ CHANNEL, SK2. for the closed than the open-channel form, and thereby shift the Heike Jager, Stephan Grissmer; University Ulm, Albert-Einstein- channel-opening equilibrium towards the closed-channel form 1. Allee 11, Ulm, 89081 The mechanism indicates ligands may exist that bind with higher Germany to the than to the closed-channel In order to define the structure of affinity open-channel forn form, the outer vestibule of small do not inhibit the receptor, but do prevent inhibitors from binding. Ca2+-activated K channels we have studied the interaction Based we between and different toxins. Directed the on the mechanism of receptor inhibition have found both SK2 peptide by results combinatorially synthesized RNA ligands2 and a cocaine of toxin on receptor the Shaker K+ channel, we used side-directed metabolite that alleviate cocaine inhibition. mutagenesis to change residues in the outer pore of the SK2 K+ channel that should render it sensitive to peptide toxins other 'Hess, G. P. & Grewer, C. (1998) Meth. Enzymol. 291, 443. than apamin or scyllatoxin (ScTX). From Shaker K+ channels and 2Hess, G. P. et al. (2000) Proc. Natl. Acad. Sci. USA. *Supported IK channels it was known that small side chains at SK2 position by an NIH-NS grant (G. P. H.) and AHA fellowship (A.G.) * Send 342 (Shaker 425, KesA 58, Kvl.3 380) and negative charges at enquiries to [email protected] SK2 position 344 and 348 were favorable to confer CTX sensitivity. Therefore we changed the amino acids at position 342, 466-Pos Board # B322 344 and 348 of SK2 to amino acids expected to improve CTX ANALYSIS OF HUMAN MUSCLE NICOTINIC RECEPTOR sensitivity even if SK2 wild type is not CTX sensitive. FUNCTION BY A DIRECT MAXIMUM LIKELIHOOD Interestingly, SK2 V342G, SK2 S344E and SK2 G348D became METHOD (HJCFIT) CTX sensitive and were also sensitive to KTX while ScTX David Colquhoun', Alan G Hawkes2; 'University College London sensitivity was reduced. Because the exchange of single amino (UCL), Gower Street, London, WCIE 6BT United Kingdom, acid residues can create new high affinity binding sites for CTX 2University College of Swansea, Singleton Park, Swansea, SA2 we conclude that there are minor structural differences between the 8PP United Kingdom outer vestibule of SK2 channels and eukaryotic voltage-gated In order to understand the function of normal receptors, and the K+channels like Shaker or Kvl.3 and the prokaryotic effect of mutations on them, it is necessary to estimate the rate KcsA channels. Supported by grants from the DFG (Gr 848/4-2) constants for binding of ligands, and for the conformation changes and the BMBF (iZKF Ulm B7). of the receptor. This requires us to postulate a physically (i.e. structurally) realistic reaction mechanism. To maximise the 464-Pos Board # B320 likelihood of a sequence of observed openings and shuttings, in NONHALOGENATED ALKANE ANESTHETICS FAIL TO which short events are missed, we use the exact solution to the POTENTIATE AGONIST ACTIONS ON TWO LIGAND- missed events problem (Hawkes, Jalali & Colquhoun -see GATED ION CHANNELS AT CLINICALLY RELEVANT Colquhoun et al., 1996 Phil. Trans. Roy. Soc. A 354, 2555). We CONCENTRATIONS have found that a limitation of such analyses is the statistical Douglas E Raines, Robert J Claycomb, Michaela Scheller, Stuart correlation between the estimates of the channel opening rate, 3, A Forman; Massachusetts General Hospital, 32 Fruit Street, and the shutting rate, a. The correlation coefficient between these Boston, Massachusetts 02114 estimates is often greater than 0.9, found from the off-diagonals of Although ether, alcohol, and halogenated alkane anesthetics the Hessian matrix of the likelihood evaluated at its maximum. potentiate agonist actions on ligand-gated ion channels (LGICs), There is a corresponding diagonal ridge in the likelihood surface. the effects of nonhalogenated alkane anesthetics have not been In physical terms this corresponds to the difficulty in reported. In this study, the authors used sequential niixing distinguishing between long openings with few interruptions stopped-flow fluorescence spectroscopy and patch clamp (small a, 1) and many shorter openings separated by very short electrophysiological techniques to assess the effects of shuttings (large a, ,). This places a limit on our ability to solve cyclopropane and butane on the apparent agonist affinities of the binding-gating problem, which can probably be overcome only Torpedo nicotinic acetylcholine receptors (nAcChoRs) in native by achieving quieter recordings. membranes and GABAARs expressed in HEK 293 cells. Neither cyclopropane nor butane increased the nAcChoR's apparent 467-Pos Board # B323 agonist affinity at clinically relevant concentrations. At a UNNATURAL AMINO ACIID MUTAGENESIS concentration equivalent to twice that required for anesthesia (2 COMPLEMENTS PHARMACOLOGY: DIFFERENCES IN MAC), neither cyclopropane nor butane enhanced GABAAR NICOTINE AND ACETYLCHOLINE BINDING TO THE currents induced by low concentrations of GABA or increased the NACHR GABAARs apparent agonist affinity. These results suggest that the Gabriel S. Brandt', Wenge Zhong2, Niki M. Zacharias', Caroline anesthetic actions of nonhalogenated alkane anesthetics do not result from M. Gibbs', Dennis A. Dougherty', Henry A. Lester3; 'Caltech, agonist potentiation of LGICs and imply that other Division of Chemistry 164-30, 2Scripps Research Institute, molecular mechanisms account for their abilities to induce anesthesia. Chemistry Department BCC-483, La Jolla, CA 92037, 3Division of Biology 156-29, Caltech, Pasadena, CA 91125 465-Pos Board # B321 Ligand-gated ion channels open in response to the binding of small NEW molecules. In the absence of three-dimensional structural INHIBITION MECHANISM FOR THE NICOTINIC information, the details of binding must be inferred from functional ACETYLCHOLINE RECEPTOR BASED ON TRANSIENT KINETICS AND studies. Traditionally, these experiments have involved varying COMBINATORIAL SYNTHESIS the structure of the ligands and investigating the response of the George P. Hess, Armanda M. Gameiro, Yongli Chen; Cornell channel. Here, this pharmacological approach has been expanded University, 216 Biotechnology Building, Ithaca, NY 14853-2703 to include simultaneous variation of both ligand and receptor. The Nicotinic acetylcholine receptors play a central role in signal nicotinic acetylcholine receptor relies on a number of aromatic transmission in the nervous system. They are inhibited by both residues to bind its positively charged agonists, attracting them to therapeutic agents and abused drugs. Electrophysiological the binding pocket via cation-7i interactions. To assay for this techniques indicated that receptor inhibition involves an entry of interaction, a series of fluorinated tryptophan residues has been inhibitors into the open receptor-channel, thereby blocking it. site-specifically introduced into the receptor, using unnatural

103a 467-471I . I - -POSTERS- ., - -.0- I%..-, %j.qiin,ri,vuiiua v

amino acid mutagenesis in Xenopusoocytes. At the same time, the Taken together, the results suggest that T339 and S340 interact ligand itself has been modified at its cationic center. Dose- with monovalent cations and calcium as these ions pass through response studies of a series of quaternary and tertiary ammonium the channel pore. compounds were carried out on modified receptors. While acetylcholine experiences a very close-range interaction with the t 470-Pos Board # B326 electrons of Trp149 in the alpha subunit of the receptor, nicotine does not. FUNCTIONAL CHARACTERIZATION OF RAT P2X7- Interestingly, a tertiary analog of acetylcholine retains EGFP FUSION PROTEINS the interaction with Trpl49, while a quaternary analog of nicotine does not acquire the ability to bind the Megan L Smart, David A Williams, David N Bowser, Rekha G typtophan. Panchal, Steven Petrou; University of Melbourne, Victoria 3010 468-Pos Board # B324 Australia [Hi]o ACCELERATES SIGNALING IN A3/B4 NEURONAL The cytolytic P2X7 purinoceptor is an ATP-gated channel that can NACHRS STABLY TRANSFECTED IN HEK 293 CELLS undergo conversion to a non-selective pore (Petrou et al 1997). As J. a prelude to future studies we characterized properties of N and C- Galya Abdrakhmanova, Dorfman, Y. Xiao, M. Morad; terminal EGFP fusions of the rat P2X7 receptor (rP2X7R). Fusion Georgetown University, 4000 Reservoir Road, Washington, DC 20007 constructs were made using Cl (EGFP-rP2X7R) or NI (rP2X7R- EGFP) EGFP fusion vectors (Clontech). ATP or BzATP were Despite vast structure/function and pharmacological literature on applied with ethidium bromide and emissions of EGFP and neuronal nAChRs, little is known of their pH regulation. Here we ethidium were imaged on a confocal microscope. cRNA encoding examine the effect of rapid changes in [H+io on the a3/(34 nAChRs. the fusion and wild-type receptors was injected into Xenopus Acidification increased the amplitude of the current induced by oocytes and electrophysiological properties were compared. nicotine (Hill coefficient-1.5), cytosine, ACh or carbachol, but did Transfection of rP2X7R-EGFP into HEK-293 and COS-7 cells not alter its reversal potential. Quantification of the currents at showed discrete membrane localization. EGFP-rP2X7R different nicotine and proton concentrations, suggested that the distribution was diffuse in COS-7 and membrane delimited in fraction of the current enhanced by e.g. pH 6 was larger at lower HEK-293 cells. The time course and extent of ethidium uptake in nicotine concentrations. Decreasing pH accelerated the decay cells expressing fusion constructs were identical to that seen in kinetics of both nicotine- and cytosine-evoked currents, but not cells expressing rP2X7R alone and no ethidium uptake was seen in that of ACh or carbachol, suggesting pKa-dependence of agonist control cells. Electrophysiological studies revealed that EGFP effect. Increased [H`]i did not alter the effect of [Hi]0 on the fusion did not affect ATP-elicited currents. In conclusion, channel current. Changes of pH from 7.4 to 6 during the activation of and pore properties of the rP2X7R and EGFP fusions are identical nicotinic current increased the current and accelerated its decay. to rP2X7R, making these EGFP fusions a powerful tool for Acidification also accelerated the decay kinetics of a "rebound" analysis of pore formation. current, recorded on withdrawal of nicotine. Our data suggest that Petrou et al. (1997) FEBS Letters 411 339-345. protons interact with multiple extracellular sites on a3/J4 nAChRs, increasing their affinity to the agonist. Nicotine appears also to act as a permeant channel blocker thus enhancing the decay kinetics of 471-Pos Board # B327 the current. We speculate that co-release of protons with ACh from CALCIUM OVERLOAD OF DYSTROPHIC MUSCLE MAY the secretory vesicles may produce accelerated transmitter DEPEND ON THE HIGH EXPRESSION LEVEL OF P2X7 signaling with minimal desensitization of the receptor. RECEPTOR. Romeo Bettol, Dorianna Sandoni2, Elena Tarricone2, Tiziana 469-Pos Board # B325 Martinello2, Laura Morbiato2, Donatella Birall; 'C.N.R., Viale POLAR RESIDUES OF TMD2 INFLUENCE IONIC Giuseppe Colombo 3, Padova, 35121 Italy, 2University of Padova, PERMEABILITY OF RECOMBINANT ATP-GATED P2X2 Viale Giuseppe Colombo 3, Padova, 35121 Italy RECEPTORS. We recently reported that a-sarcoglycan is an ecto-ATPase (Betto Keisuke Mgita, William Haines, Mark Voigt, Terrance M. Egan; et al., 1999). These enzymes control the concentration and Saint Louis University consequently the extracellular action of ATP, which, in turn, Scanning cysteine mutagenesis suggests that the side chains of modulates many cellular activities through P2 receptors. P2X4 and and residues of the second P2X7 receptors, ligand-gated cationic channels, are expressed in charged polar transmembrane domain skeletal muscles. When stimulated by ATP, P2X receptors became (TMD2) of ATP-gated P2X2 receptors face a water-accessible surface of the ion channel We measured the effect of site- permeable to Ca2@,Na' and K+.BzATP, a specific agonist of P2X7 pore. receptor, elicited intracellular calcium transients in cultured mouse directed mutagenesis within this domain on ionic permeability ratios determined from reversal FDB fibers. The amplitude of calcium transients decreased during potentials obtained under bi-ionic postnatal development, paralleled by the progressive reduction of conditions. Substitution of for D315 or no asparagine D349 had P2X7 receptor. Diversely, BzATP evoked in mdx fibers effect on PCaPCs suggesting that the negative charges at these large are not critical for calcium calcium transients even in adult fibers, where the expression of positions entry. By contrast, mutations P2X7 that increased either the hydrophobicity and/or side-chain volume receptor was high. In differentiating C2C12 cells, the of residues within TMD2 reduced The appearance of a-sarcoglycan is accompanied by increasing ecto- polar Pc./Pc,. biggest ATPase activity, inhibited by an antibody to the reductions occured when T339 or S340 were extracellular mutated to tyrosine; domain of In these mutants were to calcium. a-sarcoglycan. addition, down-regulation of a- largely impermeable Monovalent sarcoglycan expression reduces ecto-ATPase activity of transfected cation permeabilty sequences of WT and mutant receptors were also determined. The of the WT cells. The lack of a-sarcoglycan ectoATPase activity and the high permeability sequence receptor level of P2X7 receptor in dystrophic muscle lead to the was defined. and K+ were may poorly Na+, Cs+, Rb+, almost equi- uncontrolled stimulation of the permeant Li' was receptor and to calcium overload although perneability high. By contrast, both and cell death. Funded by Telethon Italy and CNR. T339Y and S340Y had the same clearly defined permeabilty sequence of Cs+>Rb+>W>Li+>Ne>TEA+>NMDG+.

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472-Pos Board # B328 474-Pos Board # B330 DIFFERENT ACTIVATING BINDING SITES FOR ATP AT COMPARISON OF MECHANISM AND SUBUNIT THE HUMAN P2X7 RECEPTOR. SPECIFICITY FOR TWO NMDA RECEPTOR PUTATIVE Manuela Klapperstuckl, Cora BUttner2, Gunther Schmalzing3, PARTIAL AGONISTS: ACPC AND D-CYCLOSERINE Fritz Markwardt'; 'JB Inst. f. Physiology, Magdeburger Str. 6, Anton Sheinin, Sara Shavit, Morris Benveniste; Tel Aviv Halle (Saale), D-06097 Germany, 2Pharmacol. Inst. f. Naturwiss., University Marie-Curie-Str. 9, Frankfurt (Main), D-60439 Germany, 3Inst. f. Pharmacological properties of partial agonists at the glycine Exp. Clin. Pharmacol., Hermann-Herder-Str. 5, Freiburg, D-79104 binding site of the N-methyl-D-aspartate (NMDA) receptor Germany established in vitro do not correlate well with their ability to In human B lymphocytes, the expression of an ATP"4-gated cation protect against neurotoxicity in vivo. This lack of good correlation channel has been shown by patch clamp measurements. In contrast may result from different mechanisms of action and subunit to the cloned P2X7, purinoceptors in B-lymphocytes rapidly specificity. Utilizing two-electrode voltage clamp on Xenopus deactivate in extracellular solutions with low Ca2+ after removal of oocytes, pharmacological properties of two putative partial the agonist and are not permeabilized to molecules as large as agonists, I-aminocyclopropanecarboxylic acid (ACPC) and D- 629 Da. To examine whether human B lymphocytes express a cycloserine (DCS) have been examined. ACPC dose-response distinct P2X subtype, RT-PCT was performed and revealed the curves analyzed for NR1I/NR2A, NRI/NR2B and NRI/NR2C presence of P2X7. Oocytes injected with P2X7 cRNA expressed a heteromeric channels were biphasic suggesting both agonist and cation current that was evoked by ATP4-. In contrast to the native antagonist actions of ACPC. NRlINR2B and NRI/NR2C lymphocytic purinoceptor, the activation by ATP4- was best containing channels had overlapping ACPC dose-response curves described by the sum of a monoexponentially and a linearly with a peak efficacy of -80% in comparison to glycine controls, increasing component. The deactivation was best approximated by but NRI/NR2A containing channels had a significantly different the sum of two exponentials. The detailed concentration-response dose response curve with an efficacy of only -60%. In contrast to analysis revealed two ATP4- binding sites for the different ACPC, DCS dose-response curves for NRI/NR2A and NR1/NR2B components at about 10 and 500 jiM, respectively. The slowly containing channels had efficacies for DCS of 35 - 68% of glycine deactivating component and one part of the exponentially controls, but NR1I/NR2C containing channels had efficacies greater activating component were activated at low concentrations of than glycine controls (192%). The reduced efficacy of DCS results ATP4-. A second part of the exponentially activating component, solely through its interaction with the glycine binding site. The the linearly activating and the fast deactivating component were efficacy of DCS for NR2A or NR2B-containing channels was pH activated at high concentrations of ATP4- only. Truncation of the sensitive, but not for channels containing NR2C. We suggest that C-terminus of the hP2X7-receptor abolished the effects of ATP at the deprotonated form of DCS may act as an agonist and the high concentrations. protonated form as a competitive antagonist at the glycine binding site for NR2A and NR2B containing channels. 473-Pos Board # B329 IDENTIFICATION OF AMINO ACID RESIDUES 475-Pos Board # B331 CONTRIBUTING TO THE ATP BINDING SITE OF A A MILLISECOND TIMESCALE FOR UNBLOCKING OF PURINERGIC P2X RECEPTOR NMDA RECEPTORS AFTER DEPOLARIZATION. Lin-hua Jiang', Francois Rassendren2, Annmarie Surprenant', R Mariana Vargas-Caballero, Hugh P.C. Robinson; University of Alan North'; 'Institute of Molecular Physiology, University of Cambridge, Downing Street, Cambridge, Cambridgeshire CB2 Sheffield, 2Institut de genetique humaine, 141rue de la Cardonille, 3EG United Kingdom Montpellier, 34396 France Synaptic NMDA receptors (NMDARs) in central neurons can be P2X receptor subunits have intracellular N- and C-termini, two activated for 100 ms or longer in a synaptic event, but experience a membrane-spanning domains, and an extracellular loop of about voltage-dependent block by extracellular Mg at the resting 280 amino acids. We expressed the rat P2X2 receptor in human potential. To assess the role of activated NMDARs in contributing embryonic kidney cells, and used alanine-scanning mutagenesis on to the upstroke of action potentials, it is essential to know how 30 residues with polar side-chains conserved among the seven rat quickly they are freed from Mg block during depolarization. We P2X receptor subunits. This identified a region proximal to the have measured the kinetics of unblock and block of single-channel first transmembrane domain which contained two lysine residues and whole-cell NMDAR currents in rat cortical pyramidal neurons that were critical for the action of ATP (K69 and K71). We (P5-12 slices) during voltage steps. Following steps from -60 mV substituted cysteines in this region (D57 to 71) and found that for to -30 or -20 mV, at which peak inward current through NMDARs S65C and 167C ATP-evoked currents were inhibited by is expected, the time constant of NMDA current activation was =5 methanethiosulfonates. At 167C, the inhibition by negatively ms at room temperature, while block upon repolarization was charged ethylsulphonate and pentylsulphonate derivatives could be faster than I ms. This could be explained if unblock involves a overcome by increasing the ATP concentration, consistent with a return to the open state of the channel along a chain of Mg-bound reduced affinity of ATP binding. The inhibitory action of the blocked states, while block only requires passage to the first of methanethiosulphonates was prevented by pre-exposure to ATP, these states. Simulations of spiking pyramidal neurons showed that suggesting occlusion of the binding site. Finally, introduction of time-dependent unblock of NMDARs greatly limits their negative charges into the receptor by mutagenesis at this position contribution to inward current during the upstroke of isolated (167E and 167D) also gave receptors in which the ATP action potentials, and plays a significant role in shaping burst concentration-response curve was right-shifted. The results suggest responses to synaptic input. Supported by the EC and the BBSRC that residues close to 167 contribute to the ATP binding site. (UK). 476-Pos Board # B332 COOPERATIVE GATING OF AMPA RECEPTOR CHANNELS: A MODEL FOR SYNAPTIC STRENGTHENING Kandiah Manivannani, Thirumalini Subramaniam2, Vishnu Suppiramaniam2; 'Southwest Missouri State University,

105a 476-480 POSTERS Sunday

Springfield, MO 65804, 2Tuskegee University, Tuskegee, AL anesthetics and neurosteroids). We have investigated the effect of 36088 the composition of the bilayer on the structure of the inhibitory Alteration of AMPA receptor channel kinetics has been implicated glycine receptor (GlyR), a paradigmatic member of the in the expression of long term potentiation (LTP). Recently, superfamily of ligand-gated ion channels which also includes AMPA receptors have been shown to cluster at the postsynaptic receptors for acetylcholine, serotonin and GABA. Homomeric domain during tetanic stimulation. Our recent findings suggest that human al GlyR was purified from baculovirus-infected insect the close proximity of AMPA receptors due to clustering may give cells. Reconstitution of the purified GlyR into small unilamellar rise to lateral interactions between them leading to cooperative vesicles by gel filtration provides the opportunity to manipulate the channel gating (Suppiramaniam et al., J. Neurochem. 74:S50A, vesicle composition and fluidity. We examined the role of the 2000). We have utilized purified and immunoprecipitated AMPA various parameters of the bilayer (i.e. phosphoryl head group, receptors reconstituted in lipid bilayers to demonstrate such cholesterol content, temperature) on GlyR structure as monitored interactions in the presence of proteoglycan components that are by circular dichroism (CD). For select phospholipids, the spectra endogenous to synapse and a cognitive enhancer ampakine. of the GlyR in small unilamellar vesicles were virtually However, the mechanism of induction of cooperative channel superimposable. However, a dramatic shift in the CD spectra of gating behavior of AMPA receptors by these molecules is not well GlyR was observed upon addition of cholesterol to these bilayers, understood. To characterize channel cooperativity, we utilize a indicating that the structure of GlyR is affected by the cholesterol simple steady-state model based on lateral interactions among concentration in the bilayer. channels (Manivannan et al., Biophys. J. 61:216, 1992 and Bull. of Math. Biol. 58(1): 141, 1996). This model involves just two 479-Pos Board # B335 parameters, one for single channel open probability and the other POSITIVE ALLOSTERIC MODULATION OF UV for "strength of interaction". Our results show that the endogenous IRRADIATION ON GABAA BUT NOT GABAC components and ampakine strongly enhance the interactive RECEPTORS EXPRESSED IN XENOPUS OOCYTES channel gating of AMPA receptors. These findings indicate that Yongchang Chang, Yi Xie, David S. Weiss; University of cooperative channel gating among AMPA receptors may be a Alabama at Birmingham, 1719 Sixth Ave S, Birmingham, AL potential mechanism for synaptic strengthening. 35294-0021 (Supported by a NIH grant NS 02018 to V.S. and a SMSU Faculty GABAA and GABAc receptors are both GABA-gated ion Research Grant to K.M.) channels, but they pocess distinct pharmacological properties. For example, GABAA receptors can be allosterically modulated by 477-Pos Board # B333 benzodiazapines, barbiturates, neurosteroids, and bicuculline, L-TYPE CA+ CHANNEL BLOCKERS DIRECTLY INHIBIT while GABAc receptors are insensitive to these compounds. In this GABAA RECEPTORS. study, we observed that UV irradiation also has differential effects Paromita Das, Cathy L Bell-Homer, Glenn H Dillon; U N Texas on recombinant GABAA and GABAc receptors. al(2, (2y2, Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX al2y2 (GABAA) or pl (GABAc) receptors were expressed in 76107 Xenopus oocytes. Dose-response relationship for each receptor was The al( 02y2 GABAA receptor is an anion channel belonging to the examined before and after 312 nm UV irradiation (1 min) using a superfamily of ligand gated ion channels (LGIC). Members of this transluminator. A two to 4-fold decrease in EC50 values for superfamily are structurally similar and often share functional al2, j2y2, and a1lo2y2 GABA receptors was observed upon UV characteristics. Previous studies have demonstrated that L-type irradiation. Furthermore, the dose-response relationship of acl2y2 Ca++ channel (Ca-L) antagonists block the 5HT3A receptor, a GABA receptors after UV irradiation can be further shifted to the cation-selective member of the LGIC superfamily. In the present left by diazepam indicating that UV and diazepam have an additive study, we tested the possibility that this class of drugs also inhibits effect. At the time this abstract was written, the authors were the anion-selective GABAA receptor. Whole-cell patch clamp absolutely clueless as to the potential mechanism. (NS35291 and recordings were obtained from human embryonic kidney cells NS36195) stably expressing rat axl 12y2 GABAA receptors. Cl-currents generated by Sm GABA were blocked by simultaneous 480-Pos Board # B336 application of nitrendipine, a dihydropyridine Ca-L antagonist. SPONTANEOUS OPENING OF THE RHOl GABA-C The block was concentration-dependent (IC5o approximately 50 RECEPTOR WITH A POINT MUTATION IN THE AMINO IiM), voltage-independent, and predominantly due to enhancement TERMINAL DOMAIN. of current decay. Nifedipine, the prototypical dihydropyridine, and Viviana I Torres, David S Weiss; Univ. Alabama at Birmingham, verapamil, a non-dihydropyridine Ca-L blocker, had similar 1719 Sixth Ave S, Birmingham, Al 35294 inhibitory effects. The inhibition was not blocked by the Recently, it has been described that a tyrosine residue in the amino benzodiazepine (BZ) antagonist flumazenil, demonstrating that the terminus of the -y-aminobutyric acid type A receptor 12 subunit site of interaction of the Ca-L blockers is distinct from the BZ site (Y62) is involved in high affinity agonist binding. In order to study of the receptor. Our data demonstrate that, in addition to blocking the role that this residue plays in the GABA-C receptor we have cation-selective LGICs, Ca-L blockers directly inhibit at least one mutated the corresponding tyrosine residue in the rhol subunit to anion-selective channel (Support: NIH ES07904 and AHA). serine (Y102S) or phenylalanine (Y102F) and expressed the receptors in Xenopus laevis oocytes. The Y102S mutant showed a 478-Pos Board # B334 very high leak current that was inhibited by picrotoxin EFFECT OF LIPID COMPOSITION ON THE STRUCTURE (ICso=0.27pM) and had an EC50 for GABA-induced activation OF THE GLYCINE RECEPTOR. 174-fold higher than the wild type. The ECso for the Y102F Michael Cascio', John F. Leite', Robert 0. Fox2; 'University of mutant was not significantly different from the wild type receptor. Pittsburgh School of Medicine, Pittsburgh, PA 15219, 2Univ. of These results suggest that replacement of this tyrosine by serine in Texas Medical Branch at Galveston, Sealy Ctr. for Struct. Biol., the rhol subunit might cause spontaneous opening of the receptor Galveston, TX 77555-0647 by mimicking the conformational change induced by GABA. The role of cholesterol in bilayer structure and fluidity is well Furthermore, the much lower ECso of the Y102S mutant for understood. It is also becoming progressively evident that lipid GABA suggests that this change might reduce the affinity by composition has an important role in the function of membrane perturbing the binding site. Supported by NIH Grant N535291 proteins and the pharmacological effects of certain drugs (e.g. (D.S.W)

106a APO.RTR.R.W U.7 A iLaj.,%bi -Tl A21 ARI-ARA -JAA.IOULILMY ~AbF,Jm I 1p * rj

481-Pos Board # B337 483-Pos Board # B339 EVIDENCE FOR CONCENTRATION-DEPENDENT TWO MUTATIONS IN THE GABAAR PORE DOMAIN SUBSTATE GATING IN THE DECAY OF FAST SELECTIVELY ALTER DEACTIVATION AND EXCITATORY POSTSYNAPTIC CURRENTS. DESENSITIZATION RATES J. R. Howe', A. Robert', T. C. Smith', M. J. Wall2, M. M. Michaela Scheller', Stuart A. Forman2; 'Klinikum rechts der Isar, Usowicz2; 'Yale University, New Haven, CT , 2Univeristy of Munich, Germany, 2Massachusetts General Hospital, CLN-3, Fruit Bristol, Bristol, United Kingdom Street, Boston, MA 02114 Excitatory postsynaptic currents (EPSCs) were evoked by Mutations L264T and S2701 in GABAAR a, subunits enhance stimulating mossy fiber inputs to granule cells in cerebellar slices. sensitivity to GABA, but the changes they cause in the gating AMPA-type channels expressed in these neurons prior to mechanism are unknown. We used rapid agonist application on synaptogenesis show concentration-dependent substate gating patches and whole HEK293 cells expressing human a,J23r2L (Smith & Howe, 2000). To determine if synaptic channels show GABAARs to measure peak currents, and rates of current this behavior, we tested the effects of the competitive antagonist activation, desensitization, and deactivation. Wild-type GABAARs NBQX on EPSC decay. The decay of EPSCs contained two were characterized by ECso = 25 4 3 pM and nH = 1.4 i 0.2. exponential components. At 34 to 37°C, the time constant and Maximal opening rate was 3000-6000 s"', desensitization was relative amplitude of the fast component were 850 ± 20 jus and biphasic with time constants of 35 + 10 ms and 800 ± 270 ms, and 0.92 + 0.01, whereas the time constant of the slow component of deactivation was biphasic with time constants of 23 ± 9 ms and decay was 8.3 ± 0.7 ms (n = 15). NBQX substantially reduced the 140 t 54 ms. Deactivation from ft-alanine had a time constant of 3 amplitude of the fast component at concentrations that did not ± 0.9 ms. These rates are similar to those reported from reduce the slow component. The concentration dependence of the mammalian neuronal membranes. The a,L264T mutation reduced block, and the effect of NBQX on EPSCs with different amounts the GABA EC50 to 0.33 pM, and dramatically slowed both of fast component, indicate that the fast and slow components do deactivation and desensitization. The aiS2701 mutation reduced not arise from channels with different NBQX sensitivities. Non- the GABA BC50 to 1.5 pM and greatly slowed deactivation, but did stationary fluctuation analysis indicated that the unitary not slow desensitization. Both mutations also slowed deactivation conductance of channels open near the peak of the EPSC is greater from 1-alanine. These results suggest that the a,L264T mutation than the conductance of channels open in the tail. In total, the selectively stabilizes the open state of GABAAR, while the a,S2701 results indicate that the AMPA receptors underlying the EPSC mutation affects a different (non-open) state between open and show concentration-dependent substate gating. resting states. Supported by the Leverhulme and Wellcome Trusts, NS 37904, and GM 58926. 484-Pos Board # B340 PHARMACOLOGICAL EVALUATION OF 7- 482-Pos Board # B338 NITROINDOLINE-CAGED NEUROTRANSMITTERS AND INTERACTIONS OF GLUTAMATE, KAINATE AND AMPA 4-METHOXY-7-NITROINDOLINE-CAGED L- WITH THE S1S2 DOMAIN OF THE GLUTAMATE GLUTAMATE RECEPTOR. Marco Canepari, Laura Nelson, George Papageorgiou, John ET Shalita Thlran', Dean Madden2, Vasanthi Jayaraman'; 'Marquette Comrie, David Ogden; National Institute for Medical Research University, 535 North 14th Street, Milwaukee, WI 53233, 2Ion 7-nitroindoline(NI)-caged L-glutamate, GABA and glycine, and Channel Structure Research Group, Max-Planck Institute of methoxy-nitroindoline(MNI)-caged -glutamate (Papageorgiou & Medical Research, Heidelberg, Germany Corrie, 2000. Tetrahedron 56:8197) are new, stable, fast caged Fourier transform infrared spectroscopy was used to investigate neurotransmitters. Whole cell clamp was used with Purkinje ligand-protein interactions between agonist glutamate, kainate or neurones in cerebellar slices and cultured hippocampal neurones to AMPA with the S1S2 domain of the GluR4 receptor subunit. To test L-glutamate and GABA, and with spinal cord neurones to test elucidate these interactions the asymmetric carboxylate vibrations glycine. Release of 20-400 gM L-glutamate by photolysis (1-ms of the ligands, the SH stretching mode of the single non-disulfide- pulse, 280-360 nm) of NI-caged or of MNI-caged glutamate bonded cysteine residue, and the amide vibrations of the protein produced fast AMPA-receptor cufrents blocked by 100 IM NBQX were investigated. These studies indicate that the binding of and slow, AP5-sensitive NMDA receptor currents. An inward glutamate and AMPA at the ligand-binding domain induces similar current due to mGluR1 receptors was activated in Purkinje but more pronounced secondary structural changes than with neurones. MNI-caged L-glutamate was 3-4 times more efficient kainate binding. These secondary structural changes include than NI-caged glutamate (maximum conversion 50-60% per flash). increases in the content of a-helices, 1-sheets, and turns for Neither NI-caged nor MNI-caged L-glutamate at 1 mM affected glutamate and AMPA while only 1-sheet content increases for AMPA or NMDA receptor iontophoretic responses, nor mGluRl kainate binding. In addition, glutamate binding enhances the receptors nor release parameters at climbing fibre synapses. At I hydrogen-bonding strength of cysteine 426 side chain in the mM the NI and MNI-caged glutamates appear pharmacologically domain while kainate does not alter this sulflhydryl environment. In inert. Photorelease of 20-150 pM GABA or glycine activated contrast, the interaction of kainate or AMPA with a binding site bicuculline or strychnine sensitive currents, respectively. However, arginine residue appears stronger and hence is closer in proximity application of 1 mM NI-GABA or NI-glycine inhibited current with each other than that of glutamate binding. These chemical and elicited by iontophoretic application of GABA or glycine, structural differences could contribute to the functional differences respectively, and identical concentrations of photoreleased GABA of these agonists acting on ionotropic glutamate receptors. or glycine evoked smaller currents at high concentrations of caged compound, indicating that NI-GABA and NI-glycine are antagonists at GABAA and glycine receptors.

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485-Pos Board # B341 Wallace et al. GABAA Receptor Subunit Mutation in Childhood CHARACTERIZATION OF MODULATORS OF a4- Absence Epilepsy and Febrile Seizures. Submitted 2000. SUBUNIT-CONTAINING HUMAN GABAA RECEPTORS THROUGH FRET-DERIVED MEASUREMENTS OF 487-Pos Board # B343 MEMBRANE POTENTIAL. OPENING OF THE GABAA RECEPTOR CHANNEL Charles E Adkins', Gopalan V Pillail, Julie Kerby', Timothy P REQUIRES ASSYMETRIC ROTATION OF ADJACENT Bonnert', Christine Haldon', Jesus E Gonz7Aez2, Kahuku Oades2, SUBUNITS Paul J Whiting', Peter B Simpson'; 'Merck, Sharp & Dohme, Myles H Akabas', Jeffrey Horenstein', David A. Wagner2, Cindy Terling's Park, Eastwick Road, Harlow, Essex CM20 2QR, Czajkowski2; 'Albert Einstein College of Medicine, 1300 Morris 2Aurora Biosciences Inc., La Jolla, California Park Avenue, Bronx, NY 10461, 2Univ. of Wisconsin-Madison, Modulators of y-amino butyric acid (GABA) receptors are used to Madison, WI 53706 treat a variety of CNS disorders, including anxiety and insomnia. GABAA receptors are formed by the assembly of 5 subunits around We have measured GABA-evoked changes in membrane potential a central ion channel. Each subunit has an -200 amino acid, using a fluorescence resonance energy transfer (FRET) based dye extracellular N-terminal domain that contains the GABA binding system in Ltk- cells stably transfected with human a4, 03, and site and a similar sized C-terminal domain that contains four either y2 or 8 GABAA receptor subunits (a403y2 or a0(38). Cells membrane-spanning segments (Ml, M2, M3, M4). a,Thr261 and were incubated with 4,uM CC2-dimyristoyl f3Thr256 are aligned channel-lining residues at the 6' position in phosphatidylethanolamine and ijM DiSBAC2(3) in low-Cl buffer, the M2 segment. In the double Cys mutant cxsT261COI3T256C, and membrane-potential-dependent changes in FRET coupling application of 100 tM copper phenanthroline (1:4) in the presence between the dyes were recorded using a VIPR. GABA depolarized of GABA locks the channels in an open, conducting state. both cell types, and was 5-fold more potent at 8-subunit-containing Application of copper phenanthroline in the absence of GABA had receptors (ECso=l.9 .M at (x4033Y2, 0.4,uM at (X4338). Both 0x433Y2 no effect on subsequent GABA-induced currents. Similar results and 04338-mediated GABA responses were blocked by picrotoxin were obtained in the single mutant czl1IT256C. Dimer formation and bicuculline. 0(4338 responses were potentiated by alfaxalone between f subunits was shown to occur only in the presence of (124% increase in GABA ECso response, ECso=lOnM) and GABA and copper phenanthroline by western blotting of epitope- inhibited by pregnanalone (-100%, ECso=lpM). a4338 receptors tagged subunits. We infer that at the level of i3Thr256 GABA- were insensitive to classical benzodiazepines but a433y2 receptors induced gating causes the neighboring f3 subunits to rotate towards were modestly potentiated by bretazenil (30%, ECso=122nM) and each other. This brings the channel-lining engineered Cys residues inhibited by DMCM (-38%, ECso=860nM). This technique into close proximity thereby allowing disulfide bond formation that provides a new means of identifying and characterizing novel locks the channels in the open state. This implies that during modulators of GABAA receptors. channel opening the subunits undergo asymmetric rotation relative to each other. Supported by NIH grants NS30808, GM61925 & 486-Pos Board # B342 NS34727. ELECTROPHYSIOLOGICAL INVESTIGATION OF A GABAA RECEPTOR SUBUNIT MUTATION FOUND IN 488-Pos Board # B344 HUMANS WITH EPILEPSY FUNCTIONAL PROPERTIES OF A PROKARYOTIC David N Bowser', Rekha G Panchal', Robyn H Wallace2, Carla GLUTAMATE RECEPTOR Marini3, Louise Harkin2, Grant R Sutherland2, John C Mulley2, Changhai Cui, Carla Glasser, Mark Mayer; NICHD, NIH, Ingrid Scheffer3, Samuel F Berkovic3, David A Williams', Steven Bldg.49/Rm5A78, 49 Convent Dr., MSC4495, Bethesda, MD Petrou'; 'University of Melbourne, VIC 3010 Australia, 2Women's 20892-4495 and Children's Hospital, North Adelaide, SA Australia, 3Austin GluRO is a recently discovered prokaryotic glutamate receptor. It is Repat Med Cent, Heidelberg, VIC Australia a glutamate gated potassium channel. Despite the fact that GluRO In a large Australian family, febrile seizures and childhood absence may be an important link between glutamate receptors and epilepsy are genetically linked to a nucleotide mutation within a potassium channels, the electrophysiological properties of this putative benzodiazepine binding domain of a GABAA receptor receptor have not been fully examined. In this study the basic subunit (Wallace et al, 2000). Here we present an channel properties of GluRO were examined in HEK cell electrophysiological analysis of this mutant subunit expressed in recombinant system. Both Na+ and tetraethylammonium (TEA), Xenopus oocytes. cRNA's encoding wildtype or mutant subunits of but not spermine, can block the receptor in a voltage dependent the human GABAA receptors were injected into Xenopus oocytes manner. Na+ blocks GluRO from the extracellular but not and two-electrode voltage clamp recordings made using standard intracellular side. Analysis of Na+ block with a Woodhull model procedures. Responses to GABA (1 gM) were indistinguishable in suggests that the binding site for Na+ is near the intracellular side oocytes expressing wild type or mutant receptors. However, of the electrical field. TEA can block GluRO from both sides of the diazepam (1 pM) potentiation of GABA currents was completely membrane with similar affinity. Most striking is the phenomenon abolished in oocytes expressing mutant GABAA, suggesting that that the receptor is spontaneously active. The ratio of the amplitude the mutation alters benzodiazepine modulation. Functional between spontaneously active current and the glutamate activated expression of the mutant subunit was then confirned. We conclude current is close to 1:1. Like eukaryotic glutamate receptors, the that the mutation does not effect expression, assembly or desensitization of GluRO can be modulated by thiocyanate. Not sensitivity to GABA, but abolishes diazepam potentiation. This only the desensitization of glutamate responses was significantly demonstrates a direct association between GABA receptor blocked but also the current amplitude was potentiated. The mutations and human epilepsies and raises the intriguing molecular mechanisms of these properties are being studied further possibility that endozepines play a role in preventing seizures. using mutagenesis.

108a Sundav.cAiinrlv- POSTERSPOSTPR 5 vRcLAO'ARQ-AQII

489-Pos Board # B345 491-Pos Board # B347 A SEROTONIN-GATED CHLORIDE CHANNEL INTRAMOLECULAR SIGNAL TRANSDUCTION IN MEDIATES EXPERIENCE-DEPENDENT MODULATION OSMOREACTIVE TRANSPORT SYSTEMS FROM OF C. ELEGANS BEHAVIOR. BACTERIA Rajesh Ranganathanl, Stephen C Cannon2, H. Robert Horvitz'; Reinhard Kraemer, Udo Burger, Hendrik Ronsch, Rene 'Massachusetts Institute of Technology, Rm 68-425 77 RtUbenhagen, Ralf Steger, Susanne Morbach; University of Koln, Massachusetts Ave, Cambridge, MA 02139, 2Harvard Medical Zitlpicher Str. 47, Koln, 50674 School, EDR 413 / Mass Gen Hospital, Boston, MA 02114 Prokaryotic and eukaryotic cells harbour mechanisms to sense and C. elegans hermaphrodites respond to a bacterial lawn (their food respond to hyper- and hypoosmotic stress. We are studying source) by slowing their locomotion rate. Animals food-deprived transport systems for compatible solutes, which function both as for 30 minutes exhibit enhanced slowing when reintroduced to a sensors and regulators responding to osmotic stress. At least two bacterial lawn. This modulatory response is mediated by serotonin signal inputs are available to trigger these systems (a) via the (5-HT). Mutations in the gene mod-i (modulation of locomotion membrane, i.e. mechanosensation, or (b) via the hydrophilic space, defective) diniinish the slowing response and also abolish the i.e. osmosensation. We are studying biophysical and biochemical ability of exogenous 5-HT to immobilize the animals. properties of osmoreactive transporters for compatible solutes in mod-i encodes a channel in the nAChR family similar to GABAA the Gram-positive soil bacterium Corynebacterium glutamicum. and glycine receptors and less similar to 5-HT3,Ab receptors. The systems BetP, a Na+ coupled betaine uptake carrier, and EctP, Nonetheless, expression studies using Xenopus oocytes showed a Na+ coupled ectoine-proline-betaine carrier, are analyzed both in that MOD-I is specifically gated 5-HT = I with a intact cells and after reconstitution in liposomes. The sensory by (ECso ,uM) function of this is reversal potential of - -20 mV. Unlike 5-HT3a channels, MOD-1 systems closely related to shifts in osmolality currents are insensitive to 10 mM [Ca2i],,, and to the 5-HT3a- and/or mechanical stress forced upon the membrane. The specific antagonists granisetron and ondansetron ( >100 gM ). regulatory response of these systems in terms of transport activity, mod-l(n3034) mutants have a missense substitution, A281V, setpoint and time course of response, is analyzed in intact cells and within the M2 transmembrane of in proteoliposomes harbouring the transporters BetP and or EctP as region. Injection A281V cRNA well as hybrid constructs of these does not produce a detectable 5-HT-gated current, and coinjection membrane proteins. of A281V with wild-type MOD-1 cRNA suppresses the current amplitude in a dominant-negative manner. The permeation properties of the MOD-1 channel were measured in HEK cells 492-Pos Board # B348 transiently expressing MOD-1. The reversal potential was sensitive HOW MICROORGANISMS RESPOND TO GRAVITY: to [Cl with a 52 mV shift per 10-fold change, and insensitive to CHLAMYDOMONAS MUTANTS DEFECTIVE IN changes in [Na'],,t or [W].w. We conclude that MOD-1 is a 5-HT- GRAVITAXIS. gated chloride channel. Kenjiro Yoshimura, Yudo Matsuo, Ritsu Kamiya; University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033 Japan Mechanosensitive Channels Many free-swimming microorganisms tend to swim upward although their densities are higher than the medium density. This 490-Pos Board # B346 phenomenon, negative gravitaxis, has been thought to arise either SENSOR PROPERTIES OF BACTERIAL from some physiological process such as gravity perception or OSMOREACTIVE TRANSPORT SYSTEMS simply from cell's physical characteristics such as the imbalance of Susanne Morbach, Udo Burger, Hendrik Ronsch, Rene the center of gravity and that of buoyancy. However, there has Riibenhagen, Ralf Steger, Reinhard Krimer; University of Koln, been little convincing evidence to support either view. To obtain ZUlpicher Str. 47, Koln, 50674 insight into the mechanism of gravitaxis, we isolated The response to hyper- and hypoosmotic stress is an essential Chiamydomonas mutants deficient in gravitaxis. These mutants, mechanism for prokaryotic and eukaryotic cells. In bacteria, highly gtxl and gtx2, showed significantly weak negative gravitaxis in the active and tightly coupled carriers for compatible solutes mediate order of wt> gtxl > gtx2. Their swimming velocities and flagellar the response to osmotic upshifts. Structural infomation on the beat frequencies were normal. Interestingly, other cellular components involved in osmosensing and coupling this responses that involve membrane excitation were also defective in information to the energy-driven uptake systems is not available. these mutants; both the photophobic response upon reception of We have elucidated the biochemical properties and the primary strong light and the shock response upon reception of mechanical structure of the whole set of four osmoregulatory, energy- stimuli were weak in the same order. In contrast, the positions of dependent uptake systems for compatible solutes in the Gram- the center of gravity and that of buoyancy in these mutants were positive soil bacterium Corynebacterium glutamicum. BetP, the the same as in the wild type cells. Based on these observations, we most important system, a Na+coupled betaine uptake carrier, is suggest that the gravitaxis in Chlamydomonas involves tightly regulated on the level of expression and activity. It is able physiological signal sensing mechanism. to switch from virtually zero activity to Vmax conditions within a second or less. It is fully active both in catalytic and regulatory 493-Pos Board # B349 terms in an in vitro system (proteoliposomes). Two domains of this STRUCTURAL AND FUNCTIONAL RELATEDNESS OF membrane protein, both at the N- and the C-terminal end of the ARCHAEAL AND BACTERIAL MS CHANNELS carrier, are directly involved in sensing osmotic stress and Anna Kloda, Boris Martinac; The University of Western modulating the carrier activity. Site-specific mutations are used to Australia, QEII Medical Center, Nedlands, WA 6907 elucidate structure-function correlation of BetP and related TMI domain of Eco-MscL was used as a genetic probe against the proteins in C. glutamicum (EctP) in terms of osmosensing, signal genomic database of the archaeon M. jannaschii. The hypothetical transduction and catalytic activity. protein MJ0170 (MscMJ) was found to harbor two MscL-like TM1 structural motifs and showed a high degree of sequence conservation with MscS homologues. The phylogenetic analysis of MscMJ and MscL homologues further revealed the common ancestry of prokaryotic MS channels. Our study indicates the evolution of prokaryotic MS channels via gene duplication of mscL-like progenitor followed by divergence. The gene duplication

109a dr..QtF--493 ztQR.f499 PO,qFR,qRPC iJJTPRS oiii'flhPRimlAnu61.

most likely occurred before separation of archaeal and bacterial confirms the relative positions of Ml domains in the closed lineage indicating antiquity and common heritage of prokaryotic conformation, whereas a significant coupling of A20C/L36C and MS channels. In addition to sequence homology MscMJ also 124C/V37C pairs shows a high probability of tilted conformations shares functional similarities to other MS channels. The free for TM domains and suggests that the channel barrel is energy of activation AGo-7kT matches the one calculated for the expandable. MscS AGo-SkT. Similar to MscS, MscMJ is activated by amphipaths CPZ and TNP and like other MS channels is blocked 496-Pos Board # B352 by gadolinium. Unlike MscL or MscS, MscMJ shows preference ON THE ROLE OF THE Sl-Ml LINKER IN THE GATING for cations over anions. In this respect the channel resembles the MECHANISM OF THE LARGE MECHANOSENSITIVE eukaryotic SA-CAT channels. Further phylogenetic studies of MS CHANNEL MSCL. channels from three domains of universal tree phylogenetic may Chien-Sung Chiang', Paul Gray', Monica Betanzos', H. Robert help to understand evolution and common biophysical principles, which transduction. Guy2, Sergei Sukharev'; 'University of Maryland, 2NIH, Bethesda, govern mechanosensory MD 20892 494-Pos Board # B350 The primary gate of MscL is formed by the hydrophobic TOWARDS AN UNDERSTANDING OF THE FUNCTION constriction in the pore, whereas the N-terminal amphipathic (SI) OF THE LARGE MECHANOSENSITIVE CHANNEL segments may form an additional cytoplasmic gate. Flexible FROM M. TUBERCULOSIS USING COMPARATIVE linkers (R13-G14-N15) connecting SI to Ml helices are predicted ANALYSIS. to pull the S1 gate bundle apart when tension distorts the channel barrel. Here we study the gating of MscL mutants with extended Joshua A. Maurer, Donald E. Elmore, Henry A. Lester, Dennis linkers. An extra glycine inserted next to G14 (GG14 mutation) A. Dougherty; California Institue of Technology, Mail Code 164- slightly reduces the activation threshold of MscL (pMsclipMscS) 30 Cr, Pasadena, CA 91125 to 1.29±0.1, and significantly increases the occupancy of short- The crystal structure of the large mechanosensitive ion-channel lived substates of low-conductance. Further extension with an (MscL) from M. tuberculosis (Tb) has provided a starting point extra alanine (GAG14) produces non-functional channels. In from which to gain insight into channel function. Nine V23D, a severe gain-of function (GOF) mutant with the disrupted homologues of Tb-MscL have been identified electrophysioloically hydrophobic lock, similar GAG14 extension increases the and some variation in their required gating tension and channel activation threshold from 0.5 to about 1 and prevents complete kinetics has been observed.' We have sought to compare and openings at any tension. The GAG14 insertion to the G22N contrast these various channels using site directed mutagenesis, mutant removes the long-lived low-conductance substate and also computational biology, and a variety of biochemical and prevents full openings. The observations support the hypothesis biophysical techniques. Site directed mutagenesis has identified that for proper gating, the length of the linkers must be tuned to the striking differences between E. coli (Ec) MscL and M. tuberculosis degree of the barrel deformation. Neither of the extensions rescues MscL, such as substantial differences in the mutagenic profile of toxic phenotypes of the severe GOF mutants, indicating that the A20 in Th-MscL compared with the aligned G22 in Ec-MscL. prinmary gate must be intact to keep MscL leak-free. MEME and regional AMPS pairwise alignment have pointed to the loop region and C-terminus of the protein as being the most 497-Pos Board # B353 divergent, suggesting these regions may be responsible for the MSCL GATING STUDIIED BY MOLECULAR DYNAMICS observed differences in channel physiology. Additionally, SIMULATIONS mutation of the loop region of Tb-MscL has shown that this region is essential for normal channel function. Comparative circular Justin R Guliingsrud, Dorina Kosztin, Klaus Schulten; University dichroism studies have also revealed structural differences between of Illinois at Urbana-Champaign, 405 N Mathews, Urbana, IL these homologues. 61801 'Moe, P.C.; Blount, P. Kung, C. Mol. Microbiol. 1998, 28, 583- We present results from molecular dynamics simulations of the 592. MscL protein embedded in a fully hydrated POPC bilayer. The simulations, totalling 3 ns in length, were carried out under conditions of constant temperature and pressure using periodic 495-Pos Board # B351 boundary conditions and full electrostatics. The protein remained THE OPEN CONFORMATION OF A in the closed state corresponding to the crystal structure, as MECHANOSENSITIVE CHANNEL STUDIED BY evidenced by its impermeability to water. Analysis of equilibrium DISULFIDE TRAPPING fluctuations showed that the protein was most immobile in the Monica Betanzos', Chien-Sung Chiang', H. Robert Guy2, Sergei narrowest part of the channel. The gating process was also studied Sukharev'; 'University of Maryland, 2NIH, Bethesda, MD 20892 with simulations of the bare protein under conditions of constant Based on the crystal structure of TbMscL, the models of E. coli surface tension. Under a range of conditions the transmembrane MscL in the closed, open and several intermediate conformations helices flattened as the pore widened. Inplications for theories of have been proposed. They predict that (1) the bundle of short N- the gating process in light of these and experimental results are terminal domains (S1) forms the cytoplasmic gate; (2) when discussed. membrane tension stretches the transmembrane (TM) region, the tilt of the TM helices increases and Ml's move away from the axis 498-Pos Board # B354 of the pore in an iris-like manner; (3) in the fully open MOLECULAR DYNAMICS SIMULATIONS OF THE M. conformation SI helices may dock to a specific site on the inner TUBERCULOSIS MECHANOSENSITIVE CHANNEL OF surface of the pore. The pairs of residues predicted to be proximal LARGE CONDUCTANCE. in either closed or open conformations were mutated to Cys, and Donald E. Kenneth D. Philipson2, Dennis A. the were tested Elmore', couplings with patch-clamp and biochemically. Dougherty'; 'California Institute of Technology, Mail Code 164-30 Spontaneous bridging of cysteines at positions 7 or 10 linked pairs of subunits and the Cr, Pasadena, CA 91125, 2University of California at Los Angeles, prevented channel from opening until the bonds School of Medicine, Los Angeles, CA 90095-1760 were reduced. The "docked" position of SI in the open state was supported by a 13C to 196C bridge, which locked the channel in The crystal structure of the M. tuberculosis mechanosensitive one of the subconducting states, preventing complete closures. A channel of large conductance (MscL) provides a unique complete cross-linking of MscL complex via 124C-G26C pairs opportunity to consider mechanosensitive signal transduction at the atomic level. An atomic resolution structure allows the use of

1lOa Sunday- I POSTERS 498-502- - %., -1 %O A&

computational techniques, such as molecular dynamics (MD), identify the contribution of this mechanism to stretch-induced which were previously inapplicable to this system. In order to arrhythmogenesis in multicellular preparations. further understand MseL dynamics, nanosecond MD simulations Supported by the British Heart Foundation, Medical Research of the channel have been performed, using an explicit lipid Council & The Royal Society. [I] van Wagoner. Circ Res membrane and water. Wildtype MscL and mutant simulations 1993/72:973-83. [2] Link et al. Circulation 1999/100:413-18. [3] have been compared to gain insight into the structural changes that Kohl et al. Cardiovasc Res 2001 (May). [4] Riemer et al. Am J lead to experimentally observed changes in gating tension and Physiol 1998/275:H431-42. kinetics. Additionally, modeling sections of the channel, such as the transmembrane domains and the C-terminal helix region, has 501-Pos Board # B357 minimized computational expense to allow screening of several POSITIVE CHRONOTROPIC WHOLE CELL CURRENT additional mutants. Results from these simulations have been ACTIVATED BYAXIAL STRETCH OF RABBIT SINO- considered in light of experimental data to gain additional insight ATRIAL PACEMAKER into channel structure-function relationships and to plan future CELLS experimental directions. Patricia J. Cooper, Ming Lei, Peter Kohl; University Laboratory of Physiology, Univ of Oxford, OXI 3PT, UK 499-Pos Board # B355 Stretch of the sino-atrial node (SAN) region has been shown to HIGH SPEED PRESSURE CLAMP increase spontaneous beating rate in vivo as well as ex vivo in isolated hearts, atria, and SAN tissue. We have recently shown that Stephen Robert Besch, Thomas Suchyna, Frederick Sachs; this response to axial stretch can be reproduced in spontaneously SUNY at Buffalo, 320 Cary Hall, Buffalo, NY 14214-3005 beating cells isolated from rabbit SAN, thereby demonstrating the We have built a high-speed pneumatic pressure clamp. The device presence of a stretch-activated positive chronotropic mechanism at provides reliable and precise control of pressure applied to patch the single cell level [Cheng, Cooper & Kohl Biophys J pipettes and was designed for the application of ±pressures to 2000/78:472A]. The nature of this mechanism, however, remained membranes as a means of mechanical stimulation. The output unknown. In this study, we have measured the stretch-induced pressure is controlled using a newly designed differential whole cell current (perforated patch clamp) in isolated rabbit SAN piezoelectric valve in a PID feedback loop. Manual and external cells subjected to axial stretch, using computer controlled carbon voltage control of the pressure set point are available. Several fibres attached to either end of a cell. Voltage-clamp step pulses enhancements over previous designs improve performance. First, (1 s) from a holding potential of -60 to +40 mV (10 mV steps) the valve uses a single piezoelectric bending element to control were applied before, during and after stretch of cells by 5-10% of both pressure and vacuum, a valve body design that significantly resting length. The stretch-induced current was obtained as the reduces internal volume, and a very low volume piezoresistive difference between currents recorded in the presence and absence pressure sensor that further reduces volume. These all improve of stretch. The current-voltage relation was best fitted by a linear response time and stability and result in a 3-fold decrease in conductance (slope 6 nS/pF) with a reversal potential near - actuation latency. Second, an optical sensor detects water entering 11 mV. This is consistent with stretch-activation of cation- the valve and rapidly increases pressure to clear the system. This selective channels and would, according to mathematical protects the valve from contamination in the event of a broken modelling (OxSoft v4.8) be sufficient to cause the observed pipette. Third, small resonant piston pumps are used to supply positive chronotropic response of SAN cells to stretch. Further pressure and vacuum and eliminate the need for compressed gas. experiments, using a newly established selective blocker of these The open loop time constant for pressure change is 2.5 ms for a channels [Suchyna et al. J Gen Physiol 2000/115:583-598], will 120 mmHg step, and the closed loop settling time is 500-600 As. confirm or refute this hypothesis. Valve actuation latency is 150 us. Supported by the British Heart Foundation, Medical Research Council & The Royal Society. 500-Pos Board # B356 STRETCH-INDUCED AFTERDEPOLARIZATIONS IN 502-Pos Board # B358 RABBIT ISOLATED VENTRICULAR MYOCYTES STRETCH-DEPENDENT POTASSIUM CHANNELS IN Long-Xian Cheng, Ming Lei, Patricia Cooper, Peter Kohl, MURINE AND CANINE COLONIC SMOOTH MUSCLE University Laboratory of Physiology, University of Oxford, OXI CELLS 3PT, United Kingdom. Sang Don Kohl, Catharine A Conley2, Kenton M. Sanders'; The mechanisms of sudden cardiac death after moderate, non- 'University of Nevada, Reno, Nevada 89557, 2NASA, SLR, penetrating precordial impact (Commotio cordis) are not well building N239, Moffett Field, CA 94035 understood. Recent experiments suggest that mechanical activation of channels Gastrointestinal muscles are able to maintain resting membrane K+ATP [1] during the early T-wave [2] is a crucial potentials during dynamic changes in length. We investigated arrhythmia-sustaining factor (3]. The nature of the trigger event, whether stretch-dependent K+ channels might contribute however, that gives rise to mechanically-induced ventricular to fibrillation remains elusive. Modelling studies suggest an myogenic regulation of smooth muscle cells. Negative pressure involvement of channels, whose applied to on-cell patches activated K+ channels that were voltage- stretch-activated cation selective independent with a slope conductance of 95 pS in symmetrical K+ activation during action potential repolarization could cause early gradients. The effects of negative pressure on open probability loo. were graded as a function of pressure. Cell elongation activated K+ channels with the same properties as those activated by negative ti-etch O. pressure, suggesting 5.|e that the channels were stretch-dependent K+ cnrol (SDK) channels. Channels with the same properties were maximally activated by patch excision, suggesting that either an e 300 Joe 9ce 12ae 15tl Tz- {nw ) intracellular messenger or interactions with the cytoskeleton afterdepolarization [41 and trigger arrhythmias. We studied rabbit regulate open probability. Cytochalasin D, an actin depolymerizer, isolated ventricular myocytes using the perforated patch clamp activated SDK channels in on-cell patches. SDK channels in technique during application of axial stretch (10-12% of resting excised patches were activated by arachidonic acid and inhibited length) by a pair of carbon fibres. Our experiments confirm, for the by decreasing pH. 4-aminopyridine, Ca2+ (10-8 to 10-6 M), and first time, that stretch of single ventricular myocytes can cause TEA were without effect on SDK channels. Nitric oxide donors afterdepolarization-like events. Further studies are required to (and cell permeable cGMP analogues) activated SDK in on cell patches, suggesting that these channels mediate a portion of the

llla 502-506 POSTERS Sundav- 7

enteric inhibitory neural response in colonic muscles. In summary, Biophysics of Ion Permeation SDK channels may stabilize membrane potential during dynamic changes in cell length and mediate responses to enteric neurotransmitters. (Supported by DK 41315) 505-Pos Board # B361 ROLE OF ANION BINDING IN DETERMINING CFTR CHLORIDE CHANNEL SELECTIVITY 503-Pos Board # B359 VISUALIZATION OF CA2+ ENTRY THROUGH STRETCH- Paul Linsdell; Dalhousie University, Halifax, Nova Scotia B3H ACTIVATED CATION CHANNELS 4H7 Hui Zou, Lawrence M. Lifshitz, Michael T. Kirber, Richard A. The relationship between anion binding and anion permeability in Tuft, Kevin E. Fogarty, Joshua J. Singer; University of Cl channel pores is not clear. This relationship was examined in Massachusetts Medical School, 55 Lake Avenue North, Worcester, the CFIR Cl channel by comparing anion binding within the pores MA 01655 of wild-type and mutant channels expressed in mammalian cell lines. Under symmetrical ionic conditions, wild type CFTR Stretch-activated channels (SACs) have been found in both showed the conductance sequence Cl > NO3 > Br > formate > F > vascular and visceral smooth muscle and are thought to be SCN C104.O Chloride currents were blocked by low involved in stretch-induced myogenic responses. Although patch concentrations of SCN, I and Cl04, implying relatively tight clamp studies have shown that Ca2+ can permeate these channels binding of these ions within the pore. Two pore mutations which when it is the only available charge carrier, it has not been clearly significantly alter the anion permeability sequence, F337S and demonstrated that Ca2+ passes through SACs in more physiological T338A, also altered the anion conductance sequence and block by solutions. By imaging the single channel Ca2+ fluorescence penneant anions, consistent with altered anion binding within the transient (SCCaFT, see Zou et al. J Gen Physiol 114: 575, 1999) pore. The effects of these mutations on anion permeability and due to Ca2+ entry through a single opening of a SAC, we now show anion conductance suggested that, for most anions, increased that Ca2+ can indeed enter the cell through SACs and increase the permeability was associated with increased conductance, and local Ca2+ concentration. Experiments were carried out using decreased permeability with decreased conductance. This single smooth muscle cells from the stomach of toad, Bufo indicates that CFR does not achieve its anion selectivity by marinus. Cells were loaded with fluo-3AM. The fluorescence was selective anion binding within the mutated region. Instead, it is simultaneously imaged using a high-speed digital imaging wide- suggested that entry of anions into the region around F337/T338 field microscope while recording unitary SAC currents from cell- facilitates their passage through the pore. In wild type CFrR, attached patches. Cells were pretreated with thapsigargin and anion entry into this crucial pore region is probably dominated by caffeine to deplete intracellular Ca2+ stores, and the only source of anion hydration energies. Ca2+ was the physiological salt solution containing 2 mM Ca2+ in the patch pipette. Localized fluorescence increases were observed Supported by MRC (Canada) and the Canadian CF Foundation. corresponding to SAC openings induced by stretching the membrane patch. 506-Pos Board # B362 A PAIR OF TM2 MUTATIONS CHANGES SELECTIVITY 504-Pos Board # B360 OF THE HOMOMERIC GABA P1 RECEPTOR FROM STRUCTURE-FUNCTION STUDY OF TREK-2, A TANDEM ANIONIC TO CATIONIC. PORE K+ CHANNEL Virginia E Wotring, T S Kaylor, D S Weiss; Univ Alabama at Donghee Kim, Yangmi Kim; Chicago Medical School, 3333 Birmingham, 1719 Sixth Ave S, Birmingham, Al 35294 Green Bay Road, North Chicago, IL 60064 Three concomitant mutations have been shown to change the TREK-2 is the most recently cloned member of the polarity of nACh a7 and glycine al receptor ionic selectivity. We TRAAK/TREK class within the tandem pore K+ channel family. investigated the effect of the corresponding mutations on the Native K+ channels with properties identical to TREK-2 have been GABA pl subunit, individually and in all possible combinations. identified previously in neuronal cells. TREK-2 is activated by Mutants were expressed in Xenopus oocytes and reversal membrane stretch, unsaturated free fatty acids and protons. To potentials (Vrev) were measured. The double mutant identify the regions involved in the activation, we studied the pAP290/T305V and the triple mutant pAP290/A291E/T305V did responses of various deletions and chimeric constructs to the three not function. When 90% of [chloride]o was replaced by activators. Deletion of the N-terminus had no effect on TREK-2 isethionate, pl and the double mutant pA291E/T305V had Vrev sensitivity to all three activators. Progressive deletion of the C- shifts of 57.7 + 0.46 and 50.6 ± 2.25 mV, respectively. This is terminus resulted in gradual reduction in sensitivity to the three consistent with chloride selectivity. Three individual mutants activators. Replacing the C-terminus of TREK-2 with that of pAP290, pT305V, and pA29lE had Vrev shifts ranging from 10 to TASK-3 (TREK2/TASK3C) abolished pH and fatty acid 28 mV, suggesting that these mutants were somewhat permeable to sensitivities without affecting mechanosensitivity. Studies using other ions. Vrev of the double mutant pAP290/A29IE was additional TREK-2/TASK-3 chimeras showed that the proximal 30 unaffected by low chloride. When 90% of the [sodium]o was amino acids was critical for pH and fatty acid sensitivities. Within replaced by choline, p, pT305V, pAP290, and pA291E/T305V had the 30 amino acid region, no single or a group of amino acids no significant shift in Vrev, suggesting insignificant sodium could be identified that confers sensitivity to free fatty acids, permeability. The pA29lE mutant had a Vrev shift of 17.62 ± 1.33 indicating that the entire 30 amino acid region is important. mV, while that of the double mutant pAP290/A291E was 32.45 ± TREK2/TASK3C channels showed TASK-3-like openings, 0.91 mV. These results show that the double mutant suggesting that the C-terminus regulates channel gating kinetics. pAP290/A291E is cation selective, while pA291E may be Our results indicate that the short 30 amino acid region of TREK- permeable to cations and anions. Supported by NIH MHI 1793 to 2 C-terminus near the fourth transmembrane segment is critical for V.E.W. activation by free fatty acids and low pH, and that the transmembrane segments of TREK-2 provide the mechanosensitivity.

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507-Pos Board # B363 state and moves out of the way during channel opening. A HIGHLY REFINED STUDY OF THE HYDRATION OF K+ Experiments employing macroscopic K currents in the Shaker AND NA+ AND ITS IMPLICATIONS ON THE channel have suggested a cytoplasmic localization for the gate. The SELECTIVITY MODELS OF ION CHANNELS. crystallographic structure of the KcsA K channel indeed shows a Mauricio Carrillo Tripp', Humberto Saint-Martin2, Ivan Ortega- cytoplasmic constriction, implying that the gate and selectivity 2UNAM filter are formed by distinct structures. However, the behavior of Blake2; 'UNAM/UAEM, Mexico, short-lived subconductance levels observed during open-closed An intriguing feature of the potassium channel is its ability to transitions for the drkl K channel, suggested a strict coupling translocate K+ ions with a selectivity margin of 104 over Nae. The between gating (channel opening) and permeation. The KcsA recent determination of the X-ray structure of the KcsA channel selectivity filter contains two K ions, which appear to be tightly from Streptomyces lividans has led to various proposals based on bound in a four-fold symmetric oxygen cage. This has prompted us the architecture of the pore trying to explain this phenomena. to investigated a model in which the selectivity filter acts as a gate. However, it also has been found that there is a degree of flexibility Monte Carlo simulations were performed of a model filter, which in the filter indicating that it is not rigid enough to distinguish alternates stochastically between two states: a high-affinity state between the two cations [1]. Moreover, the selectivity has been which selectively binds K ions (but not Na ions) and traps them, ascribed to the different solvation of the ions, and obviously the and a completely non-selective, low-affinity state which allows results of the simulations depend on the model of interaction used. both Na and K ions to permeate. The results of these simulations The analytical potentials so far employed do not reproduce even indicate that an affinity-switching selectivity filter allows efficient the experimentally determined coordination numbers [2]. In this permeation withoutjeopardizing ion selectivity. work we use a potential derived from an ab initio interaction surface. This model takes into account flexibility, polarizability 510-Pos Board # and many body effects [3], to make a study of the hydration of K B366 and The differences on the shell structures ION PERMEATION THROUGH NARROW CHANNEL. Na+. hydration may play WHAT an important role on the selectivity mechanism. POTENTIAL LANDSCAPE DOES IT SEE? [I] Charlotte E. et al, J. 78, 2929, Maria G Kurnikova', Artem Mamontov', Rob Coalson', Capener Biophys. (2000). Abraham Nitzan2; 'University of PIttsburgh, Chevron Science [21 Toby W. Allen et al, Biophys. J. 77, 2502, (1999). Center, Pittsburgh, PA 15260, 2university of Tel Aviv, Tel Aviv, [3] H. Saint-Martin et al, J. Chem. Phys. (In press) Israel Despite recent advances in computational approaches to ion 508-Pos Board # B364 transport through biological pores, current-voltage properties of a THE PORE MUTATION G431S SIGNIFICANTLY narrow channel such as Gramicidin A are not completely REDUCES CURRENT THROUGH THE MHCN2 understood. Several BD and PNP studies were performed recently. CHANNEL. One limitation that both theories share is that polarizability of the Vincenzo Macri, Eugene Agranovich, Catherine Proenza, Deirdre environment is assigned ad hoc. A conventional model for Brink, Eric Accili; Simon Fraser University, Burnaby, BC V5A dielectric environment of a biological channel and its vicinity is to 1S6 Canada assign a dielectric constant of two to the membrane and protein Hyperpolarization-activated cyclic nucleotide-gated (HCN) region, and dielectric constant of 80 to water everywhere. channels are voltage-gated mixed cation channels similar in However, in this model a huge dielectric barrier for the ion in the primary structure to voltage-gated K channels, especially HERG channel arises resulting in unrealistically small currents. For and KATI channels. As with most K channels, HCN channels example, our simulations showed that even with dielectric constant have a GYG motif located in the pore region between the S5 and of 15 on the protein and 40 in the channel the dielectric barrier is S6 transmembrane domains. We mutated the second glycine of this still too big, that is, the ion flow is too small in comparison to the motif to serine (G43IS) in the mouse HCN2 (mHCN2) isoform to experimental results. Our goal in this work is to construct a model determine the importance of this site in ion conduction. This potential, which takes into account inhomogeneous polarizability mutation eliminates current in HERG and KATI. CHO cells were of the media around the ion as well as any specific interactions that transfected with either wild-type (wt) or mutant mHCN2, and might arise in the channel due to the steric restrictions for the water currents were measured using the whole-cell patch clamp molecules in the first solvation shell. BD and PNP modified with technique. In a high external K solution, hyperpolarizing pulses the dielectric potential (PNP-DP) simulations are also performed from -35 to -140 mV produced average time-dependent current on Gramicidin A and on a cylinder model channel to explore values of -917 +/- 194 pA (n = 19) and -12.5 +/- 9.8 pA (n = 6) in current dependence on the potential profile. cells expressing wt and mutant mHCN2, respectively. Increased linear leak current was also observed in cells expressing wt 511-Pos Board # B367 mHCN2. The slopes of the leak I-V relation were 3.16 nS (n = 7) SIMULATIONS OF PROTON TRANSFER IN and 1.23 nS (n = 6) in cells expressing wt and mutant mHCN2, BIOLOGICAL MEMBRANES respectively. These results indicate the importance of the second Alexander M Smondyrev, Gregory A Voth; University of Utah, glycine in the GYG motif for current flow in HCN channels and 315 South 1400 East, Salt Lake City, UT 84121 that a suggest constant fraction of channels remain open at all The proton transfer across biological membranes is of great potentials. importance to the normal functioning of most living cells. A Supported by HSFBC/Yukon. recently developed multistate empirical valence bond (MS-EVB) model for an excess proton in liquid water (U.W.Schmitt and 509-Pos Board # B365 G.A.Voth, J.Chem.Phys., 111, 9361-9381, 1999) was employed to MONTE CARLO SIMULATIONS OF AN AFFINITY study possible mechanisms of proton diffusion across membranes. SWITCHING SELECTIVITY FILTER One possibility, is that protons permeate through a water wire Mark L Chapman, Antonius Michael VanDongen; Duke spanning a lipid membrane. Another mechanism involves proton University, C141 LSRC Bldg, La Salle str e, durham, nc 27710 transfer through a water pore confined within a protein channel A universal property of ion channels is their ability to alternate such as influenza M2 channel. It is likely that a limiting stage for stochastically between two permeation states, "open" and "closed". the two mechanisms described above is a proton diffusion along The prevailing model assumes the existence of a mechanical the surface of membrane through a hydrogen bonded network. a structure that These mechanisms were tested using molecular dynamics "gate", physical impedes ion flow in the closed simulation technique.

113a -1512-516AL ~ -' AL W -POSTERS1%.01 -I - -- - -I U.,Sundav"lA%w64 T mmm.

512-Pos Board # B368 channels actually open and close (ie. gate). Unfortunately the SIGNIFICANT CONTRIBUTION OF ION-WATER timescale of the gating processes in ionic channels is too slow INTERACTION TO THE ION SELECTIVITY OF A DE (upto the order of milliseconds) to be studied effectively by usual NOVO SYNTHESIZED HYDROPHOBIC CHANNEL: A molecular dynamics simulations. In order to address this issue we MOLECULAR DYNAMICS STUDY have performed non-equilibrium molecular dynamics simulations Zhi Qil, Masahiro SOKABE2; 'Nagoya University School of in order to artificially promote motion of the protein associated Medicine, Japan, 2ICORP Cell Mechanosensing Project, JST, with the gating process. The starting point for these simulations is 466-8550 the crystallographic structure of the potassium channel, KcsA. This Nagoya, Japan structure is likely to be a representative conformation of the Previously, we have performed electrophysiological analysis (1) channel in the closed state. The underlying reasoning is that and molecular dynamics (MD) simulations (2,3) on a de novo movements observed in the protein in these simulations will be at synthetic hydrophobic ion channel. In the present study, MD least similar to movements on a longer timescale. A variety of simulations have been carried out on K, Na+ and Li' permeation different studies that reveal like motions may indicate an through the channel in order to understand its ion selectivity accelerated view of the gating process. Our results provide insight mechanism. The potential energy profiles for these ions across the into the general manner in which potassium channels not only gate, channel could quantitatively account for the selectivity sequence, but also how they are able to accomodate internal blocking ions K+ > Na >> Li', observed in our electrophysiological experiment. such as the tetra-alkyl ammonium ions. Decomposition of the total interaction energy of the ion in the channel demonstrated that the selectivity sequence is mainly due to the difference between the ion-water interaction energy at the 515-Pos Board # B371 narrowest of the channel and that in the bulk solution. Present SIMULATIONS OF SELECTIVITY AND CURRENT FLOW part IN MODEL CHANNELS. study may help us understand ion selectivity mechanism of natural channels because a channel filled with water is a prerequisite for David D. Busathl, Dezso Boda2, Paul S. Crozier', Richard L. the ion permeation. Rowley', Nathan B. Holladay3, Douglas Henderson'; 'Brigham (1) Z. Qi, M. Sokabe, K. Donowaki, and H. Ishida. 1999. Biophys. Young University, Provo, UT 84602, 2University of Veszprem, J. 76:631-641. 3Univ. of Texas Southwestern Z. M. Sokabe. 1998. Chem. 71:35-50. Monte Carlo (MC) simulations using an infinite cylinder and (2) Qi, Biophys. molecular M. dynamics (MD) simulations of constant voltage current (3) Z. Qi, Sokabe. 1999. Biophys. Chem. 82:183-193. flow through an atomistic cylindrical channel in a rigid membrane were undertaken to explore the origins of valence selectivity in 513-Pos Board # B369 calcium channels and the role of electrodiffusion in channel entry. PERMEATION ENERGETICS OF THE KCSA CHANNEL In the first study, a cylinder containing trapped anions SELECTIVITY FILTER (representing Glu side chains) was equilibrated with a surrounding Stefano Garofoli, Peter C. Jordan; Brandeis University, P.O. Box bath by MC steps. The free electrolyte (IM NaCl in an E"=78.5 5491 10, Waltham, MA 02454-9110 bath) with increasing additions of CaC12, was allowed to enter the We use the semi-microscopic approach (Dorman et al., Biophys. J. channel. As Ca++ was added in small concentrations, it replaced 70:121 [19961) to study aspects of alkali cation energetics in the Na+ in the channel because it could provide the same KcsA K-channel. The aqueous extra- and intra-cellular regions neutralization of the channel anions with less volume and the mid-channel water pool are viewed as dielectric continua, displacement. In the second study, a voltage was applied to a while selectivity filter waters, ions, and the carbonyls forming the water/membrane/channel system either by charged electrodes at ions' coordination cages are described explicitly. Dielectric the boundaries or through a constant field applied to all charged relaxation of these groups is treated rigorously. Crucial charged atoms. In both MD systems, multiple ion crossings were observed features near the filter are also included: a-helices directed at the with a preference for Na+ over Cl- passage at moderate voltages. water pool, the pool ion, Thr7 hydroxyls and acidic residues near In the constant field case, periodic boundaries allowed continuous the peptide-water boundary. The role of individual electrical flow. Over 600 ns of simulations at three concentrations and two elements is separately assessed. Interaction between the filter voltages yielded superlinear current-voltage relations with contents and the a-helices, the pool ion and/or the TIhr75 hydroxyls saturation. The net electrical potential across the membrane is is cooperative; in contrast, interaction with the acidic residues is significantly reduced during ion flow due to series resistance in the additive. Since recent theoretical studies (Berneche and Roux, baths. The saturation implies a limit to the capacity of the channel Biophys. J. 78:2900 [2000]) raise the possibility of surprising to conduct ions. occupancy configurations, we analyze states other than those observed crystallographically. For ions separated by a single 516-Pos Board # B372 water, the double occupancy penalty is least in the GRAMICIDIN A CONDUCTANCE IS ENHANCED BY "crystallographic" occupancy scenario. We include the Born free PHLORETIN IN PHOSPHOLIPID BUT NOT energy, and find that: effective K/Na biasing occurs at both MONOGLYCERIDE BILAYERS "external lock-in" and "enhancement" sites; discrimination Steven Moothl, Kelsey Thompson', Robert Duffin', Mark Garrett', between K, Rb or Cs is relatively weak. J. Walter Woodbury2, Timothy A. Cross3, David D. Busathl; 'Brigham Young University, 2Univ. of Utah, Salt Lake City, UT, 514-Pos Board # B370 3Florida State Univ., Tallahasee, FL NON-EQUILIBRIUM MOLECULAR DYNAMICS STUDY A phosphatidyl choline bilayer like DPhPC has a large interfacial OF KCSA GATING dipole potential (IDP) compared to a monoolein (GMO) bilayer. Phil C Biggin', Indira H Shrivastava', Graham R Smith2, Mark SP Phloretin is a polar dye that absorbs to the bilayer surface and Sansom'; 'Oxford University, Rex Richards Building, South Parks reduces the interfacial dipole potential. From differences in Road, Oxford, OXON OXI 3QU United Kingdom, 2lmperial fluorination effects on gA conductances in DPhPC and GMO Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A bilayers, we concluded that the IDP inhibits conductance 3PX United Kingdom significantly in DPhPC bilayers, but not in GMO bilayers. We Ion channels are responsible for the conduction of ions through therefore hypothesized that phloretin would enhance gA biological membranes. Despite significant crystallographic conductance in DPhPC bilayers, but not in GMO. In advances in recent years, relatively little is known about how these DPhPC/decane bilayers, with IM NaCl at 100 mV, the gA conductance was increased from 13.0±0.5(3) pS to 16.5±0.6(3) or

114a Sundav- 7 POSTERS 516-520." JL 1%.F ., ~ %.,

19.8±0.1(3) pS by addition of phloretin to a concentration of 8.3 or 519-Pos Board # B375 41.7 jg/ml in the bath. No increase was found for gA channels in THREE-DIMENSIONAL POISSON-NERNST-PLANCK GMO/hexadecane bilayers. This supports the hypothesis that the SIMLATION OF OMPF PORIN lower gA conductance found in phosphocholine bilayers is due to Trudy A. van der Straaten', Robert S. Eisenberg', John M. the IDP. Supported by Al 23007. Tang', Umberto Ravaioli2, Narayan Aluru2,; 'Rush Medical Center, 1750 W Harrison St, Chicago, IL 60612, 2University of 517-Pos Board # B373 Illinois at Urbana-Champaign, 405 N Mathews Ave, Urbana, IL A UNIFIED MODEL OF PROTON CONDUCTION 61801 THROUGH GRAMICIDIN A, B AND M. Current voltage (IV) curves for individual molecules of the trimer Mark F. Schumaker', Joseph A. Gowen2, Jeffrey C. Markham2, ompF porin have been computed from a three dimensional (3-D) Sara E. Monison2, Timothy A. Cross3, David D. Busath2; Poisson-Nernst-Planck (PNP) model. The model is based on the 'Washington State University, 2Brigham Young University, self-consistent solution of Poisson's equation, describing coulomb 3Florida State University interactions and a continuity equation for each ion species, Gramicidin A (gA) has 8 tryptophans. In gramicidin B (gB), describing permeation down an electrochemical gradient. The phenylalanine (Phe) is substituted for 2 of these, and in gramicidin permanent charges residing on the porin structure were used to M (gM), Phe is substituted for all 8. These substitutions are construct a 3-D charge density distribution. PNP was solved using believed to increase the electrical potential energy of cations in the PROPHET (http://www-tcad.stanford.edu/), a rapid prototyping pore. We present proton current measurements for all 3 channels over a wide range of concentrations and voltages. Progressing W from gA to gB and gM, these show progressively extended , 150 I 1- regimes in which conductance is nearly linearly proportional to 100I concentration. The gA data are compared with a configuration space diffusion model water 50 .F using proton and reorientation u potentials of mean force (PMFs) based on those of Pomes and o Roux. Two parameters, controlling proton entrance and exit rates, H -50 are optimized to achieve a fit. The model PMFs are then perturbed _I00 by either a constant (similar to Anderson et al.) or trapezoidal *150 (similar to Dorigo et al.) potential modeling the electrostatic .200 -150 -100 -s50 so 100 150 200 contribution of the Phe substitutions. Two parameters associated with the entrance process are again optimized to achieve fits with Applied Bias (mV) the gB and gM data. The extended regimes of linearly computational platform used in the TCAD engineering community. proportional conductance seen experimentally correspond in the Calculated and experimentally measured IV curves are compared models to domains of single ion conductance. These extend to for the full trimer. Water, protein and membrane are treated as higher concentrations as the electrical potential in the pore interior uniform background media with e = 80, 20, 2, respectively. Both increases. simulation and experiment were performed with symmetric IOOmM KCl bath concentrations (activity = 77mM). Asymmetry in 518-Pos Board # B374 the IV curve reflects the longitudinal asymmetry inherent in the A MULTI-SCALE APPROACH TO COMPUTING FLUXES porin structure. The agreement between calculated and IN ION CHANNELS. II. THE MESOSCOPIC SCALE OF experimental IV is satisfactory given that PNP uses two adjustable RESOLUTION: BROWNIAN DYNAMICS. parameters (DK+ = Dci- = 8.0 xlO4cm2sl). IV curves are calculated Yuzhou Tang, Jay Mashl, Jim Schnitzer, Eric Jakobsson; Univ. of in a few hours, demonstrating that TCAD tools can simulate ion Illinois at U-C, Urbana, IL 61801 channels. In this paper we utilize output from molecular dynamics and electrostatics descriptions of the KcsA bacterial channel from 520-Pos Board # B376 Streptomyces lividans as input to Brownian dynamics (BD). The CALCIUM ION PERMEATION THROUGH THE resulting BD simulations closely emulate experimental data for CALCIUM RELEASE CHANNEL (RYANODINE single channel current-voltage and current-concentration RECEPTOR) OF CARDIAC MUSCLE. relationships in KcsA, and for radiotracer flux ratio observations DUAN CHEN1, Le Xu2, Bob Eisenberg', Gerhard Meissner&; for other K channels (we know of no radiotracer data for KcsA). 'Rush University, 1750 W. Harrison Street, Chicago, IL 60612, Since the simulations reproduce the electrophysiology reasonably 2University of North Carolina, Chapel Hill, North Carolina 27599 accurately, the observed ionic trajectories may provide a detailed Single channel current-voltage (IV) relations were measured from picture of the permeation process in KcsA. We see that the the calcium release channel (RyR2) of cardiac sarcoplasmic selectivity filter is usually occupied by two K ions in preferred reticulum in mixed solutions of 250mM alkali metal ions Na+, K+, sites separated by about seven angstroms. The most common and Cs' with sarcoplasmic reticulum lumenal Ca++ ranging from significant movement observed is a "three-ion shift" mechanism. 5mM to 50mM. Using the already published permeation properties In this motion one ion leaves the selectivity filter almost for monovalent cations (Chen, D.P., L.Xu, A.Tripathy, simultaneously with its place being taken by the ion that was in the G.Meissner, and B.Eisenberg 1999. Selectivity and permeation other preferred site in the selectivity filter, as a third ion enters the through the calcium release channel of cardiac muscle: selectivity filter from the other side. Occasionally the selectivity Monovalent alkaline metal ions. Biophys. J. 76:1346--1366.), the filter is occupied by only one ion, which has a preferred position measured IV relations were analyzed by the extended Poisson- intermediate between the two preferred positions in double Nernst-Planck (PNP) formulation to give the permeation properties occupancy. Supported by an NSF grant to EJ, with computer time of Ca"+ --- the diffusion coefficient (D(Ca)=8.4xlIO8 cm /sec) and provided by NCSA. the "excess" chemical potential (p,,(Ca)=-9lmV). The model is

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used to predict the calcium ion activity profiles in the RyR2 pore 523-Pos Board # B379 and the calcium ion fluxes in solutions of physiological interest. NO EFFECT OF LAMBERT-EATON SYNDROME ANTIBODIES ON CLONED alH CHANNELS OF THE T- Non-L-Type Voltage-gated Ca Channels TYPE FAMILY. In Kwon Park, Eric H. Middlekauff, Janine M. Pociluyko, 521-Pos Board # B377 Edward Perez-Reyes, Yong I. Kim; University of Virginia School DIFFERENTIAL EXPRESSION OF VOLTAGE- of Medicine, Charlottesville, VA 22908 DEPENDENT CALCIUM CHANNEL GAMMA ISOFORMS Lambert-Eaton syndrome (LES) antibodies specifically inhibit (GAMMA-2, -3, AND -4) IN DEVELOPING BRAIN P/Q-type Ca2+ channels and have relatively no effects on the other Maureen W. McEnery', Terry D. Copeland2, Courtney L. Vance3, types of high voltage-activated Ca2+ channels. However, Sonali Sundarraj3; 'Dept. Physiology and Biophysics and pathogenic action of LES IgG on T-type Ca2+ channels is not clear. Neuroscience, Case Western Reserve University, 'ABL-NCI, It has been reported that LES IgG reduces the T-type currents in 3Department of Physiology and Biophysics, Case Western Reserve murine DRG neurons (Proc. Nati. Acad. Sci. 93:9264, 1996). We University studied the effects of LES antibodies on cloned alH channels of The dynamic regulation of voltage-dependent calcium channels the T-type family expressed in HEK-293 cells; in situ (VDCC) from component alphal, alpha2/delta and beta subunit hybridization data suggest that alH is the predominant T channel isoforms has been shown to modulate the localization and function in DRG neurons (J. Neurosci. 19:1895, 1999). The cells were of the complex. Recently, a new neuronal VDCC subunit (gamma- treated with LES and control sera (24 hrs, 1 mg/ml IgG) and 2) had been revealed (Letts, et al., 1998) in the neurogenetic whole-cell T-type Ca2+ currents (ICaT) were recorded. Our data analysis of the epileptic and ataxic mouse, Stargazer. The loss of show that there is no significant difference between the two gamma-2 protein expression in Stargazer (Sharp, et al., 2000), groups: average peak value of "IcT was -83.9 ± 4.5 pA/pF (n=112 normally expressed in hippocampus and cerebellum, underscores cells) for the LES and -84.5 ± 3.6 pA/pF (n=1 13) for the control the importance of gamma to normal neuronal function. Molecular cells. No apparent modification in overall I-V characteristics could cloning has revealed the presence of additional gamma-2 be observed. These results suggest that calH channels are not homologues (gamma-3 and -4) that are also differentially localized affected by LES IgG. However, in view of the fact that there are at in mature brain. Using anti-peptide antibodies to these gamma least two other members of T-type channels besides alH, it is yet isoforms, we identified and characterized gamma isoform to be determined whether T-type channels are implicated in the expression in forebrain and cerebellum during development in pathogenesis of LES (supported by NIH NS38159 and MDA). normal and diseased mice. The results of gamma isoform expression were contrasted with the developmental acquisition, 524-Pos Board # B380 pattern of expression, and subcellular localization of high-voltage- ALTERED EXPRESSION AND LOCALIZATION OF activated VDCC alphal (A, B, C, D, E) subunits and other CALCIUM CHANNEL SUBUNITS IN ALPHAlA KNOCK- synaptic and non-synaptic markers. These findings indicate unique OUT MICE. characteristics of isoforms and reinforce the role of gamma A. H. gammas as critical determinants of Sharp', 0. Ikusika', S. Sundarraj', C. F. Fletche&, N. G. synaptic specialization. Copeland2, N. A. Jenkins2, T. D. Copeland2, M. W. McEnery'; 'Dept. Physiol. & Biophys., Case Western Reserve Univ., 10900 522-Pos Board # B378 Euclid Ave., Cleveland, Ohio 44106, 2NCI, Frederick, MD IDENTIFICATION OF KEY AMINO ACID RESIDUES NECESSARY FOR FAST INACTIVATION OF R-TYPE Mutations in the alphalA pore-forming subunit of P/Q-type CALCIUM CHANNELS. voltage-dependent Ca channels (VDCC) result in neurological C disorders in humans and mice including epilepsy, ataxia and Stephanie Stotz, Jawid Hamid, Michelle I Arnot, Gerald W Purkinje cell neurodegeneration. Mice genetically null for alphalA Zamponi; University of Calgary, 3330 Hospital Drive N.W., have been shown to AB T2N (knock-out) previously increase functional Calgary, 4N1 Canada expression of N-type (alphalB-mediated) VDCC in both We have recently shown that the domain I-IIlinker, IIS6 and 1S6 hippocampus and cerebellar granule cells. As the distribution regions are essential for fast inactivation of alE calcium channels patterns of alphalA and alphalB in normal mouse brains overlap, (Stotz et al., 2000. J.Biol. Chem. 275: 24575). Comparison of the we investigated the pathologic features of alphalB expression that amino acid sequences in the domain 11S6 regions of the slowly arise from the loss of alphalA. In control mice, alphalB was inactivating alC channel and the rapidly inactivating alE channel localized to punctate structures associated with both neuronal cell reveals a differences of 7 amino acids. Using site-directed bodies and neuropil. Regionally selective alterations in cellular mutagenesis, each of the different residues in domain II of the disposition of alphalB were apparent in alphalA knock-out mice. wild-type alC calcium channel have been mutated to the In the hippocampus, alphalB immunoreactivity was lost from all corresponding alE residues, and the effect of the substitutions on cell layers. Furthermore, alphalB expression in axons of the the inactivation rate was assessed by whole-cell voltage-clamp. corpus callosum was dramatically increased. In the cerebellum, Substitution of an uncharged polar amino group for a non-polar however, no alterations were apparent in alphalB distribution at amino group in two positions of the domain IIS6 segment was the light microscopic level. These changes in the cellular capable of dramatically increasing the inactivation rate of alC, and distribution of alphalB in the alphalA knock-out offer a first a double substitution of these residues resulted in inactivation rates glimpse at cell- and isoform-specific mechanisms that underlie close to those of wild-type alE channels. Substitution of the key VDCC expression and targeting in diseases of alphalA origin. alC residues with several different amino acid residues yielded an inactivation rate rank order of Ser>Arg>Cys>Glu=Thr=Asp>Phe, that 525-Pos Board # B381 indicating polarity is essential in determining the rate of EXPRESSION AND IMMUNOFLUORESCENT inactivation. our data Overall, indicate that the inner vestibule of LOCALIZATION OF VOLTAGE-DEPENDENT CALCIUM the Ca2+ channel pore contains highly localized determinants of fast inactivation. CHANNEL (VDCC) SUBTYPES IN A DIFFERENTIATING HUMAN NEUROTYPIC CELL LINE Ya Chen', Anne Marie R. Yunker2, Sonali Sundarraj2, Kirsten A. Brown2, Terry D. Copeland3, Robert W. Brown', Alan H. Sharp2, Maureen W. McEnery2; 'Dept. Phys., CWRU, 2Dept. Physio. &

116a SundavLi"in%d" y NivFPOSSTERSAPOSTFRi 5AO.;A 575-579

Bioph., CWRU, 10900 Euclid Ave., Cleveland, OH44106,, ABLU K+ from 2 to 7 mM induced seizure-like activity in hippocampal NCI pyramidal cells. Fluoxetine at 1 1tM shortened the depolarization N-type and P/Q-type VDCCs function to mediate calcium entry shift by 40% in average, thus markedly inhibited the epileptiform and contribute as scaffolding proteins for the organization of pre- activity. Similar effect was observed with inhibitors of voltage- synaptic docking sites for synaptic vesicle proteins. T-type VDCC, gatedCa2+ channels (NiCl2, nifedipine). hi hippocampal pyramidal although not coupled to neurotransmitter release, are essential to cells,FtM1 fluoxetine decreased the high-voltage activatedCa2+ establishing the resting potential and contribute to neuronal currents almost to 50%. This effect was due to the inhibition of L- oscillations. Human neuroblastoma (IMR32)cells respond to and N-typeCa2+ channels by fluoxetine. The drug inhibited the T- treatment with cAMP analogs by acquiring a neuronal phenotype typeCa2+ currentICCswith value of about 7 uM. These results accompanied by marked changes in the density of neuronal N-, provide evidence for inhibition of different types of voltage-gated P/Q-, and T-type VDCC currents (Carbone, et al., 1990). We Ca2a channels by fluoxetine at therapeutically relevant previously used this model of neuronal differentiation to examine concentrations. This effect and the attenuation of highK+-induced differentiation-specific changes in expression of alphalB and beta seizures may explain the recently reported anticonvulsant action of subunits (McEnery, et al., 1997). We have now extended these fluoxetine. studies, using specific anti-peptide antibodies in western blotting and immunofluorescence studies, to examine levels of expression 528-Pos Board # B384 and subcellular localization of alphal isoforms that constitute N-, ROLE OF THE C-TERMINUS OF THE CA CHANNEL P/Q- and T-type VDCC at various times (4,8,12,16d) after ALPHA-i SUBUNIT IN SELECTIVEINHIBITION BY G- initiation of differentiation. Expression patterns were compared PROTEIN COUPLED RECEPTORS with those of markers for plasma membranes, synaptic vesicles, Arthur A.Sinen, Chong C. Lee, Birgitte B. Simen, Vytautas I. and cytoskeleton. Our data reveal dynamic regulation of VDCC Bindokas, Richard J. Miller; University of Chicago, Chicago,I1 alphal subunit expression patterns in neuronal differentiation. 60637 Voltage dependent inhibition of N-type Ca channels is believed to occur through the direct interaction of G-protein beta-gamma 526-Pos Board # B382 subunits with the alpha-l subunit of the channel. However, DIFFERENTIAL EXPRESSION OF LOW VOLTAGE- inhibition appears to depend on the class of G-alpha molecule to ACTIVATED CALCIUM CHANNEL SUBUNITS IN BRAIN which a receptor is coupled. We now demonstrate that this DEVELOPMENT selectivity is due to structural determinants in the C-terminus of the Anne Marie R. Yunker, Sonali Sundarraj, Rachael B. Liu, alpha-I subunit. N-type channels showed robust inhibition by Kirsten A. Brown, Maureen W. McEnery; Dept. Physiology and Gi/Go coupled galanin receptors (GalR), but not by Gq coupled Biophysics, Case Westem Reserve University, 10900 Euclid galanin receptors (GalR2). However, deletions in the C-terminus of Avenue, Cleveland, OH 44106 alphalB-l produced Ca channels that were inhibited following Two families of voltage-dependent calcium channels mediate activation of both GaIRl andGalR2.Imaging studies using GFP neuronal calcium signaling. High voltage-activated calcium fusions of the C-terminus of alphalB demonstrated that activation channels (HVAs) participate in spike-induced calcium entry and of the GalR2 receptor caused translocation of the C-terminus of neurotransmitter release.Low voltage-activated calcium channels alphalB-1 to the membrane and co-localization with Galpha-q, but (LVAs) mediate calcium entry close to resting potential, not with the C-terminus of a C-terminal truncated splice variant contributing to rhythmic oscillations and burst firing. HVA alphalB-2. Inmunoprecipitation experiments demonstrated that expression is developmentally regulated, yet little is known Galpha-q interacts directly with the C-terminus of the alphalB regarding LVA subunit distribution and expression. As LVAs are subunit, and fluorescence resonance energy transfer (FRET) crucial for neuronal function and important antiepileptic experiments showed that the C-terminus interacts with theVII therapeutic targets, we generated affinity-purified anti-peptide loop. Our data are consistent with a model in which Galpha-q antibodies against pore-forming alphal LVA subunits. AlphalG, modifies inhibition of N-type Ca channels through a binding site in alphalH, or alphall antibodies recognized appropriate molecular the C-terminus of the alphalB subunit and the C-terminus in turn weight proteins differentially expressed in adult brain regions and modifies the function of theI/II loop. revealed unique expression patterns of LVA alpha subunits in development. Immunostaining verified widespread distribution of 529-Pos Board # B385 LVA alpha subunits and revealed that some regions express EXPRESSION OF T-TYPE CALCIUM CHANNELS IN multiple LVA alpha subunits, whereas other LVA alpha subunits CULTURED CELLS USING ADENOVIRAL VECTORS. were selectively expressed. Regional differences in LVA Leanne L. Cribbs, Kenneth S. Ginsburg, Donald M. Bers; Loyola subcellular localization were observed. Taken together, the data Univ. Chicago, Maywood, IL 60153 an anatomical basis for underlying LVA heterogeneity and provide activated channels are in may identify specialized roles for LVAs in contributing to Low voltage (LVA) T-type Ca2+ present activity. many different cardiovascular cell types. In contrast to L-type epileptiform Ca2+ channels, relatively little is known about the functional roles of T-type channels. Cloned T-type Ca2' channels provide a useful 527-Pos Board# B383 alternative to study their properties by expressing them in cultured INHIBITION OF THE POTASSIUM-INDUCED cell systems. We are interested in studying the contribution of T- ELECTROGRAPHIC SEIZURES BY FLUOXETINE type channel activity in native cardiac and vascular smooth muscle Ferenc Deak, Balint Lasztoczi, Krisztina Nadasy, Pal Pacher, myocytes, but these cell types are resistant to methods commonly Valeria Kecskemeti, Andras Spat; Semmelweis Univ. Dept. used to transfect recombinant plasmid DNA into established cell Physiol. and Parmacol., P.O.Box 259., Budapest, 1444 Hungary lines. Since adenoviral vectors allow efficient delivery and high Fluoxetine, a selective serotonin re-uptake inhibitor antidepressant expression of foreign DNA sequences to a wider variety of drug, is thought to reduce seizure activity. Recently, inhibition of mammalian cell types, we constructed recombinant adenoviruses different voltage-gated Na+ and K+ channels by fluoxetine was that express alH, a T-type Ca2+ channel cDNA isolated from reported. We examined the effect of fluoxetine on the high human heart. We infected HEK-293 cells with "AdEasy-HH1", a potassium-induced electrographic seizure and on voltage-gated recombinant that contains the full length coding sequence of alH, Ca2, channels using patch-clamp technique in whole-cell resulting in high efficiency expression of LVA calcium currents. configuration in rat hippocampal cells. Elevation of extracellular These currents had kinetics and voltage dependence typical for alH, and were blocked by Ni2' with a KD of 12 AM, as previously

117a 529-534.5IAd-P 34. -POSTERSPOSTERSbFAL&-JI%- u reported. We are currently studying the functional consequences 532-Pos Board # B388 of overexpressing these channels in neonatal, adult, and vascular CHARACTERIZATION OF HUMAN Al, SUBUNIT SPLICE smooth muscle myocytes in culture. VARIANTS OF T-TYPE CALCIUM CHANNELS AS EXPRESSED IN NG 108-15 CELLS 530-Pos Board # B386 jean CHEMIN, Arnaud MONTEIL, Stephan J. DUBEL, Joel EVIDENCE FOR INTRINSIC G-PROTEIN INDEPENDENT NARGEOT, Philippe LORY; IGH-CNRS UPRI 142, Montpellier PREPULSE FACILITATION OF N-TYPE CA2+ CHANNELS FRANCE CONTAINING alBI At least three genes encode T-type Ca2e channel a, subunits and Huijun Zhong', Stefan Herlitze2, William A Catterall', Todd molecular diversity of these channels is also enhanced by Scheuer'; 'University of Washington, Mailstop 357280, Seattle, alternative splicing mechanisms. Alternative splicing also WA 98195-7280, 2University of Tubingen, Ob dem Himmelreich contribute to increase diversity of T-type currents, as shown for the 7, Tubingen, 72074 Germany a,G subunit (Chemin et al. Biophys. J. submitted). Recently, we have characterized a cDNA coding for the human a,, subunit, P/Q (Cav2.1) and N-type (Cav2.2) Ca channels trigger designated all.a, (Monteil et al. J. Biol. Chem. 275:1653-16535, neurotransmitter release at many synapses. Activation of 2000) and we describe here the properties of a novel splice variant presynaptic G protein-coupled receptors (GPCRs) releases G for this subunit. The two isoforms compared in this study harbor protein fy subunits and shifts channels from an easily activated distinct C-terminal regions: 13 aa encoded by exon 33 and I aa "willing" (W) state to a more difficult to active "reluctant" (R) encoded by exon 36 are absent in a,l,. These two a,1 isoforms state. Gpy inhibition can be reversed by strong depolarization, were expressed in Xenopus oocytes, HEK-293 cells and neuronal resulting in prepulse facilitation. Two variants of Cav2.2 termed NG 108-15 cells. Calcium current kinetics were faster in neuronal alB, and alBil have different properties, and it has been proposed cells, compared to other expression systems (NG 108-15HEK-293>X. oocytes). Interestingly the aQ1lb isoform and had positively-shifted IV curves without GPCR activation. exhibited even slower current deactivation. These results suggests This was not due to tonic GPCR activity since N-ethylmaleimide that expression in neuronal cells provide a better cellular system (NEM) which blocks the action of PTX-sensitive GPCRs had no for these a,, subunits to yield more classical T-type channel kinetic effect. Also, somatostatin (SST) which activates endogenous SST behavior. receptors in tsA-201 cells did not further reduce the current or increase facilitation of alB, N-type Ca channels. Control 533-Pos Board # B389 experiments with P/Q type Cav2.1 channels confirmed modulation FUNCTIONAL CHARACTERIZATION OF A TWO- by SST and its blockade by NEM in these cells. These results DOMAIN FORM OF THE A12.1 SUBUNIT OF THE support the view that the R state of alBi is intrinsic to the channel VOLTAGE GATED P/Q TYPE CALCIUM CHANNELS and independent of G-protein modulation. Jyothi Arikkathl, Ricardo Felix', Christopher Ahern2, Roberto Coronado2, Kevin Campbell'; 'Howard Hughes Medical Institute / 531-Pos Board # B387 University of Iowa, University of Iowa College of Medicine, Iowa THE A1H CA2` CHANNEL IS THE PREDOMINANT LVA City, IA 52242, 2University of Wisconsin, 1300 University Ave., SUBTYPE IN BOVINE AND RAT ADRENAL ZONA Madison, WI 53706 GLOMERULOSA. We have previously demonstrated the existence of a two domain, Edmund M. Talley, Hongge Wang, Andrew M. Schrier, Edward 95kD form of the a,2.1 subunit of the voltage gated P/Q type Perez-Reyes, Paula Q. Barrett; Univ. Virginia School of Medicine, calcium channel in the brain (J. Neuro.,18, 641, 1998). To examine Pharmacology the potential physiological function of such a protein, we Zona glomerulosa (ZG) cells of the adrenal cortex have robust low engineered a construct encoding the first two domains and part of voltage-activated (LVA) Ca2+ currents that regulate aldosterone the II-II1 loop ofthe rabbit a 12.1 subunit (aal-1218). The construct secretion. We used in situ hybridization and whole cell recordings produces a protein of the predicted size (-136kD) in transiently to identify the LVA Ca2+ channel family members (alG,alH,alI) transfected cultured mammalian cells. The protein interacts with in rat and bovine adrenal glands. Multiple 33-mer antisense probes the ( subunit and is trafficked to the surface membranes when co- specific to each of the LVA channel mRNAs were designed from expressed with the B subunit. The functional properties of the sequences of their putative I-II loops. Bovine sequences were protein were investigated by expression of the cDNA in dysgenic obtained from ZG cells and brain cerebellum RNA using myotubes. The protein generated a high density of non-linear degenerate primers that recognize highly conserved charge movements that could be fit by the Boltzmann equation transmembrane regions flanking the I-II loop. High levels of with Qmax=3.1+0.57fC/pA. The midpoint of the charge movement expression of alH mRNA transcripts were detected in both rat and was V1/2=+1.4+3.9mV and the slope factor k=1 1.6+1.1mV. There bovine adrenals localized to the aldosterone producing ZG. By was no detectable Ca 2+ current. These results demonstrate a contrast, a weak level of alH mRNA expression was observed in functional role for the two-domain protein as a voltage sensor. We the zona fasiculata that was species dependent (bovine). aiG propose that the 95kD form of the a,2.1 protein may possess mRNA transcripts were present at extremely low levels throughout similar activity and may hence be involved in modulating and fine the rat adrenal only, compared to alI mRNA transcripts that tuning neuronal responses via protein - protein interaction. remained undetectable in both species. Whole cell recordings of isolated bovine ZG cells showed native LVA current to be 534-Pos Board # B390 inhibited by NiCI2 with an IC50 of 6.4 ± 0.2 jtM. Since this high INHIBITION OF CLONED T-TYPE CA2+ CHANNELS BY sensitivity to NiCk2 is specific to the alH subtype, we conclude NEUROLEPTIC DRUGS that the alH is the predominant LVA Ca2e channel expressed in the adrenal ZG that regulates aldosterone secretion. Celia M. Santl, F. S. Cayabyab2, J. E. McRory', K. G. Sutton', D. Parker2, K. Hamming', J. Mezeyova2, T. P. Snutch'; 'University of British Columbia, Vancouver, B.C. V6T 1Z3 Canada, 2NeuroMed Technologies Inc., Vancouver, B.C. V6T 1Z4 Canada T-tpe Ca2 channels play critical roles in cellular excitability and Ca + influx, and are also implicated in epilepsy. Here, we studied

118a v,lindlAoul& ay4IVL%. . PO)STFERSLJdLL 514_53RI r .

the effects of T-type Ca2' channel blockers (Ni2+, amiloride) and KD. We found no evidence for an anomalous mole fraction effect at several anti-psychotic agents on cloned T-type channels from rat 70 mM. In contrast, at 5 mM the unitary current amplitude and brain. Whole-cell patch clamp recordings were obtained from HEK conductance of single channels showed minima at Ba2+/Ca2+ ratio cells expressing T-type al-subunits using Ca2+ (alG, all) or Ba2+ of 1:4. Thus, at conditions close to the physiological (alH) as charge carriers. Ni2+ blocked currents as follows: alH concentrations of the charge carrier, pore occupancy and ion-ion (IC50 - 12 gM); alG (- 161 gM); all (- 201 pM). Amiloride interactions play significant roles in the ion throughput rate. (500 FM) inhibited alH by -90% and aiG and all by only -40%. Finally, we will provide data to show that modal gating in L-type The neuroleptic agents pimozide, haloperidol, penfluridol, and Ca2+ channels in hair cells is regulated by the type and flunarizine all reversibly blocked the three T-type channels. concentration of the permeant ion. Supported by NIDCD, AOS and Pimozide (50 nM) and haloperidol (1 MM) inhibited aIG, alH, and CONACyT-Mdxico. all to similar levels (-50%), while penfluridol (10 nM) more potently inhibited these currents (-80-90%). Higher sensitivity to 537-Pos Board # B393 flunarizine (I MM) was observed for alG (-73%) and all (-65%) EFFECTS OF MEMBRANE STRETCH ON than for alH (-27%). Pimozide (50 nM) shifted the steady-state RECOMBINANT N-TYPE CALCIUM CURRENTS. inactivation curves of and all -12.7 and -6.1 alG, alH, by -7.1, Barbara Calabrese, Peter F Juranka, Catherine Elizabeth Morris; mV, respectively. Similar results were obtained with the other Our indicate that the of Ottawa Hospital Research Institute, 725 Parkdale, Ottawa, Ontario neuroleptics. findings sensitivity T-type KIY 4K9 Canada channels to neuroleptic agents requires drug interaction with regions mediating channel inactivation. Mechanical stimuli enhance native voltage gated calcium currents in smooth and cardiac muscle, but no recombinant calcium 535-Pos Board # B391 channels have been examined for mechanosensitivity. We report PURINERGIC EFFECTS ON TWO TYPES OF INWARD on N-type calcium currents (ICa,N) in transiently transfected HEK CURRENTS IN MURINE COLONIC MYOCYTES. cells under whole cell clamp with and without inflation-induced membrane stretch. Cell geometry was monitored both by Kevin Monaghan, Kenton M. Sanders, Sang Don Koh; University videomicroscopy and by repeated checks of membrane of Nevada, Reno, Nevada 89557 capacitance. Barium (20 mM) was the current carrier for the ATP is a major inhibitory neurotransmitter in GI smooth muscles. channels and intracellular calcium was clamped with EGTA or ATP release induces hyperpolarization and muscle relaxation. ATP BAPTA. 'Ca,N reversibly increased whenever positive pressure activates SK channels in murine colonic myocytes associated with applied via the patch pipette resulted in detectable cell inflation. inhibitory junction potentials. ATP effects on inward current in GI (On release of pressure, cell volume returned to normal). The myocytes are unknown. We reported two types of voltage- increase in peak inward ICa,N (20-120% increase; V2= +10 mV) dependent inward currents: a negatively activating current was always coincident with inflation. Incorporation of new characterized as a non-selective cation current (IvNscc), and the channel-containing membrane can be excluded because there was high threshold voltage-activated Ca2+ current (L-type Ca2' current; no detectable change in the total capacitance. There is no evidence IckL). External application of ATP (1mM) did not affect IvNscc or that stretch shifted the I/V relations. Over time, the inactivation IcaLin cells dialyzed. However, ATP increased IvNscc and time constant of ".a.N decreased and the steady-state inactivation decreased IC,L in cells using the perforated whole cell curve shifted to the left. Stretch appeared to hasten these configuration. UTP (1 mM) caused the same effects as ATP. The irreversible effects. Unlike the reversible effect on peak current, P2 receptor inhibitor (PPADS) had no effect on either current, and the irreversible effects on the kinetics of inactivation did not in the presence of PPADS, ATP continued to increase IvNscc and require that a detectable inflation of the cell occur. Supported by decrease Ica. The phospholipase C inhibitor (U-73122, L M) also NSERC Canada had no effect on inward currents, and U-73122 did not affect the ability of ATP. Pretreatment with protein kinase C inhibitor 538-Pos Board # B394 (Calphostin C) , however, inhibited the effects of on IvNscc and MODULATION OF N-TYPE CA+ CHANNEL Ic.L. These data suggest that the effect of ATP occurs through G- PERMEATION BY NON PORE RESIDUES and activation of kinase C. In protein coupled pathway protein Zhong-Ping Feng1, Jawed Hamid', Clinton Gregory M conclusion, ATP released by neural stimulation may induce Doerin'g, relaxation activation of SK channels and Bosey2, Emmanuel Bourinet3, Terry P Snutch, Gerald W by decreasing Ic.L. 'University of Calgary, 3330 Hospital Drive Activation of IvNscc by ATP may affect the pattern of inhibitory Zamponil; NW, junction potentials, particularly the repolarization phase. Calgary, Alberta T2N 4N1 Canada, 2University of British (Supported by DK 57169) Columbia, 3IGH-CNRS UPR 1142, France It is widely accepted that divalent cation permeation of high 536-Pos Board # B392 voltage activated Ca++ channels is critically dependent on four CALCIUM CHANNELS IN HAIR CELLS EXHIBIT glutamate residues in the narrow pore region. However, despite DISTINCT PERMEATION AND GATING PROPERTIES IN the notion that these residues are conserved across all types of PHYSIOLOGICAL CONCENTRATIONS OF CHARGE high-voltage activated Cae channels, these channels do not display CARRIERS. equal permeation profiles, suggesting that other residues may be important for permeation. One candidate region is a putative EF Ebenezer Nketia Yamoahl, AdriAn Rodriguez-Contreras2; hand motif located within the domain mIS5-S6 linker region which 'University of California at Davis, 1544 Newton Court, Davis, is formed by a highly conserved glycine residue flanked by three California 95616, 2University of Cincinnati, 231 Bethesda Avenue, negative charges. We have thus combined site directed Cincinnati, OH mutagenesis with single channel patch clamp recordings to test the The patch-clamp technique was used to study the permeation and effect of this region on permeation. Replacement of residues gating properties of single L-type Ca2, channels in frog hair cells. E1321, D1323, and E1332 with arginine reduces the Ba++:Ca++ By varying the concentration of permeant ions (2-70 mM), we conductance ratio from 1.54 to 1.17 by decreasing the permeability estimated the apparent affinity constants (KD, in mM: Ba2+ = 7.4 ± for Ba++ and increasing Ca++conductance. This effect was not 1.0; Ca2+ = 7.1 + 2.2; Sr2+ = 4.0 ± 2.4; n = 6) and maximal dependent on a charge substitution as a replacement of G1326 conductances (y.,,, in pS: Ba2+ = 29.2 ± 1.1; Sr2+ = 16.2 ± 2.0; Ca2+ residue with proline to perturb the structure of the putative EF = 13.6 ± 1.1; n = 6) of the channel. To study mechanisms of ion hand motif mediated an even larger reduction in the Ba++:Ca++ permeation, we used mixtures of Ba2+ and Ca2+ with total conductance ratio (0.82). These results suggest that N-type Ca++ concentrations above (70 mM) and close (5 mM) to the apparent channel permeability is modulated by residues outside of the

119a w -1 5 -1 POSTERS Sundav- y

narrow region of the pore and may involve an extracellular Cae+ 541-Pos Board # B397 binding domain. PROTON MODULATION OF THE T-TYPE AlG CALCIUM CHANNEL. 539-Pos Board # B395 Andrei Kozlovl, Yves Maulet', Robert Cannon2, Anne Feltz', ENHANCEMENT OF P/Q-TYPE CALCIUM CHANNEL Regis C Lambert'; 'CNRS, 5, rue Blaise Phscal, Strasbourg, 67084 CURRENTS BY NITRIC OXIDE INVOLVING France, 2UCL, Gower Street, London, WC1E 6BT United OXIDATION. Kingdom Jianguo Chen', Stefan H. Heinemann2, Toshinori Hoshil; 'The Proton blocking effect on high voltage activated Ca channels has University of Iowa, Bowen 5660, Iowa City, IA 52242, 2The been instrumental to our understanding of the pore functioning. University of Jena, Jena, D-07747 Germany Mutational studies have indicated that the protonation site is We previously reported that oxidation regulates cloned P/Q-type superimposed to the four EEEE selectivity filter. From the high pK neuronal voltage-dependent Ca2+ channels expressed in Xenopus value of this locus, it was concluded that the glutamate carboxylate oocytes (Li et. al. 1998, J. Neurosci 18, 6740). We further side chains project in the pore and constitute a cation chelating site. examined potential physiological roles of oxidative regulation of We have investigated if the same characteristics hold for low P/Q-type Ca2+ channels in mammalian cells. External H202 (0.005- voltage-activated Ca channels which have a homologous EEDD 0.03%) enhanced the whole-cell Ca2ecurrents in PC12 cells in a motif. We studied effects of extracellular protons on the cloned T- voltage-dependent manner when 20 mM Ba2+ was used as the type Ca channel subunit alG expressed in HEK-293 cells. charge carrier. In BHK cells stably expressing alA/02a P/Q-type Extracellular acidification shifted Vin of activation and channels, H202 increased the whole-cell Ca2+ channel currents at inactivation to more positive potentials with pKs differing by two +20 mV when 40 mM Ca2+ was used as the charge carrier. This orders of magnitude. Deactivation was found to be relatively pH- increase was maintained after wash but reversed by the reducing insensitive. A pK of 6.5 was determined for inhibition of agent dithiothreitol (DTT, 2 mM). Bath application of nitric oxide macroscopic currents. The same 50% inhibition was obtained at (NO) donors, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 100 this pH with either divalent or monovalent ions as charge carrier. ,tM), sodium nitroprusside (SNP, 100 itM) and diethylamine Single channel data indicated a microscopic conductance close to 8 NONOate (1 mM) increased the whole-cell Ca2+ currents by more pS in 50 mM Ca2+ which was not changed with acidification from than 50 % at +20 mV and the effect was reversed by DTT. pH 7.4 to pH 6.5. After substituting aspartate HI to a glutamate, the Following adenovirus-mediated expression of eNOS activated by same magnitude of inhibition was observed at pH 6.5. These data Ca2+, the Ca2+ ionophore A23 187 increased the Ca2+ current. These indicate a distinct molecular arrangment of the pore in the T-type results suggest that P/Q-type Ca2+ channels, Ca2+ influx, activation channel. of NOS, NO may constitute a positive feedback loop mediated by oxidation. (Supported by NIH GM57654). 542-Pos Board # B398 NOVEL HUMAN AIG CALCIUM CHANNEL SPLICE 540-Pos Board # B396 VARIANTS USING THE JELLYFISH CALCIUM CHANNEL TO Aaron M Beedle, Jawed Hamid, Gerald W. Zamponi; University STUDY THE PUTATIVE PORE OF YEAST CCH1. of Calgary lustin V Tabarean, Peter F Juranka, Catherine Elizabeth Morris; a, subunits encoding T-type Ca2+ channels have recently been Ottawa Hospital Research Institute, 725 Parkdale Avenue, Ottawa, cloned and alternative splicing has been described. Here we report Ontaria K1Y 4K9 Canada the finding of novel exon deletion variants of a,G. With As the only putative voltage gated calcium channel (VGCC) in the hybridization cloning, a 4 kb aiG fragment was identified in human yeast genome, CCHI is of evolutionary interest but to date, it will fetal brain cDNA and shown to lack exons 20, 23 and 24. In not express functionally in Xenopus oocytes or mammalian cells. addition, RT-PCR of undifferentiated retinoblastoma cell mRNA The putative pore of CCH1 lacks two residues conserved in all also detected a Aexon 23 variant. Interestingly, these exon classical Ca channels. In an attempt to mimic the putative CCHI deletions are all located within putative transmembrane segments pore, we made point mutations (E339N and W341F) in the most of domain Im, including the 11S2, HISs and pore forming regions, primitive cloned VGCC, that of the jellyfish, Cyanea capillata. and thus, are unlikely to produce functional calcium channels. To Injected in Xenopus oocytes, W341F mRNA did not generate determine the tissue specific expression of these variants, an functional channels. E339N mRNA, however, yielded channels RNase protection assay designed to detect the deletion of exons 23 with permeation properties differing sharply from wild-type and 24 was carried out. The wild-type ai, fragment was expressed jellyfish VGCCs. Either 40 mM Ba or 110 mM Li were used as in all sources of human brain mRNA tested, as well as in RNA external charge carriers. Inward Li and Ba currents were much isolated form retinoblastoma cells. The exon 23 deletion was smaller than in wild-type channels, while outward currents, detected in fetal brain, adult brain, thalamus, substantia nigra, presumably carried by K ions, were larger, so whole-cell I-V cerebellum, and in retinoblastoma cells, whereas the Aexon 24 relationships displayed strong outward rectification. The changes variant was detected in fetal brain, adult brain, amygdala, caudate in ion selectivity were studied by measurements of the reversal nucleus, and retinoblastoma cells. These results raise the potential, E,,v With 40 mM Ba Erev was 64 + 4 mV (n=6) in the possibility that the deletion variants may indeed be expressed in E339N mutant and 93 + 5 mV (n=5) in the wild type. With 100 human brain, their precise function, however, remains subject to mM Li Er., was 26 +5 mV (n=4) in the E339N mutant and 43 + 4 further investigation. mV (n=2) in the wild type. Thus, a pore region mutation in jellyfish channel that mimics a key difference between jellyfish 543-Pos Board # B399 and yeast channels reduced both PB,/PK and PI,PK , thereby PROMOTER CLONING AND TRANSCRIPTIONAL rendering the jellyfish less like a calcium-selective channel and REGULATION OF THE SKELETAL MUSCLE DHPR AlS- more like a cation-selective channel. CCH1 may be a voltage- SUBUNIT. gated cation channel. Zhenlin Zheng', Patricia A Powers2, Osvaldo Delbono'; 'Wake Supported by NSERC, Canada Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157, 2University of Wisconsin, Madison, 425 Henry Mall, Madison, WI 53706 Previous studies have shown that Ca2+ (Renganathan et al., Pfltigers Arch. 438:649,1999), trophic factors (Wang et al., J.

120a Sundav- -7 POSTERSAL %-, %-F JL A-d.AL%U-0 54d-54L7J-rj-.J-r 1

Physiol. 516.2:331,1999) and aging (Wang et al. Biophys. J. Similar to IHC currents, SVI activated at more negative voltages (- 78:1947,2000) can regulate DHPRalS expression. To define the 41mV) than alC (-22mV). Peak IBa was elicited at test potentials regulatory elements that control transcription, the 2.0-kb 5'- to -2.8 mV (VO.5.a,=-14mV). Unlike in IHC, IBa inactivated during flanking region fragment immediately upstream of the mouse test pulses (pulse length, %inactivation: 400ms,29; ls,41; 2s,60) DHPRalS gene was isolated and sequenced. Sequence analysis of and was slowed, but not removed, by coexpression of ,B2a this region revealed that the proximal promoter region contains a (400ms,13, ls,23; 2s,40). SV1 exhibited typical L-type TATA box, GC box, CAAT box, Octamer element, and enhancer pharmacology (IBa stimulation 3.1-fold by 5AM BayK8644, >90% and silencer elements. RNase protection assays (RPA) suggests inhibition by 5AM isradipine, h.p. -9OmV). Splice variant 2 that DHPRalS gene transcription is initiated at a single start site containing exon 8B did not yield IBa or protein in tsA201 cells. 126-bp upstream of the ATG translation start point. Luciferase Our data show reporter constructs driven different of that alD(SV1) accounts for the low activation by promoter regions mouse threshold but not the lack of inactivation observed in DHPRaIS gene were used for transient transfection assays in IBa HIC. Exon C2C12 cells. In this preparation, DHPRalS mRNA is expressed in 8 may serve as a switch for alD subunit expression. differentiated myotubes, but not in myoblasts. The -280 region of Support: FWF (P12641,P12689), EC (HPRN-CT2000-00082), 5'-flanking DHPRaIS gene is essential for basal DHPRaIS gene OeNB, Legerlotz Foundation. transcription. Furthermore, a region located between -1076 to -700 is associated with enhanced transcriptional activity. The 546-Pos Board # B402 characterization of the promoter will allow for a better ABSENCE OF REGULATION OF SKELETAL MUSCLE T- understanding of DHPRaIS gene transcription regulation. TYPE CALCIUM CHANNEL BY DIHYDROPYRIDINE RECEPTOR SUBUNITS IN VIVO. 544-Pos Board # B400 Christine Berthier1, Patricia A Powers2, Ron G Gregg3, Roberto IMMUNOGLOBULIN G FROM LAMBERT-EATON Coronado2, Caroline Strube'; 1CNRS ERS 2019, UCB-Lyonl, MYASTHENIC SYNDROME (LEMS) PATIENTS BINDS TO Bat 401B, Villeurbanne, 69622 France, 2University of Wisconsin, HEK 293 CELLS TRANSIENTLY TRANSFECTED WITH Madison, WI 53706, 3University of Louisville, Louisville, KY SPECIFIC CA+ CHANNEL SUBTYPES. 40202 Kristin M Huntoon, Tobi L Limke, Ravindra K Hajela, William During prenatal myogenesis, skeletal muscle fibers express a T- D Atchison; Michigan State University, B338 Life Sciences, East type Ca + current, which disappears 2 to 3 weeks after birth. This Lansing, MI 48824 T-type Ca2+ channel has some biophysical and pharmacological LEMS is a paraneoplastic neuroautoimmune disorder causing properties that are similar, and other dissimilar, to those of the alH dysfunction of Ca2+ channel function in presynaptic nerve subunit expressed in heterologous systems. The differences cannot terniinals at neuromuscular junctions. Biochemical and be explained by the concomitant expression of a1, or a,, subunits electrophysiological evidence suggests that LEMS antibodies which are also encoding for T-type channels. We therefore target the a, and maybe a f subunit of the Ca2+ channels at these investigated a possible regulation of the skeletal muscle T-type terminals. LEMS antibodies have been shown to decrease Ca2+ channel by dihydropyridine receptors (DHPR) subunits which conductance via P/Q-type Ca2+ channels. However, there is also are abundantly expressed in myofibers. Since T2, y4 and ys subunits suggestion for a presynaptic unmasking of an L-type Ca2+ current regulate aiG in heterologous systems, the skeletal type DHPR Ty not normally present, raising the question if LEMS antibodies also subunit was considered a good candidate for alH regulation. We bind to L-type Ca2+ channels. We addressed this question using tested this possibility in fetal myotubes freshly dissociated from Ty immunocytochemical probes on human embryonic kidney cells knock-out mice. We also tested DHPR als and Pia subunits using (HEK 293) transiently transfected with distinct a1 subunits along dysgenic (als-null) and ,B, knock-out mice. We show that the with an a25, B subunit and jelly fish green fluorescent protein absence of als, 1 or y, subunits does not significantly affect the (GFP) reporter gene clones. Transfected cells were allowed to electrophysiological properties of T-type Ca current in skeletal grow for 72 hrs prior to labeling. al subunit specific antibodies muscle. decorated only cells transfected with the gene for that specific a, subtype and showed no cross reactive binding. Affinity purified 547-Pos Board # B403 immunoglobulin G (IgG) from two LEMS patients bound to cells DIFFERENCES IN CA2+ CURRENTS IN ACTIVE AND transfected with all (alA, aIB, aicX or a1E) subtypes. This suggests INACTIVE PITUITARY MELANOTROPES that the LEMS antibodies may be directed against multiple types of Wim Scheenen', Michiel Langeslag2, Bruce Jenks', Eric Roubos'; Ca2+ channels or may also be directed against the a28 or f 'University of Nijmegen, Toernooiveld 1, Nijmegen, 6525 ED subunits. Supported by NIH grant ES05822. All cDNA clones Netherlands, 7The Netherlands Cancer Institute, Plesmanlaan 121, were generously provided by SIBIA Neurosciences, now Merck Amsterdam, 1066 CX Netherlands Research Laboratories. The amphibian Xenopus laevis is used to study neuro-endocrine cell-activation processes. The animal adapts its skin colour to the 545-Pos Board # B401 background light condition, a process in which the pituitary PHARMACOLOGICAL AND BIOCHEMICAL melanotrope cells are involved. On a black background, the PROPERTIES OF CLASS D CA2+ CHANNEL SPLICE melanotropes become activated and secrete a-MSH; adapting to a VARIANTS. white background will inactivate the melanotropes and a-MSH Daniel Reimer, Alexandra Koschak, Josef Platzer, Irene G Huber, secretion is inhibited. The result of this activation or inactivation is Manfred Grabner, Jorg Striessnig; University of Innsbruck, Peter- a change in cell size: active cells have an extended biosynthetic Mayrstr. 1, Innsbruck, Tyrol 6020 Austria apparatus and are approximately 3 times larger than inactive cells. alD It is known that active melanotropes in vitro display spontaneous (Ca,1.3)-containing Ca2+ channels in cochlear inner hair cells Ca2+ oscillations, with a (IHC) activate at unusually negative voltages and do not inactivate. characteristic stepping pattern in the rising We cloned phase due to bursts of Ca2+-driven action potentials. Inactive cells different human alD splice variants and investigated display Ca2, oscillations, but with a different the biophysical properties of Ba2+ (20mM) inward currents (IBa) stepping pattern. We after investigated kinetics of Ca2+ currents of active and inactive expression (with ac2&+-B3) in tsA201 cells using the patch- melanotropes. When the melanotrope changes its activity state, clamp technique. Splice variant 1 (SV1) contained exons 8A, but different lacked classes of voltage-activated Ca2' channels appear on the exons 32 and 44 (in contrast to a previously cloned human cell membrane: active cells have a pronounced N-type Ca2' current alD cDNA containing exons 8A,32,44; genebank: M76558). that is absent in inactive cells. Moreover, the kinetics of the

121a 547-552--I POSTERSJL %.P S.F AL A-djL%.%-f wiindnv'R.;intiqv-

individual currents changes with the activity change. We postulate 550-Pos Board # B406 that the differences in the Ca2" currents account for the differences DIFFERENTIAL INTERACTION OF TTX AND STX WITH in Ca2+ oscillations observed. T-TYPE CALCIUM CHANNELS IN DOG ATRIUM Hui Sun, Denis Chartier, Stanley Nattel, Normand Leblanc; 548-Pos Board # B404 Montreal Heart Institute, 5000, BelangerE, Montreal, Quebec HIT RELIEF OF GJ3y INHIBITION MAY UNDERLIE 1C8 ANGIOTENSIN II STIMULATION OF NEURONAL CA2+ Two types of voltage-gated Ca channels have been identified in CURRENT heart: high-(IcL) and low-threshold (ICaT) Ca channels. A recent Jenafer Evans, Colin Sumners, Craig H. Gelband; University of study reported that the low-threshold1C in guinea-pig ventricular Florida, Department of Physiology, Gainesville, FL 32610 myocytes consists of ICaT and a tetrodotoxin (ITX)-sensitive Ica PKC dependent phosphorylation has been shown to stimulate N- component (Ia-rx). In this study, we reexamined the nature of type Ca2+ currents in neurons by relieving Gf3y inhibition of these low-threshold1C in dog atrium. Ca currents were recorded using channels. In neurons cultured from neonatal rat hypothalamus and the whole-cell patch clamp technique in Cs-loaded cells bathed in brainstem, angiotensin 11 (Ang II) causes an increase in Ca2+ Na-free solution. At a Vh of -90 mV, a transient inward current current in a PKC-dependent manner (Sumners, et al. Am J. activated near -50 mV, peaked at -30 mV and reversed around +40 Physiol, 1997). The objective of this study was to examine if the mV. It was unaffected by 30 jIM TTX or low concentrations of AngII-mediated increase in Ca2+ current may be due to relief of external Na, but was inhibited by 50 jiM nickel (by -90%) or 5 PM inhibition. Non-L Ca2+ current and R subtypes) mibefradil (by -50%), consistent with the reported properties of G(3y type (N, P/Q, Addition of 30,uM in was 60.8±6.3% of the total inward current while 39.2±6.3% (n=10) ICaT. TTX the presence of nickel enhanced the current to 41% of control shifted the curve of the current was L-type. Ca2+ current facilitation was examined level, dose-response of nickel block to the right (ECso from 7.6 to 30 pM). STX at 1 pM using a triple-pulse protocol yielding a facilitation ratio 1.17±0.03 abolished (n=l 1). A facilitation ratio >1.0 is indicative of voltage-dependent the current left in 50 pM Nickel. In the absence of inhibition. nickel, STX potently blocked 'CaT (EC5o=185 nM) and modestly Ca2+ current Recovery of inhibition following a reduced These occurs with a time constant of 39+0.01 IcL (EC5o=1559 nM). findings bear important depolarizing voltage pulse for our consistent with rates of implications understas.ding of structure-function ms (n=5), Gfy reassociation. Ang H (100 of in and that sodium and and a which relieves relationships 'CaT heart, suggest 'CaT nM) depolarizing prepulse (+80 mV, voltage- channels share a common dependent G protein inhibition) caused similar increases in the likely phylogenic ancestor. peak of the current voltage relationship (n=10). Application of Ang II (n=5) or a depolarizing prepulse (n=l 1) had no effect on the 551-Pos Board # B407 voltage dependence of Ca current activation. These data suggest INHIBITION OF NATIVE P-TYPE CA2+ CHANNELS BY (-)- that Ang II, via the AT, receptor, may increase Ca2e current by (S)-BAY K 8644 AND Ql-CONOTOXIN GVIA relieving tonic G protein inhibition in a PKC dependent manner. Elizabeth Tringham, Jonathan Dupere, Maria Usowicz; University Supported by an NRSA predoctoral fellowship, MH-12031. of Bristol, University walk, Bristol, BS8 ITD United Kingdom The functional classification of native neuronal Cae channels into 549-Pos Board # B405 different types (L, N, P, Q, R and T) has relied, in part, on IDENTIFICATION OF CAv3.2 (alH) AS A MAJOR pharmacological studies. In electrophysiological experiments, COMPONENT OF LOW VOLTAGE-ACTIVATED dihydropyridines and co-conotoxin GVIA (co-CgTx) are routinely CALCIUM CHANNELS IN RAT MAJOR PELVIC used at micromolar concentrations to identify the presence of L- GANGLION NEURONS type channels and N-type channels, respectively. However, this assumes that do not affect other Kyoun - they types of Ca2 channels. In this Seong-Woo Jeong', Jung-Ha Lee2, Byong-Gon Park', study, we have assessed the effect of the individual enantiomers of Han Kim2, In-Deok Kong', Joong-Woo Lee', Hyoweon Bang, K 8644 and of J 'Yonsei Univ. Col. Med., 2Sogang Bay o-CgTx on P-type Ca2+ channel currents (5 Sungwon Hong4; Wonju mM in the soma of adult cells. Univ., 3Chung-Ang Univ. Col. Med., 4Dong-Guk Univ. Col. Med., Ba2e) Purkinje Cerebellar slices Korea, of (250 jm) were isolated from young adult rats (male Wistar, >6 Republic weeks) and currents were evoked in cell-attached We patches by examined the low voltage-activated (LVA) Ca2+channels voltage ramps. Alternate recordings with or without drug in the expressed in male rat major pelvic ganglion (MPG) neurons using pipette indicated that (-)-(S)-Bay K 8644 inhibited -83% of the both electrophysiological and molecular biological techniques. current, with an IC50 of 30 nM. In contrast, (+)-(R)-Bay K 8644 channel currents Ca2+ were measured using the whole-cell variant had little effect. eo-CgTx inhibited -76% of the current, with an of the patch-clamp technique from enzymatically dissociated MPG IC5o of 2 pM. These results demonstrate that Bay K 8664 and neurons. When co- 10 mM Ca2+was a charge carrier, the LVA Ca2+ CgTx are not selective for L-type or N-type channels. Furthermore, currents were first activated and peaked around -50 mV and -20 the actions of Bay K 8644 at P-type channels are enantioselective. mV, respectively. Application of mibepradil, a nonhydropyridine Interestingly, the (-)-(S)-enantiomer, which activates L-type T-type Ca2+ channel blocker inhibited the LVA Ca2+ currents with channels, inhibits native P-type Ca2+ channels. a low potency (IC5o=3 eM, n=4). Furthermore, LVA Ca2+ currents were highly sensitive to Ni2+, an inorganic T-type Ca2+ channel 552-Pos Board # B408 blocker (ICso=10 OM, n=6). The molecular approach using RT- PCR DIVALENT AND MONOVALENT CATIONS revealed that alH isoform was predominant in the MPG PERMEABILITIES IN THE neurons. Taken T-TYPE CA7+ CHANNELS together, these data strongly suggest that the LVA CLONED FROM Ca2+ channel currents in male rat MPG neurons are primarily (aclH) HUMAN HEART attributed to the alH Toshihiko Kaku', T. S. Lee', E. Perez-Reyez2, K. Ono'; 'Oita among T-type channel isoforms (aIG, alH, Medical and alI). Supported by grant 2000-2-21300-008-3 from the University, 2Loyola University Medical Center KOSEF to Seong-Woo Jeong. Low voltage-activated Ca2e channels (T-type Ca2+ channels) are known for their contribution to the pacemaker potentials and conduction system in the heart, although their channel properties are still not clear. Recently al subunit of T-type Ca2+ channel (alH) was cloned from human heart, which provided direct recording of T-type Ca2ecurrent without any current contaminations. In this study, we analyzed alH whole cell currents

122a ORslntinvLiluaX POSTFRRSx %-IFL&IliI - -JL - 557-5156 recorded from transfected HEK cells using Ca2+, Ba2+( 1, 10, 50 bound population. The slow component of facilitation may mM ) and other cations as charge carriers. Transfected alH represent an increased rate of P3 binding to alB during the currents showed activation threshold at -60 mV and reached the depolarizing prepulse. maximum at -30 mV in I mM Ca2+ as the charge carrier, where midpoints (VIm) of activation and inactivation curves were -46.5 Intracellular Communication mV and -58.3 mV, respectively. These midpoints were shifted by 5 mV to depolarized direction in 10 mM Ca2+ and by 5 mV to 555-Pos Board # B411 hyperpolarized direction in 1 mM Ba2e. Frequency-dependent DIRECT MEASUREMENT OF [CA2l CHANGES IN THE reduction in the peak currents were observed at 5 Hz stimulation EXTRACELLULAR MICROENVIRONMENT OF AN but not at 0.2 Hz either in Ca2e or Ba2e as the charge carrier. INTACT POLARIZED EPITHELIUM Relaxation process of CICT were voltage-dependent and were slower in Ca2, than those in Ba2e, unlikely to the Ca2e-dependent Rosseila Caroppol, Lucantonio Debellis', David I. Soybel2, inactivation modulation in the L-type Ca2' channels. Aldebaran Marie Hofer2, Silvana Curcil; 'Universith di Bari, 165/A via Amendola, Bari, 70126 Italy, 2Harvard Medical SchooV West Roxbury VAMC 553-Pos Board # B409 MONOVALENT CATIONS ACCELERATE RECOVERY We recently proposed that extracellular Ca2+ ions participate in a OF N-CURRENT FROM OMEGA-CONOTOXIN GVIA novel forn of intercellular communication involving the BLOCK extracellular Ca2+-sensing receptor (CaR). In this model, Ca2+ extrusion from stimulated cells results in local fluctuations in Haoya Liang, Keith S Elmslie; Tulane University Health Science [Ca2i,,xt sufficient to activate CaR on nearby cells. Here we have Center, 1430 Tulane Avenue, New Orleans, LA 70112-2699 measured the profile of agonist-induced (Ca2+]..t changes in the w-Conotoxin GVIA (w-CGVIA) has been shown to be a virtually interstitial spaces of a polarized CaR-expressing epithelium, the irreversible blocker of N-channels. One exception is that high intact amphibian gastric mucosa. Single or double-barreled [Ba2+]0 accelerates the dissociation of w-CGVIA from the channels microelectrodes were inserted either in the extracellular space (Boland et al, 1994), as if Ba2+ interacts with the toxin binding site close to basolateral membrane of oxyntic cells or in the restricted on the channels. This predicts that in the absence of external space of the gastric gland lumen, respectively. Following divalent cations, unbinding of tr-CGVIA from N-channels should stimulation with the Ca2e-mobilizing agonist carbachol (100 ;tM), be negligible. o-CGVIA block was compared in zero Ba (110 mM basolateral [Ca2e],t decreased transiently by 350 ± 60 ItM, methylammonium as charge carrier) and 3 mM Ba2e. At 3 ttM, (S.E.M.; n=9, p<0.001). In contrast, [Ca2?] in the lumen of single c-CGVIA blocked similar amount of whole-cell current in zero Ba gastric glands started from a higher resting value (1.63 mM ± 0.16 and 3 mM Ba2e. As expected, the development of block in zero Ba mM vs. 1.4 mM basal), and increased by 510 ± 60 uM (n=6, was faster (T=2.6±1.1 s, n=15) than that in 3 mM Bae (Xr=38±8 s, p<0.OO) following carbachol treatment. Changes in transepithelial n=6). Surprisingly, the recovery of current in zero Ba was also fast potential induced by carbachol could not account for the observed and followed a single exponential time course (X=58±30 s), changes in [Ca2+i..t. Vectorial transport of Ca2+ in a polarized whereas the recovery in 3 mM Ba2e was so slow that no epithelium may therefore result in [Ca2?]6.t alterations sufficient to satisfactory fit could be obtained. Therefore, we measured current modulate resident CaRs. isochronically at 3 minutes after the initiation of toxin washout to gauge recovery. In zero Ba, 63±14% (n=3) of blocked current 556-Pos Board # B412 recovered, compared to 3±2% (n=3) in 3 mM Ba2+. These findings INTERCELLULAR COMMUNICATION IN SUPPORTING suggest that o-CGVIA binding site on N-channels changes in the CELLS OF THE ORGAN OF CORTI: FREQUENCY absence of divalent cations. DEPENDENCE AND MODULATION BY INTRACELLULAR CALCIUM 554-Pos Board # B410 Laura Lagostena', Bechara Kachar2, Fabio Mammano'; DIFFERING CONCENTRATION-DEPENDENCE OF 'Biophysics Sector and Istituto Nazionale di Fisica della Materia, CALCIUM CHANNEL B SUBUNIT EFFECTS ON International School for Advanced Studies, via Beirut 2-4, Trieste, EXPRESSION AND MODULATION OF AlB VOLTAGE 34014 Italy, 2Section on Structural Cell Biology, NIDCD, NIH, DEPENDENT CALCIUM CHANNELS Bethesda, MD Carles Canti, Anthony Davies, Annette C. Dolphin; University Supporting cells (Deiters' and Hensen's cells) are joined through College London, Gower Street, London, WC1E 6BT gap junctions and have been proposed to play a glial-like function The P subunits of voltage-dependent Ca2+ channels regulate their in the organ of Corti, the sensory epithelium of the cochlea. In plasma membrane expression and biophysical properties. Here we whole-mounts of the guinea-pig organ of Corti, we used confocal have injected a range of concentrations of 13 cDNA into Xenopus microscopy to visualise the network formed by gap junctions oocytes, with a fixed concentration of alB and a261 cDNA, and immunolabelled with an antibody against connexin 26. We also have shown that this results in a P3 cDNA concentration- investigated electrical coupling and its modulation by intracellular dependent increase in 13 protein expressed. A number of 13- free calcium concentration, performing simultaneous patch-clamp dependent processes have been studied. Firstly, the dependence of recording and calcium imaging in an isolated cochlea preparation. the maximum conductance on 13 occurs over a lower We observed a calcium-dependent and ATP-mediated reduction of concentration range than for the other processes, and may represent gap junction permeability, which were maintained in zero very high affinity binding of a 1 subunit responsible for trafficking. extracellular calcium. Finally, in a series of double patch-clamp Secondly, the effect of 13 on the voltage-dependence of steady- recordings we injected sinusoidal current waveforms in one cell state inactivation may be fully explained by the presence of two and measured the response in the coupled cell. Cell coupling ratios populations (A and B), representing alB without or with a decreased and phase lags increased with increasing frequency, (possibly additional) 13 subunit, bound with lower affinity, with revealing a frequency-dependent attenuation of the electrical the former population decreasing and the latter increasing with 13 communication with a cutoff frequency around 220 Hz. These data concentration. Thirdly, the rate of prepulse facilitation of the G suggest that hearing sensitivity may be affected by calcium- protein-modulated alB currents can also be separated into two dependent mechanisms that modulate ion transport as well as gap components, compatible with two identical populations of alB junction communication in the organ of Corti. channels, with the rapid facilitation rate attributed to the P subunit-

123a -11557-561-1 I -.j m x POSTERSAL N-IF %.P L JL-J.AL'%. k.0 SundavW.# y

557-Pos Board # B413 The aim of the present study was to reveal mechanisms by which SYNTHESIS OF TNF-ALPHA BY GRANULOCYTES IS rhl23 induces these effects. HL-60 cells were incubated with MEDIATED BY PROTEIN NITRATION-DEPENDENT AND rhl23 for 24 hours in the dark. Flow cytometry was used to -INDEPENDENT MECHANISMS. determine the cell cycle distribution and to estimate the expression Bashir Mnene Matata, Manuel Galiianes; University of of czclin D3, E and cyclin dependent kinase inhibitors p2IWAF' and Leicester, Clinical Sciences, Glenfield Hospital, Groby Road, P27 Ip', which were detected immunocytochemically. Apoptosis LE3 United was detected by the TUNEL assay. Expression of total Leicester, 9QP Kingdom retinoblastoma protein (pRB) was estimated by Western blot. Our We investigated the role of protein nitration on TNF-alpha(a) results demonstrated that rh123 arrested cells in G1. There was a synthesis by granulocytes obtained from blood samples of 14 significant increase in the expression of cycin D3, a decrease in healthy individuals. A million cells in 1 mL DME-F12 medium cyclin E and no marked change in expression of p21 and p27. were treated with the following agents: ONOO- (200ytM), FeTPPs Expression of total pRB was unchanged compared to control cells. (100IM) as a ONOO- scavenger, azide (100MM) which inhibits Cells were dying by apoptosis predominately in the Giphase. myeloperoxidase activity, and the combination of FeTPPs and These results indicate that the point of the cell cycle arrest in G,by azide. Cells were incubated at 37 deg C in air/5%C02 and rhl23, independent of excitation by light was past the point of stimulated with 1 ig/mL LPS. Following a 6h incubation, protein initiation of cyclin D3- and prior to intiation of cyclin E- synthesis. nitration (nmolI/lg) and TNF-a (pg/mL) levels were determined in The arrested cells appeared to preferentially undergo apoptosis. cell homogenates by ELISA assays. The results show that ONOO- induced a 2-fold increase in protein nitration in both stimulated (SC) from 14±3 to 28±3; P<0.05 and unstimulated cells (UC) from 560-Pos Board # B416 13±3 to 27±3; P<0.05. This increase in nitration did not affect GREEN PLANTS: ENVIRONMENTAL BIOSENSORS. TNF-a levels in SC (180±14 to 191±22,P-NS) and UC cells John Mwesigwa, Maia I. Volkova, John Mwesigwa; Oakwood (169±24 to 197±16, P=NS). In contrast, FeTPPs caused a 2-fold College, 7000 Adventist Blvd., Huntsville, Alabama 35896 reduction in nitration in SC (7±1; P<0.05) and UC cells Plants are exposed to a diversity of continuously varying (5.3±0.8;P<0.05) and an increase in TNF-a synthesis in SC perturbations, including acid rain, air and soil pollution, attack by (390±31;P<0.05) and UC cells (282±43;P<0.05). Azide alone did insects and pathogens, nutrient deficiencies and surpluses, drought, not affect protein nitration in SC (14±1;P=NS) and UC cells variations of temperature and illumination. Uncouplers, pesticides (12.6±1 ;P=NS), however, it increased significantly TNF-a levels in and defoliants induce fast action potentials and decrease the resting SC (290±25;P<0.05) and UC cells (313±42;P<0.05). Azide and potential in soybeans. The speed of the propagation of action FeTPPs in combination did not have an additive effect on TNF-a potentials in soybean induced by adding an aqueous solution of synthesis. In conclusion, these results suggest that the synthesis of FCCP to soil reaches 40 m/s. This is the first attempt of high-speed TNF-a by granulocytes is modulated by protein nitration- automatic measurements of action potentials in green plants. The dependent and -independent pathways. cells of many biological organs generate an electric potential that may result in the flow of electric current. Electrical impulses may 558-Pos Board # B414 arise spontaneously or they may result from stimulation. Once MECHANISMS OF BLOCK OF EPITHELIAL SODIUM initiated, they can propagate to adjacent excitable cells. The CHANNELS BY MG2+ AND OTHER DIVALENT CATIONS. change in transmembrane potential creates a wave of Ping Zhang, Cecilia Canessa; Department of Cellular and depolarization, or action potential, that affects the adjoining, Molecular Physiology. Yale University School of Medicine resting membrane. Thus, when the phloem is stimulated at any point, the action potential is propagated over the entire length of Divalent cations (Mg2+, Ca2+, Ba2+) applied to the extracellular side the plasma membrane and along the phloem with a constant of ENaC channels decrease the amplitude of the unitary currents. voltage. Once initiated, the action potential has a stereotyped form The effect is dose- and voltage-dependent and is competed by Li+ and an essentially fixed amplitude - an "all or none" response to a and Nae. However, the block differs according to the subunit stimulus. The propagation of each impulse is followed by the composition of the channels: cxfy, ay or a(. The voltage- absolute refractory period during which the fiber cannot transmit a dependence is larger in ac (8Mgz+=0.5) than in acfy or cay second impulse. The high sensitivity of protoplasm and all cell (mg2,+=0.3) indicating that divalents penetrate deeper in the pore of organelles to any natural and chemical effects is the basis for c4P channels. In addition to decreasing the conductance, Mg2+ and excitability. Ba2+ change the kinetics of ac channels by inducing discrete closures. These closures become more frequent at hyperpolarizing 561-Pos Board # B417 voltages, at increasing concentrations of the blocking cation, and ACID RAIN: BIOPHYSICAL AND when the concentration of the permeant ions is decreased. The ELECTROPHYSIOLOGICAL EFFECTS IN GREEN mechanism mediating the closures is not occlusion of the open PLANTS. pore but rather a change in the channel kinetics. The kinetics are Shaunda best described by a two-step reaction, in which the closed state in Kely, D'Jahna Thomas, Anthony Labady, John a3 channels is entered from the Mwesigwa, Maya Volkova, Tatiana Shvetsova, Alexander George only Mge-blocked state. We Volkov; Oakwood College, 7000 Adventist Blvd., Huntsville, conclude that divalent cations enter the pore of ENaC channels and Alabama 35896 block the conduction pathway by two processes: decreasing the conductance and inducing closures. Acid rain is caused by emissions of sulphur dioxide and nitrogen oxides. Once released into the atmosphere, they are chemically converted into secondary pollutants such as nitric acid and sulfuric 559-Pos Board # B415 both of PERTURBED MITOCHONDRIA FUNCTION IN HUMAN acid, which dissolve easily in water. The resulting acidic HL-60 LEUKEMIA CELLS RESULTS IN CELL CYCLE water droplets can be carried long distances by prevailing winds, ARREST returning to earth as acid rain, snow, or fog. In this research we show that acid rain induces fast action potentials and decreases the Roxanne Sperry, Frank Traganos, Zbigniew Darzynkiewicz; New resting potential in green plants. Strong dependence of pH York Medical College, 19 Bradhurst Avenue, Suite 2400, variation in soil on electrochemical signaling in green plants was Hawthorne, New York 10532 found. The automatic measurements of the electrical potential It has been reported that treatment of several tumor cell lines with difference can be effectively used in environmental plant rhodamine 123 (rhI23), a mitochondrial transmembrane potential electrophysiology, for studying the molecular mechanisms of ion probe, perturbs cell cycle progression and results in cell death. transport and the influence of external stimuli on plants.

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562-Pos Board # B418 was also inhibited. HeLa cells expressing Cx43 or Cx45 were PLANT ELECTROPHYSIOLOGY: FCCP INDUCES FAST plated at low density on glass-gridded coverslips. Dye coupling ELECTRICAL SIGNALING IN SOYBEAN. was quantified after microinjecting Lucifer Yellow (LY) or John Mwesigwa, Tatiana Shvetsova, Alexander George Volkov; Neurobiotin (NB). Chemical gating was tested using TPA at Oakwood College, 7000 Adventist Blvd., Huntsville, Alabama 500nM for 60 min. Heterotypic junctions were formed after co- 35896 culturing cells expressing Cx43 or Cx45 previously stained in red or green. Each group of data comes from 2-4 experiments and 10- Carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) 50 injections and it is expressed in % coupling ± SEM. After TPA, induces ultra fast action potentials and decreases the variation tested with LY between potential in a soybean. The speed of the of dye coupling homotypic Cx43 channels propagation action was reduced from 93±3 % to 16±8%. Similar potentials in a soybean induced by FCCP reaches up to 40 n/s. results were obtained The duration of single action potentials after treatment by FCCP is using NB (82±6 to 26±5). TPA had no effect on dye coupling 0.3 ms. Adding FCCP to soil reduces variation potential to zero. through homotypic Cx45 (72±6% to 66±12%). Dye coupling in During the experiments, the spatial distribution of protons around coverslips with co-cultured cells after TPA was 13±12 for the soybean root was photographed. All experiments showed that heterotypic Cx43-Cx45, 56±29 for homotypicCx45, and 17±16 for proton concentration around the plant surface was higher than in homotypicCx43. Cx45 channels seem not to have a chemical gate, the rest of the bulk solution. Three zones were observed around the as they do not respond to TPA or low pH (Stergiopoulos et al. plant in FCCP and control culturing solutions: orange, yellow, and Circ. Res. 84.1999). Therefore, TPA uncoupling of Cx43-Cx45 green, which correspond to pH values of 5, 6, and 7, respectively. channels suggests that Cx43 connexons are the ones gating, and The difference in color intensity of the orange zone was slightly that the mechanisms of chemical and fast voltage gating are lighter around FCCP plants than around control plants. Day/night independent. conditions did not to influence the results of measurements. FCCP does not influence turgor pressure, Ho efflux, or K+/H+ antiporter in 565-Pos Board # B421 the root of a soybean; however, it was found to reduce the rate of DIFERENTIAL REGULATION OF MOUSE AND CHICK plant maturation. The mechanism by which FCCP decreases plant CX43 GAP JUNCTIONS maturation most likely includes depolarization of the plasma Irina Vilktorovna Sokolova, William H. Fletcher; Loma Linda membrane, retardation of photosynthetic water oxidation and Loma Linda, CA 92354 respiratory electron transfer. University, Unitary currents of GJs expressed in Neuro2A cells were recorded Channels using a dual whole-cell voltage-clamp method with 140 mmoll Gap-Junction KCI in the pipettes. Fitting the amplitude histograms to Gaussian distributions revealed 3 conductance states of both mCx43 and 563-Pos Board # B419 chCx43 GJ channels: a main state of about 90 pS and much rarer MAGNESIUM BLOCK OF HCX46 HEMI-GAP- 26 pS and 106 pS states. The relative frequency of the 106 pS state JUNCTIONAL CHANNELS. L. EBIHARA, X. LIU, J.D. PAL. in mCx43 transfectants, determined as the areal ratio under the 106 Lisa Ebihara, Xiaoqin Liu, Jay D Pal; FUHS/The Chicago pS Gaussian peak relative to that under the 90 pS Gaussian peak, Medical School, 3333 Green Bay Rd., North Chicago, IL 60064 was significantly higher than in chCx43. However, the frequency In this study, we have characterized the effects of extracellular of the 106 pS state of mCx43 channels became equal to the magnesium (Mg) and holding potential (HP) on hCx46 hemi-gap- frequency of the 90 pS state after forskolin treatment (20pmol/, 30 junctional currents in Xenopus oocytes. Injection of hCx46 cRNA min.). The same treatment did not affect chCx43 GJ channels. To into oocytes induced large hemi-gap-junctional currents that test whether the differences in basal states and after forskolin are activated on step depolarization. Increasing Mg or hyperpolarizing due to the differences in the C-terminals of the two Cxs, chimeric the HP slowed the time course of activation. The time course of Cxs were constructed in which the carboxyl tails of mouse and activation had a fast and a slow component. The fast component chick Cx43 were swapped. The resulting chimeras formed GJs had a time constant of -100 ms at 20 mV. The time course of the with basal properties and a response to forskolin reflecting the C- slow component failed to reach a steady-state level, even for pulse terminal grafts. These data indicate that the differences between durations as long as several minutes. The relative contribution of mCx43 and chCx43 GJs are determined by their C-terminal the slow component to the overall time course of activation domains. Specifically, we propose that this results from the increased for higher Mg or more negative HP's. These results can 362PPS364 sequence in chick, which contrasts to the 363PSS365 be interpreted in terms of a sequential model of channel activation sequence in mouse. that had 2 kinetically distinct steps: an extremely slow activation phase corresponding to transitions between remote closed states; 566-Pos Board # B422 and a fast activation phase which corresponded to transitions ELECTRICAL PROPERTIES OF GAP JUNCTIONS IN between a nearby closed state and the open state. At higher Mg CANINE VENTRICULAR MUSCLE concentrations and/or hyperpolarized HP's, the channel resided predominantly in remote closed states and was unavailable for Jianan Yao', Penelope A Boyden', Nicholas S Peters2, Andrew L opening on subsequent application of a short depolarizing test Wit', ; 'Columbia University, 630 W. 168th Street, New York, pulse. Replacing asparagine 63 by serine in the first extracellular New York 10032, 2St. Mary's Hospital and Imperial College loop enhanced Mg block. School of Medicine, London, United Kingdom Cardiac myocytes are electrically coupled through gap junctions 564-Pos Board # B420 (GJ) composed of connexins (Cx). Cx channels expressed in CARDIAC CONNEXINS: DIFFERENTIAL EFFECTS OF oocytes and mammalian cell lines are sensitive to transjunctional HETEROTYPIC DOCKING ON VOLTAGE AND voltage (VJ). Similar voltage dependence has not been established CHEMICAL GATING. in mammalian cardiac cells due to difficulty in accurately measuring GJ conductance We Damon M Abaray', Agustin D Eric C (G,). characterized GJ channels in Martinez2, Beyer2, Alonso P canine ventricular cell pairs using a dual patch clamp method with Moreno'; 'Krannert Inst. of Cardiology, 1111 West 10th Street, large-tipped which Indianapolis, IN 46202, 2University of Chicago, 5841 S Maryland pipettes greatly minimized series resistance. MC 4060, Chicago, IL 60637 The averaged G, was 81±14 nS (mean±SE, n=31) at Vj=10 mV. At Vj beyond ±40 mV, transjuctional current (I) showed rectification Heterotypic docking of Connexin 43 (Cx43) with Cx45 inhibits (Fig.A). At to +100 fast gating of Cx43 Vj=-100 mV, instantaneous IJ-VJ relationship connexons. We investigated if chemical gating was linear (-, Fig.A inset), but steady state I, (o) decreased at VJ

125a r%r%-r%7nJtt_-JIVCbL3 P('.ql.RRP f .QlinAinx.VuiwiAo

to 6.3. Truncating the C-terminal at amino acid 294 caused a loss +100 ~~~ ~ ~ ~ ~ ~~~0 of pH, sensitivity. There were no significant changes in single channel current amplitude or voltage dependence of macroscopic A~~~~~~~~~B~~~A steady-state junctional conductance. 569-Pos Board # B425 CHARACTERIZATION OF GAP JUNCTION CHANNELS 0 (1~~~~~~~~~~~3 FORMED BY CO-EXPRESSED CX40 AND CX43. Virglnijus Vailunas', Eric C. Beyee, Joanna Gemel2, Robert Weingart3, Peter R. Brink'; 'SUNY at Stony Brook, Nichols Rd., r~~~~~~~~ID8X~~~~~~A1w4D-W -2 20 DZ 6D 8IO Stony Brook, NY 11794, 2University of Chicago, Chicago, IL -10mV 1SVJs6 60637, 3University of.Ben, Bern, .CH 3012 Switzerland beyond ±50 mV. GJ channels had a symmetric bell shaped Gjp-Vj In many tissues, co-expression of multiple connexins may result in relationship (Fig.B, n=7, G(,=steady state G,), which can be the formation of mixed (heteromeric) gap junction channels, with described by a two-way Boltzmann function, with Vos (half- different biophysical properties. We studied HeLa cells stably maximum Vj) of -68 and +65 mV. Therefore, voltage dependence transfected with Cx40 and Cx43 DNAs to compare the gating of GJ channel in ventricular cells is similar to those of Cx43 properties of gap junctions in pairs of co-transfected cells to channels, which may play a role in conduction changes when homotypic (Cx40-Cx40, Cx43-Cx43) and heterotypic (Cx40- membrane potential is depolarized in diseased conditions. Cx43) gap junctions. Cell pairs were studied using a dual whole- cell patch-clamp method, which allowed control of the voltage 567-Pos Board # B423 between cells (Vj), measurement of intercellular current (Ij), and KIETIC MODELS FOR THE GATING OF CX46 AND determination of gap junction conductance (gj) and single channel CX32E143 GAP JUNCTION HEMICHANNELS. conductance (yj ). Many of the single channel conductances Xiang Qian, Xinge Hu, Karl L. Magleby, Gerhard Dahl; resembled those of homotypic or heterotypic channels. We found University of Miami School of Medicine, Miami, FL 33101 4630 that co-expression of Cx40 and Cx43 led to a gj,.= f(Vj) with a The gating mechanisms of hemichannels of lens connexin cx46 reduced Vj-sensitivity when compared with homotypic gap and the chimera cx32E143 expressed in oocytes were studied by junctions, and/or an asymmetrical Vj-dependence reminiscent of analysis of single-channel current records. The gating of cx46 heterotypic gap junctions. These data could be fit by a combination typically involves both slow (10-20 ms) ramp-like transitions and with mixture from homotypic and heterotypic channels, provided also rapid stepwise transitions between the fully closed and fully the heterotypic form was most prominent. We conclude that the open levels, as well as to and among 4-5 apparent subconductance gating properties of co-expressing cells may be understood by levels. In contrast, the gating of cx32E,43 typically involves rapid studying mixtures of homotypic and heterotypic forms and that the stepwise transitions between two levels, with the maximum heteromeric channels are functionally insignificant to voltage- conductance being - 20% that of cx46 (Hu & Dahl, 1999, PEBS dependent gating. Letters). To test the hypothesis that the differences in gating of these two channels might arise from differences in the number of 570-Pos Board # B426 subconductance states entered during gating, we developed kinetic INHIBITION OF HUMAN CONNEXIN37 HEMICHANNELS models to describe the gating using QUB software that can take BY EXTRACELLULAR DIVALENT CATIONS subconductance levels into account (www.gub.buffalo,ed). The Michael C. Puljung, Viviana M. Berthoud, Eric C. Beyer, gating activity of cx46, including both the slow and rapid Dorothy A. Hanck; The University of Chicago, 5841 S. Maryland transitions, was approximated by a two-tiered model with six open Avenue, MC 6094, Chicago, IL 60637 each with different states, equally-spaced conductance levels, and Connexins, which assemble as dodecamers, form gap junctional seven discrete closed states. The gating activity of cx32E143 was with two and three closed channels, aqueous pores between cells that allow for exchange of simpler, typically open states, suggesting small solutes. Each dodecamer is made up of two connexons or that cx32E143 may gate by entering a subset of the states involved in the of cx46. AR32805 and hemichannels. Some connexins can form functional hemichannels, gating (NIH GM48610). as demonstrated in both native cells and in heterologous systems. Connexin37 (Cx37) is a gap junctional protein that is found in 568-Pos Board # B424 many tissues and is particularly abundant in endothelial cells. The FUNCTIONAL ROLE OF THE CARBOXYL TERMINAL gap junctional channels of human Cx37 (hCx37) show pronounced DOMAIN OF HUMAN CX50 GAP JUNCTIONAL voltage dependence and multiple conductance levels when CHANNELS expressed in pairs of communication-deficient Neuro2A cells. We Xiaorong Xul, Eric C. Beyer2, Lisa Ebihara'; 'FLJHSIThe Chicago have shown that hCx37 forms functional hemichannels in single Medical School, 3333 Green Bay Road, North Chicago, IL 60064, Xenopus laevis oocytes in solutions with low divalent cation 2University of Chicago, Chicago, IL 60637 concentrations. Unlike the gap junctional currents, which rectify, We investigated the macroscopic and single channel properties of the hemichannel currents are largely time independent and ohmic wild-type and mutant hCx5O gap junctional channels in N2A over a large voltage range. We show here that hCx37 neuroblastoma cells using the dual whole-cell patch clamp hemichannels are inhibited by low concentrations of extracellular technique. Our results show that wild-type hCx5O formed calcium ions, with a Kd of 66pM. The time course of calcium functional gap junctional channel. The macroscopic Gj, - VJ block is slow and dependent on holding potential. The relationship was well described by a Boltzmann equation with a hemichannel currents are also inhibited by extracellular slope factor (A) of 0.10, a half-inactivation voltage (Vo) of 43.8 magnesium in the millimolar range. This ionic sensitivity puts mV and minimum conductance (Gji,) of 0.23 (n=10). The single hCx37 in the class of connexins whose hemichannels are channel conductance of wild-type hCx5O channels was 212 ± 5 pS extremely sensitive to divalent block, and it is likely to have a very (n=10). Multiple long lasting substates were observed with small open probability under physiological conditions. This work conductance ranging between 31 and 80 pS. The hCx5O gap junctional channels were reversibly blocked when pH, was reduced

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was supported by HL-ROI-45466 (ECB), HL-POI-20592 (DAH), sufficient to initiate Vj-gating. The negative gating of Cx32 T32-DA-07255 (MCP). homomeric channels is believed to reflect the positive valence of the voltage sensor corresponding to the charge of the unmodified 571-Pos Board # B427 N-terminal methionine residue. A negative charge substitution at GATING POLARITY OF 'FAST' AND 'SLOW' GATES OF either the 2nd, 5th and 8th through 10th amino acid residue can CX43 GAP JUNCTIONS. reverse the polarity of Vj-gating presumably by changing the Feliksas Buusas, Michael V.L Bennett, Vytas K. Verselis; valence of the Vj- sensor. To further explore the mechanism by Albert Einstein College of Medicine, 1300 Morris Park ave, Bronx, which the polarity of Vj-gating is reversed we determined the NY 10461 gating polarity of double mutations containing either positive or negative charges at the 2nd and 5th residues. Our results indicate Gap junctions have at least two distinct gating mechanisms; one that the charge of the amino acid at the 2nd position has a greater that closes channels to a substate via fast transitions in response to effect in determining the polarity of Vj-gating of homomeric Cx32 transjunctional voltage, 'fast V, gating', and one that fully closes hemichannels. them via slow transitions in response to Vj and chemical factors, termed 'slow 'or 'loop' gating. Previously we showed that attaching Bacterial enhanced green fluorescent protein (EGFP) to the C-terminus of Channels Cx43 blocks fast but not slow Vj gating. Here we used Hela cell pairs expressing Cx43-EGFP and wild type Cx43. Cx43- 574-Pos Board # B430 EGFP/Cx43 heterotypic junctions showed fast Vj gating only for EFFECTS OF POLYAMINE VENOM TOXINS ON THE relative negativity on the Cx43 side; thus, the polarity of fast BACTERIAL PORIN OMPF. gating is negative. Zn2+ injected intracellularly on one side of Arnaud Basl6, Anne H. Delcour; University of Houston, 4800 Cx43-EGFP or Cx43 homotypic junctions slowly reduced Calhoun, Houston, TX 77204-5513 junctional conductance, gj. Extracellular Zn2+ had no effect. When The effect of polyamines on E. coli porins has been previously the Zn2e-containing side was made negative, gj slowly recovered, documented. Spermine, a polyamine based on a 12-carbon motif, but slow and fast gating were blocked for this polarity of VJ. When was found to be one of the most effective modulators of OmpF Vj was reduced, g, decreased again, but Vj gating could be seen for activity. We have extended this study by using two polyamines Vj positive on the n2+ side before block returned; Cx43 junctions from animal venoms: Philanthotoxin-433 from wasp (PhTX-433) showed both fast and slow Vj gating, while Cx43-EGFP junctions and Joro spider toxin (JSTX-3). Both contain a large showed only slow gating. Thus, both fast and slow Vj gating are benzopropanamide group and a 12-carbon chain polyamine, induced by relative negativity on the cytoplasmic side, the same as making them larger than the 600 Da-size exclusion limit of OmpF. loop gating of unapposed Cx46 hemichannels. Patch-clamp experiments were performed on OmpF-containing outer membrane fractions reconstituted into liposomes. We found a 572-Pos Board # B428 concentration and voltage-dependence of PhTX-433 in the same EFFECT OF TRANSIENT OR SUSTAINED INCREASES IN range as spermine, with an onset of inhibition at 10-100 nM. The CALCIUM CONCENTRATION ON GAP JUNCTIONAL toxin induces a high degree of flickering in the OmpF-mediated PERMEABILITY. current, but very few long closures. This kinetic signature is Monica M. Lurtz, Grant C. Churchill, Charles F. Louis; distinct from the pattern obtained in the presence of spermine. University of Minnesota, Minneapolis, Minnesota 55455 Preliminary data indicate that Joro toxin may be less effective than Addition of the Ca2e ionophore ionomycin or the agonist ATP PhTX-433. The OmpF mutant DI 13A was previously shown to effect an increase in intracellular Ca2+ (Ca2ei) in primary sheep lens resist spermine inhibition. To assess whether PhTX-433 and epithelial cell cultures. Addition of ionomycin in physiological spermine use the same molecular determinants, the effect of PhTX- medium containing 11.8mM Ca2+ resulted in a sustained increase 433 on D113A is also investigated. Supported by NIH grant in Ca2e, (>lIpM, vs. 0.lIpM in the absence of ionomycin) that AI34905. resulted in significant inhibition of AlexaFluor 594 dye transfer (transfer to 5.0±0.7 cells, vs. 16.1±0.5 cells in the absence of 575-Pos Board # B431 ionomycin). This ionomycin-mediated decrease in cell coupling MODEFICATION OF PORE-FORMING PROPERTIES OF was prevented by the addition of the calmodulin inhibitor THE ENTEROBACTER AEROGENES PORIN: A NEW calmidazolium (10 pM. Addition of the purinergic agonist ATP BACTERIAL STRATEGY AGAINST THE PENETRATION (10pM) to the lens cell cultures in physiologic media resulted in a OF ANTIBIOTICS. transient Caei increase followed by a delayed closure of gap Emmanuelle Del, Arnaud Basle', Michel Jaquinod2, Monique junctions (transfer to 5.0±0.7 cells by 4 min after agonist addition); Mallea3, Nathalie Saint', Jean Marie Pags3, G6rard Molle; dye transfer returned to control levels by 25 min. Addition of the 'UMR 6522 CNRS, IFRMP 23, Mont Saint Aignan, 76821 France, protein kinase C (PKC) inhibitor staurosporine, or the 2IBS, Grenoble, 38027 France, 3CJF 9606 INSERM, Marseille, phospholipase C inhibitor U73122 prior to ATP addition prevented 13385 France the transient closure of gap junctions but not Ca2e, elevation. Enterobacter aerogenes is responsible of respiratory tract These data indicate that the ATP-mediated reduction in lens nosocomial infections and presents an increasing multidrug epithelial cell coupling requires PKC activation, whereas the resistance. Radiolabelled antibiotic uptake rates determined with ionomycin-mediated reduction in cell coupling likely requires the wild type strain and a clinical isolate showed for this latter a calmodulin. Supported by NIH grant EY-05684. decrease of outer-membrane permeability towards antibiotics. Alterations of the major porin were identified by immunological experiments. We reincorporated in planar lipid bilayers the 573-Pos Board # B429 purified porins from E. aerogenes wild type and the resistant POLARITY REVERSAL OF VJ-GATING OF GAP isolate in order to analyze their channel parameters. A 70% JUNCTION IIEMICHANNELS reduction of single-channel conductance (from 1900 to 630 pS in Seunghoon Oh, Shira Rivkin, Vytas Verselis, Ihaddeus Andrew IM NaCI), a three fold-increase of cation selectivity and the lack Bargiello; Albert Einstein College of Medicine of voltage-closure sensitivity were observed when the resistant isolate porin is compared to the wild type protein. This reduction Our previous work has shown that a transjunctional voltage sensor of diffusion properties was confirmed by swelling is contained within the N-terminus of Cx32 and that the movement liposomes of a voltage sensor at this position in an individual subunit is

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experiments. MALDI-MS spectrometry allowed to identify three study forms a basis for a study of the uptake of antibiotics via sequence heterogeneities between the two porins and one, located porin channels. in the putative Loop 3, could be discriminating. These results indicate (i) the E. aerogenes major porin belongs to the OmpC- 578-Pos Board # B434 OmpF family (ii) the clinical isolate synthesizes a modified SECA AND PRECURSOR PROTEINS ARE INVOLVED IN channel causing a restrictive diffusion of cephalosporin. Thus, this THE PROTEIN-CONDUCTING CHANNEL OF structural modification appears as an original resistance ESCHERICHIA COLI MEMBRANES mechanism developed by bacteria aginst 8-lactams. Lian Zuo, Chun Jiang, Phang C. Tai; Georgia State University, 24 Peachtree Center Ave., Atlanta, GA 30303 576-Pos Board # B432 secretion AMPICILLIN TRANSLOCATION THROUGH OMPF The general pathway in Escherachia coli requires SecA STUDIED ON A SINGLE CHANNEL and ATP hydrolysis that allow precursor proteins to pass through LEVEL protein-conducting channels in the membranes. Voltage clamp Ekaterina M Nestorovichl, Mathias Winterhalter2, Sergei M. techniques in Xenopus oocytes were used to study the properties of Bezrukov'; 'NICHD, NIH, Bethesda, MD 20892, 2IPBS-CNRS, such protein-conducting channels. The injection of SecA and E. 205, rte de Narbonne, Toulouse, P-31077 France coli inverted membrane vesicles yielded outward currents, which were permeable to either Na or K+, but not Cl. The currents were OmpF is a general diffusion porin from the outer cell wall of significantly stimulated by precursor proteins, proOmpA, and by Gram-negative bacteria of Escherichia coli. It is the preferential functional synthetic, but not defective, LamB-signal peptides. The pathway for antibiotics like ampicillin. In the cell wall as well as channel activity is completely blocked by 3 mM sodium azide or in reconstituted systems the OmpF channel is trimeric and slightly by 5 mM AMP-PCP, a non-hydrolyzable ATP analog, both of cation-selective. Here we reconstitute single trimeric OmpF into a which are effective inhibitors of SecA protein. These results show planar lipid membrane and show that addition of ampicillin into that SecA and ATP hydrolysis are involved in the protein- the membrane-bathing solution decreases ion current through the conducting channels through which protein translocation across channel in a concentration-dependent manner. Time-resolved membranes occurs and signal peptides can open it. The properties conductance measurements reveal single-molecule events of of channel formation are consistent with those of biochemical channel blocking. One ampicillin molecule reduces the channel reactions of protein translocation with E. coli membrane vesicles. conductance by 1/3 corresponding to a complete closure of one Thus, the involvement of SecA and precursor proteins in the monomer in the trimer. The power spectra of current fluctuations protein-conducting channel of E. coli membrnes can be studied as well as statistical analysis of time-resolved blocking events electrophysiologically in Xenopus oocytes, and such a system show characteristic times of hundred microseconds. This allows provides a new means of dissecting the mechanism of protein us to draw conclusions about the underlying mechanism of translocation across bacterial membranes. antibiotic permeation. Pure diffusion would lead to a translocation time in the nanosecond range. We suggest that ampicillin glides 579-Pos Board # B435 along the channel surface electrostatically interacting with the MG2* FACILITATES OPENING OF POTASSIUM residues ofOmpF. CHANNEL OF STREPTOMYCES LIVIDANS AT PHYSIOLOGICAL PH 577-Pos Board # B433 Sudipto Das, Rosetta N. Reusch; Michigan State University, 283 A STUDY OF PORIN CHANNELS USING WATER. Giltner Hall, East Lansing, MI 48824 SOLUBLE NON-ELECTROLYTES. APPLICATION TO ANTIBIOTIC PERMEABILITY Study of the potassium channel of S. lividans has contributed Edward J.A. Lea, Khidja Mosbahi, Hamid Mobasheri; significantly to our understanding ofstructure-function relationship University of East Anglia, Norwich, NR4 7TJ United Kingdom of ion channels. Openings of the purified channel reconstituted in As a preliminary to the study of the permeability of porin channels planar lipid bilayer could only be observed at < pH 5 in the to antibiotics, we have studied first the effect of series of water absence of Mge whereas at pH 7.2, the presence of Mg2e at the soluble polymers, which have been used successfully to probe cytoplasmic side was necessary for opening of the channel in membrane channels [Merzlyak et al (1999) Biophys.J. 77,3023- liposome-protoplast vesicles. We report here that Mg2e 3033, and Bezrukov & Rostovtseva, (2000). Biophys.J.,78, 405A). significantly influences the electrophysiological characteristics of We have studied porin channel activity in planar lipid bilayers in the purified channel reconstituted in egg PC/cholesterol bilayer at the presence of penetrating and non-penetrating polyethylene physiological pH at millimolar concentrations. Under these glycols (e.g. PEG's 600 and 4000 respectively) and also in the conditions, the channel shows long stretches of openings and presence of antibiotics. In the presence of PEG 600, channel size is multiple subconductance states. The fully open state has a reduced from approximately 850pS to 250pS in IM NaCI as conductance of about 236 ± 8 pS with subconductance states at expected. The channel gating characteristics and voltage 186 ± 11 pS, 104 ± 6 pS and 43 ±5 pS in 200mM KCl, 5mM dependence are virtually unchanged. In the presence of PEG 4000, MgC12, 10mM HEPES, pH 7.4. The openings of the channel size is various unchanged, but there is little gating and the subconductance states are voltage-dependent, the fully open state events seem to be predominantly co-operative showing channel is observed less at negative openings as dimers or firquently potentials. When the trimers. This further suggests that the channel is purified without any Mgi or treated with EDTA, the explanation for gating does not lie in the part of the channel lumen channel openings are rarely observed at physiological pH. The accessed by PEG's. strong influence of Mg+ on channel opening may be explained by In similar experiments with antibiotics, we report that streptomycin stabilization of the recently reported polyphosphate molecule induces a steady membrane conductivity as well as altering the within the channel complex. porin channel characteristics, including reducing channel size. This

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580-Pos Board # B436 582-Pos Board # B438 PROPERTIES AND FUNCTIONS OF NON-SPECIFIC ION CHANNEL FORMATION OF TOLAASIN, A CATION CHANNELS IN MICROORGANISMS AND PSEUDOMONAS TOXIN PLANTS Young-Kee Kim, Kwang-Hyun Cho; Chungbuk National Hermann Bihler', Adam Bertl', Clifford L. Slayman2; University, Gaeshin-dong 48, Cheongju, Chungbuk 361-763 'Universitaet Karlsruhe, Kaiserstrasse 2, Karlsruhe, 76128 Korea, Republic of Germany, 2Yale School of Medicine, 333 Cedar Street, New Tolaasin is a 1.9 kDa peptide produced by Pseudomonas tolaasii Haven, Connecticut CT 06510 and is known to disrupt the membranes of various cells by forming Recently, non-specific cation channels (NSC's) have emerged as pores. In order to understand the molecular mechanism of tolaasin- major paths for accumulation of cations--both beneficial (e.g., K+, induced cell disruption, we investigated the pore formation of NH44) and toxic (Na+, Li')--by plants, fungi, and other organisms tolaasin and tolaasin-induced hemolysis. During the purification of which operate with very large membrane voltages. A functional tolaasin by gel permeation chromatography, we observed the prototype for NSC's has been described in the yeast Saccharo- hemolytic activities in the fractions of molecular mass between 2 myces cerevisiae (FEBS Leu. 432:59; J. Bact. 181:291), where it and more than 10 kDa. Therefore, we have suggested the accounts for many older observations on cation exchange multimerization of tolaasin molecules before the formation of (Biochem. J. 39:368; J. Gen. Phys. 64:608; B.B.A. 552:322; B.B.A. membrane pores. In the electrophysiological investigation of 936:421). This channel, designated NSC1, opens at negative tolaasin in lipid bilayer, four types of ion channels were observed membrane voltages and in ensemble can admit nanoAmp currents based on the gating behavior and conductance. The slope into single cells, at normal resting membrane voltages (-200 mV)-- conductances of these channels were 150, 250, 400, and 500 pS. sufficient to swamp the normal cytosolic ion composition in -2 The ion channels were successfully obtained at the concentration msec. NSC1 is blocked by -100 FM extracelluar divalent ions of tolaasin above 0.6 unit. However, membrane fluctuations of (Ca++, Mg++, Ba++), which also enhance the cells' resistance to the spike-shaped openings were observed at below 0.6 unit and toxic effects of high Na+ and Li'. Yeast deleted of the normal K+ tolaasin-induce leakage of microsomal 45Ca2' was also measured transporters (Trklp, Trk2p) can nevertheless grow well on on the 45Ca2+-stored microsomes at the concentration of tolaasin as moderate [K+]O (-10 mM), when NSCI is present and extracellular low as 0.1 unit. These results suggest that, while tolaasin forms divalent ions are low. NSClprobably represents an adventitious membrane channel by multimerization, less-multimerized tolaasins function, in low [Ca44., of a protein having a different primary are still able to disturb membrane although they may not form a function. A systematic search is being made for the gene(s) complete membrane channel. encoding NSC1. The following genes have been ruled out: ALRI-2, AQYI, BAP2, CCHJ, FPS1, GAL2, HOLI, HXT1&3, 583-Pos Board # B439 KHA1, MEPI-3, MID], PMP3, PMRI, PORI-2, TOKJ, & TRKI- MEMBRANE STRUCTURE OF 425-442 REGION OF 2. COLICIN El PORE FORMING DOMAIN: A SITE DIRECTED SPIN LABELING STUDY. Peptide & Toxin Ion Channels Maryann S. Vogeisang', Lukasz Salwinski2, Wayne L. Hubbell'; 'Jules Stein Eye Institute and Department of Chemistry and 581-Pos Board # B437 Biochemistry, University of Califormia, Los Angeles, CA 90095, ION CHANNELS WITH DIFFERENT SELECTIVITY FORMED 2UCLA-DOE Laboratory of Structural Biology and Molecular BY TTR - Transthyretin (thyroxine binding protein aka Medicine, University of Califormia, Los Angeles, CA 90095 prealbumin) is an amyloydogenic protein present in blood plasma Colicin El, a bacterial toxin, binds to membranes (pH <4.5) to and cerebrospinal fluid which has been implicated in transport of form voltage-gated channels. To elucidate the structure of the thyroxine and retinol in association with the retinol-binding membrane-bound form, Site-Directed Spin Labeling (SDSL) was protein. Fibril formation by ITR occurs in familial amyloid employed to identify a transmembrane helix in the sequence 402- polyneuropathy and other amyloid diseases. The three dimensional 424 (Salwinski and Hubbell, Protein Sci. 8, 562, 1999). In the structure of TTR consists of two ,3-sheets with four 1-strands each, present work, a spin labeled side chain (RI) was introduced and the association of four subunits to form a central channel with through 425-442, part of the voltage-dependent region (Merrill and two thyroxine-binding sites .We have examined the channel Cramer, Biochem. 29, 8529, 1990). The sequence-dependent forming activity of TTR in lipid bilayers, were formed from mobility and solvent accessibility of RI reveals that residues 428- azolectin or POPE/POPG mixtures. TTR at concentrations of -40 434 form a regular a-helix in solution, consistent with helix Vb of mcg/ml increased the conductance of membranes by forming ion the crystal structure (Elkins et. al., Structure 5, 443, 1997). Upon channels with several different conductances (10-300 pS). At pH binding to negatively charged membranes at pH -3.8, helix Vb 4.7, channel forming activity was higher than at pH 7.4. Membrane increases in length to at least 16 residues, extending from 427-442. conductance was voltage dependent at pH4.7. Like other amyloid The accessibility of the RI residues to collision with polar and peptides, channels formed by TTR at pH 7.4 could be reversibly non-polar paramagnetic reagents suggests that the helical segment blocked by 0.5-1 mM concentration Zn-ions. Selectivity of is localized at the water/membrane interface, with residues 428, channels changed from anion-selectivity at pH 4.7 to slight cation- 430, 434 and 437 facing the lipid phase. Although the pH- selectivity at pH 7.4. Such channels in the neuronal membrane may triggered membrane insertion of Colicin El is irreversible, be a trigger for neurodegenerative processes . TTR Channels show mobility and accessibility changes of RI reveal a reversible pH- similarity to other amyloid channels in that they are heterogeneous, dependent conformational switch in the membrane protein that long-lived, relatively non-selective, inhibited by Congo red and may be related to channel opening and closing. blocked by zinc. Yutaka Hirakura, Rushania Azimova, Rustam Azimov, Bruce Kagan; UCLA Medical School

129a .ju-r-.JuuISR4-,SRR JLPOSTE%-f Ui L JLJJLI%LiRS LiRnnd.qvL&&I%A" Y

584-Pos Board # B440 examined the interactions of a pro-apoptotic peptide derived from ION TRIGGERED INTERACTION OF A PEPTIDE the low affinity neurotrophin receptor (p75NTR) with SEQUENCE FROM SEA URCHIN PROTEIN BINDIN phospholipid vesicles. Fluorescence experiments demonstrate that WITH PHOSPHATIDYLCHOLINE MEMBRANES the peptide induces leakage from large unilamellar vesicles. The Steffen Serowy', Peter Pohl', Olaf Zschdrnig2; 'Martin-Luther- vesicle leakage is dependent on the presence of anionic lipid in the University of Halle, StraBe der OdF 6, Halle/Saale, D-06097 vesicles. Circular dichroism studies show that the NTR365 has Germany, 2University of Leipzig, Liebigstr. 27, Leipzig, D-04103 greater helical conformation in the presence of anionic lipids. Germany Leakage and helix formation exhibit similar temperature The interaction of a 18 amino acid peptide sequence (B18) derived dependence, suggesting a correlation between the two. from the sea urchin protein bindin with neutral phospholipid membranes was investigated. Monolayer techniques and 587-Pos Board # B443 ultracentrifugation based binding assays indicate that the amount SEQUENCE DETERMINANTS FOR THE FOLDING OF of bound peptide is negligible. The presence of Zn2+ does not GRAMICIDIN CHANNELS change the amount ofpeptide that is bound to phosphatidiylcholine Denise V. Greathouse', S. Shobana2, Patrick C.A. van der Wel', membrane surfaces. In contrast to these observations, PC-vesicles Roger E. Koeppe, II', Olaf S. Andersen2; 'University of Arkansas, are destabilized by B18. Catalysed by Zn2 , the peptide induces Fayetteville, AR 72701, 2Weill Medical College, New York, NY leakage of fluorescent dyes that are entrapped into the liposomes. 10021 The membrane insertion of B18 is facilitated by a transmembrane Aromatic amino acids, especially Trp and Tyr, frequently are potential. The current fluctuations observed across a voltage- located at interfacial positions of membrane proteins, where they clamped planar bilayer are irregular in size. At physiological may act as anchors. The 4 tryptophans in gramicidin A are peptide concentrations, defect formation was observed in the essential for stabilizing the membrane-spanning channel, a right- presence of Zn2+ but it was not detected when Ca2+ was added handed, single stranded (RH,SS) 36-3 N-N helical dimer. instead. Interchanging the C-terminal "Trp-D-Leu" domain of gA from (Trp-D-Leu)3Trp to (Leu-D-Trp)3Leu in gLW and V9gLW alters 585-Pos Board # B441 the position, chirality, and number of Trps. Single channel analysis EXPRESSION AND BIOCHEMICAL demonstrates that gLW forms three interconverting channel types: CHARACTERISATION OF THE BACILLUS 1) left-handed (LH),SS 16.3 helices 2) RH,SS 16-3 helices and 3) THURINGIENSIS CRY4B Al-A5 PORE-FORMING long-lived, double-stranded (DS) helices. For gLW and V9gLW the FRAGMENT DS and LH,SS channels are favored over RH,SS channels by -7- Theeraporn Puntheeranurak, Somphob Leetacheewa, Gerd 22 kJ/mol. Circular dichroism, NMR spectroscopy and size- Katzenmeier, Chartchai Krittanai, Sakol Panyim, Chanan exclusion chromatography confirm a predominant LH,SS 36-3 Angsuthanasombat; Institute of Molecular Biology and Genetics, conformation for gLW. These results suggest the "handedness" of Mahidol University, Institute of Molecular Biology and Genetics, gA channels may be determined by the Trp chirality, whereas the Mahidol University, Salaya Campus, Nakormpathom 73170 occurrence of DS channels is influenced by the number, position Thailand and packing of the Trps (and other residues). Tryptic activation of the 130-kDa Bacillus thuringiensis Cry4B 6- Interestingly, a single, conservative Ala to Val replacement in endotoxin produced protease-resistant products of ca. 47 kDa and V5gLW "switches" the predominant conformation to a non- ca. 21 kDa. The 21-kDa fragment was identified to be the N- conducting RH,DS form. Molecular modeling suggests that steric terminal five-helix bundle (al-aS) which is a potential candidate crowding of V5 is responsible for the "switch" to the non- for membrane insertion and pore formation. In this study, we have conducting DS form. constructed the recombinant clone over-expressing this putative pore-forming (PPF) fragment as inclusion bodies in Escherichia coli. The 588-Pos Board # B444 partially purified inclusions were composed of a 23-kDa IDENTIFICATION OF THE KINETIC MODEL FOR A protein which cross-reacted with Cry4B antibodies and whose N- VOLTAGE-DEPENDENT GRAMICIDIN CHANNEL. terminus was identical to that of the 130-kDa protein. Dissimilar to inclusions, the PPF inclusions were soluble when Shigetoshi Oild', Roger B. Koeppe 12, Olaf S. Andersen3; 'Fukui protoxin only Med. Univ., Japan, 2Univ. Arkansas, Fayetteville, AR the carbonate buffer, pH 9.0 was supplemented with 6 M urea. 72701, After renaturation via stepwise dialysis, the refolded PPF protein 3WeillMed. Coll. Cornell Univ., New York, NY 10021 appeared to exist as an oligomer and was structurally stable upon Heterodimeric gramicidin channels formed between Val' trypsin treatment. The refolded protein was able to release gramicidin A (gA) and F6Val' gramicidin A (F6VgA) exhibit the entrapped glucose from liposomes and showed higher activity than typical features of voltage-dependent channel proteins: voltage- the full length activated toxin, although it lacks larvicidal activity. dependent conductance transitions in the ms time range; and weak These results therefore support the notion that the PPF fragment rectification of the open-channel i-V curves (Oiki, Koeppe, consisting of al-a5 of the activated Cry4B toxin is involved in Andersen. 1995. PNAS 92: 2121-2125). The unpaired F6Val membrane pore-formation. residue is crucial for emergence of the voltage-dependent gating and conductance rectification as neither the gA nor the F6VgA 586-Pos Board # B442 homodimeric channels display those properties. The apparent PORE FORMATION BY A APOPTOTIC CYTOPLASMIC gating dipole, as evaluated from the open probability using a two- PEPTIDE OF THE NEUROTROHIN RECEPTOR P75NTR state Boltzmann function, is 20 Debye. The unpaired F6Val with Ann the dipole moment of 1.7 Debye can account for only a small Leigh Plesnilak, Martha L. Medina', James P Bolender', fraction of the gating dipole. To investigate the gating mechanism, Barbara S Chapman2; 'University of San Diego, 5998 Alcala Park, San CA a systematic kinetic analysis was performed on a single channel Diego, 92110, 2California State University, San Marcos data set recorded between -350 mV and +400 mV. Neither a two- Peptides that induce apoptosis have potential as anti-cancer state model, nor three-state models with three different therapeutics. Design of safe, effective cancer therapeutics peptides conductance states, worked well. Maximum likelihood was requires characterization of the physical and chemical properties achieved by global optimization, including two non-conducting that influence activation of cell death in neoplastic cells. We have states and two intermediate conductance states.

130a %Siin4T&vSundav POST'ERSPOSTERS 58-593I.589-593

589-Pos Board # B445 dimerization/dissociation, a process that can be detected via DISRUPTING TRYPTOPHAN'S ABILITY TO FORM fluorescence resonance energy transfer (FRET) measurements. We HYDROGEN BONDS INCREASES THE have prepared gramicidin derivatives with C-terminal fluorescent CONFORMATIONAL ACCESSIBILITY OF GRAMICIDIN labels (gram-R6G, gram-Cy5, gram-PyMPO, gram-ROX) that CHANNELS serve as resonance energy donor/acceptor pairs. These derivatives Shobana Sundaram1, Haiyan Sun2, Aung-Kyaw Chi', Denise V. have single channel properties in planar bilayers that are similar to Greathouse2, Olaf S. Andersen', Roger E. Koeppe, 12; 'Corell native gramicidin. FRET is observed in vesicles together with Univ., Weill Med. College, 1300 York Avenue, New York, NY channel formation. Efforts to detect channel formation optically in 10021, 2Univ. of Arkansas, Univ. of Arkansas, Fayetteville, AR planar bilayers are underway. 72701 Formation of hydrogen bonds between amphipathic aromatic 592-Pos Board # B448 residues and the membrane/solution interface is important for the A DISCRETE CONDUCTANCE SUBSTATE IN helical organization of membrane-spanning gramicidin A (gA) GRAMICIDIN M CHANNELS channels. We investigated this further using three gA analogues Jeffrey C. Markham', Jason Merrell', Timothy A. Cross2, David D. where TT9,1,3and15 were substituted to 'N-methyl-Trp: 1) ['Me- Busathl; 'Brigham Young University, Provo, UT 84602, 2Florida Trp9Xl3"l]gA, 2) ['Me-Trp9"'JgA, and 3) ['Me-Trp'3"5IgA. State Univ., Tallahassee, FL 32306-4005 Circular dichroism spectroscopy and size exclusion Gramnicidin A (gA) channels predominantly conduct at one level, chromatography in DMPC and DOPC vesicles reveal that all three although a broad distribution of lower conductances is also analogues form double-stranded (DS) and single-stranded observed. The primary conductance of gramicidin M (gM) conformers. The DS fraction increased in the order: 'Me- channels is known to be -8-fold lower for the conditions studied to Trp9""',13,15> 'Me-Trp9"' > 'Me-Trp'3,15. For all analogues, the DS date, but nothing has been reported about alternative conductance fraction is larger in DOPC than in DMPC. Electrophysiological states. In over ten conditions of permeant ion and concentration studies show that each analogue forms three distinct channel examined we find that -30% of the channels have conductances populations (- 45 pS, - 20 pS and - 5 pS), with lifetimes 10-20- falling between 35 and 40% of the primary level in a well-defined fold longer than for native gA channels. These results show that peak of width similar to the primary peak. The enantiomer gM- 'Me-Trp substituted gA analogues can exist in multiple, stable displays the same behavior. Because the difference between gM conformations. The results suggest that the lack of hydrogen and gA is that all gA Trps are replaced by Phe, we conclude that: bonding between aromatic residues and the membrane/solution a) The Trps cause the broad dispersity in gA; and b) Some non-Trp interface increases the conformational space accessible to the related discrete conformational variant in gM channels, perhaps membrane-spanning channels. related to backbone conformational variants, is responsible for the discrete substate in gM and gM-. Supported by A123007 590-Pos Board # B446 THE EFFECTS OF THE SIMPLE PEPTIDE CHANNEL 593-Pos Board # B449 GRAMICIDIN ON THE STRUCTURAL PARAMETERS OF ELECTROSTATIC INTERACTION GOVERNS ARTIFICIAL PHOSPHOLIPID MEMBRANES REDISTRIBUTION OF GRAMICIDIN CHANNELS IN Jay Szule, Peter Rand; Brock University, St. Catharines, Ontario BILAYER LIPID MEMBRANE INDUCED BY L2S3A1 Canada POLYLYSINE BINDING Gramicidin (gD) is an iontophoretic antibiotic of prokaryotic Yury Antonenko, A Krylov, T Rokitskaya , E Kotova , A origin. It is a simple transmembrane channel well suited for the Yaroslavov; Moscow State University study of protein channel-lipid interactions. Decapentapeptide Clustering of membrane proteins, in particular of ion channels, monomers in each monolayer of the membrane form channels plays an important role in their functioning. To further elucidate when the monomers dimerize by hydrogen bonding. The effects of the mechanism of such ion channel activity regulation, we lipids on the gating properties associated with this dimerization performed experiments with a model system comprising have been well characterized. In this study we focus on the effects negatively-charged gramicidin analog, O-pyromellitylgramicidin of gD on lipid structure using small angle x-ray diffraction. 4 (OPg), forming ion channels in bilayer lipid membrane (BLM) and mol% gD fully induces the lamellar phase (La) of polycations. The effect of polylysines on the kinetics of ion dioleoylphosphatidylcholine (DOPC) to form the inverse channels formed in BLM by OPg was studied by the method of hexagonal phase (H11). The lattice dimension, dhc,x, of this Hi, phase sensitized photoinactivation. The addition of polylysine to the decreases with increasing amounts of gD which implies that gD bathing solutions of BLM led to the deceleration of the adds negative curvature to the monolayers. Quantitatively, the photoinactivation kinetics, i.e. to the increase in the characteristic results indicate that gD adds more negative curvature than does time of photoinactivation (O) for OPg. The increase in the ionic diacylglycerol. Phosphatidylcholine (PC) and strength of the medium shifted the concentration dependence of C] phosphatidylethanolamine (PE) membranes containing gD have to higher polylysine concentrations and decreased the maximum been analysed to determine several biophysical parameters such as value of [. It was shown that in the intermediate range of monolayer bending modulus, temperature coefficients of lattice polylysine concentrations the photoinactivation kinetics displays dimensions, and gD's own intrinsic curvature. systematic deviations from the monoexponential curve and is well described by a sum of two exponentials. The experimental data 591-Pos Board # B447 were well described by theoretical dependencies of energy changes FLUORESCENT GRAMICIDIN DERIVATIVES FOR (E) occurring upon polylysine-induced formation of domains of SINGLE MOLECULE STUDIES OPg calculated according to Denisov et al. (1998). In terms of this Andrew WooUey, Tyler Lougheed, Christine Hand, Vitali model, the biexponential character of the channel kinetics at the Borisenko; University of Toronto, 80 St. George St., Toronto, intermediate polylysine concentrations can be explained by the Ontario M5S 3H6 incomplete involvement of the channels into the domain phase and/or domain size heterogeneity. The gramicidin channel can serve as a useful model system for simultaneous optical and electrical studies of channel gating. Channel gating is believed to be due to peptide

131a 594-598 POSTERS Sunday

594-Pos Board # B450 Federation, 3Utah State University, Logan, 4St. Petersburg State LIPID CHARGE EFFECTS IN ALAMETHICIN-INDUCED University, Petergof, Russian Federation CONDUCTANCE Channel-forming activity of phytotoxins produced by the plant Vicente M Aguileila', Sergey M Bezrukov2; 'Universidad Jaume bacterium Pseudomonas syringae pv. syringae: syringopeptin 22A I, Castellon, 12080 Spain, 2NICHD/ NIH, Bldg. 9, Rm. IE-122, (SP22A), syringomycin E (SRE) and methylated syringomycin E Bethesda, MD 20892 (CH3-SRE) was studied in planar lipid bilayers of different Membrane surface charge modifies conductance of ion channels by composition. The threshold for membrane activity of the changing electric potential and redistributing ionic composition in phtotoxins (SP22A>SRE>CH3-SRE) was independent from both their vicinity. We have studied the effects of lipid charge on the membrane surface charge and the concentration ofNaCl (pH 6) conductance of a multi-state channel formed in planar lipid in the membrane bathing solution (in the range of 0.1-1.0 M). At bilayers by a peptide antibiotic alamethicin. The channel the same time membrane surface charge and the electrolyte conductance was measured in a neutral (DOPE) and a negatively concentration affected properties of single channels formed by charged lipid (DOPS). The charge state of DOPS was manipulated these toxins. In uncharged membranes the ion channels are opened by the pH of the membrane-bathing solution. We find that when with negative (from the side of the toxin addition) transmembrane salt concentration in the membrane-bathing solution is decreased, potentials and closed with positive ones. The same response was surface charge manifests itself as an increase in conductance of the observed for the negatively charged membranes modified by toxins first two channel levels. Our theoretical analysis shows that both and bathed in I M NaCl. When the negatively charged membranes salt and pH dependence of the surface charge effect can be were bathed in 0.1 M NaCI the opposite response was observed. In rationalized within the nonlinear Poisson-Boltzmann approach. all systems the voltage-current curves of the ion channels formed Given channel conductance in neutral lipids we use different by the toxins used were similar. (Supported in part by the Russian procedures to account for the surface charge but only one Fund for Basic Research, grant # 00-04-49386). adjustable parameter: an effective distance from a nearest lipid charge to the channel mouth center. We show that this distance 597-Pos Board # B453 varies by 3.1 to 3.9 A upon channel transition from the minimal GATING AND CONDUCTANCE OF SYRINGOMYCIN E conducting aggregate (Level 0) to the next larger one (Level 1). CHANNELS AS MANIFESTATIONS OF AN This conclusion is in accord with a simple geometrical model of ASYMMETRICAL CHIANNEL STRUCTURE alamethicin aggregation. Valery V Malev1, Ludmila V Schagina2, Jon Y Takemoto3, Ekaterina M Nestorovich4, Sergey M Bezrukov4; 'St.Petersburg 595-Pos Board # B451 State University, Russian Federation, 2lnstitute of Cytology, USE OF PLANAR LIPID BILAYER METHOD FOR THE St.Petersburg, Russian Federation, 3Utah State University, 4NIH STUDY OF THE SELF-ASSEMBLY OF RATIONALLY Syringomycin E (SRE) channels demonstrate voltage-dependent DESIGNED CHANNEL-FORMING PEPTIDES gating: when the antibiotic is added from one side of a membrane Luiz Carlos Salay, Amalia Aggeli, Neville Boden, Peter Frank only, the channels are predominantly closed at zero potential, but Knowles, Malcom Hunter; University of Leeds, Woodhouse Lane, are opened by either positive or negative potentials depending on Leeds, West Yorkshire LS2 9JT the membrane surface charge. For negatively charged bilayers the Natural transmembrane ion channels play a vital role in processes inversion in the sign of the applied potential opening the channels like nerve transmission, homeostasis and control of blood flow to is observed under increasing salt concentration or decreasing pH. the heart, among others. There is a great reduction in structural These observations suggest that the Coulomb forces involved in complexity in switching from channel proteins to channel-forming the work of channel opening act directly on the asymmetrical peptides, so the study of latter provide a powerful approach to channel structure that includes lipid molecules. Conductance of an understanding the ion conduction in pores and the structure- open single SRE channel also shows an asymmetrical organization function relationship of ion channels proteins. Moreover, to create of the channels. We assume that some dipole constituents resulting artificial transmembrane ion channels using rationally designed from the inclusion of lipids into the channel structure are important self-assembling peptides with controllable properties is a challenge in the gating process. The observed effects of a dipole-modifying that may lead to biomedical and nanotechnological applications. agent, phloretin, strongly support this assumption. The estimates based on the data allow us to We have been exploiting protein self-assembly as a route to analysis partially reconstruct the SRE novel functional nanostructures. Here we channel structure. (Supported by the Russian Fund for Basic designing present the ion Research, grant # channel activity of four systematically-altered de novo channel- 00-04-49386). forming peptides (DNI, DNI-QF, DNI-2E and DN1-30RNIQ). Under the same experimental conditions (pH 7.5, Rpi= 11500, 598-Pos Board # B454 450mM KCI cis: 150mM KCI trans) those peptides show different INFLUENCE OF WATER-SOLUBLE POLYMERS ON channel current levels and forms that reflect not only a different VOLTAGE DEPENDENCE OF SYRINGOMYCIN E ION channel pore structure but also different assemblies states of CHENNELS transmembrane peptides. Characterisation of the peptide Yuri A Kaulin', Ludmila V. Schagina2, Jon Y Takemoto3, John H conformation in model membranes, as well as change of the Teeter', Joseph G Brand'; 'Monell Chemical Senses Center, 3500 experimental conditions are under way. Monell Chemical Senses Center, Philadelphia, PA 19104-3308, 2lnstitute of Cytolog, Tikcoretsky ave, 4, St. Petersburg, 194064 596-Pos Board # B452 Russian Federation, Utah State University, Logan COMPARISON OF THE CHANNEL-FORMING ACTIVITY We demonstrated earlier that syringomycin E (SRE) forms OF THE PHYTOTOXINS, PRODUCED BY voltage-dependent ion channels in planar lipid bilayers. Single PSEUDOMONAS SYRINGAE PV. SYRINGAE IN PLANAR channel conductance increases two fold when the applied voltage LIPID BILAYERS is changed from -50 to -200 mV. To clarify the origin of the Philip A Gurnev', Yuri A Kaulin2, Jon Y Takemoto3, Valery V voltage dependence of SRE channels we studied the influence of Malev4, Ludmila V Schaginal, Joseph G Brand2; 'Institute of the neutral polymers, poly(ethylene glycol)s (PEGs), of different Cytology RAS, Tikhoretsky ave, 4, St. Petersburg, 194064 Russian molecular weight on the conductance of SRE channels at different Federation, 2Monell Chemical Senses Center, 3500 Monell applied potentials. Small PEGs (200 - 600), which are small Chemical Senses Center, Philadelphia, PA 19104-3308 Russian enough to penetrate the channel, blocked channel current at both low and high applied voltages. However these PEGs blocked

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channel current less at high negative voltages, where the channel 601-Pos Board # B457 conductance is higher. The results are discussed by considering the PROBING THE LOCATION OF A CONSTRICTION IN A geometric asymmetry of the SRE channels. PROTEIN PORE BY TARGETED COVALENT ATTACHMENT OF POLYMERS. 599-Pos Board # B455 Liviu Movileanu, S. Cheley, S. Howorka, 0. Braha, H. Bayley; PHLORETIN AFFECTS PROPERTIES OF Med. Biochem. & Gen., The Texas A&M University System SYRINGOMYCIN E CHANNELS FORMED IN LIPID Health Science Center, College Station, TX 77843-1114 BILAYER MEMBRANES We used cysteine mutagenesis and chemical modification with Ludmila V Schaginal, Philip A Gurnevy, Yuri A Kaulin2, Jon Y sulfhydryl-reactive polymers to locate the constriction in the lumen Takemoto3, Joseph G Brand2, Valery V Malev4; 'Institute of of the a-hemolysin pore, a model protein of known structure. The Cytology RAS, Tikhoretsky ave, 4, St. Petersburg, 194064 Russian rates of reaction of polymeric reagents with the individual single Federation, 2Monell Chemical Senses Center, 3500 Monell cysteine mutants (along the lumen axis) were determined by Chemical Senses Center, Philadelphia, PA 19104-3308 Russian macroscopic current recording. The rate of reaction of the 5 kDa Federation, 3Utah State University, Logan, 4St. Petersburg State polymer dropped dramatically at Cys- 111 and positions distal to University, Petergof, Russian Federation Cys- 111, whether the polymeric reagent was applied from the cis As demonstrated earlier, syringomycin E (SRE) added at the cis- or trans side of the bilayer. This semi-quantitative analysis sufficed side of a lipid bilayer forms voltage-sensitive ion channels. In to demonstrate that the constriction is located at the midpoint of negatively charged bilayers divided by 0.1 M NaCl (pH 6) the pore lumen, as predicted by the crystal structure. While the solution, the channels are opened by cis-positive voltages and constriction allows a 2.5 kDa polymer to pass, transport of the 5 closed by negative ones. The inversion in the voltage response is kDa molecule is greatly restricted. A single PEG chain gave a observed in 1 M NaCl (pH 6) solutions. The SRE channels formed greater reduction of conductance when covalently attached to a in uncharged bilayers in 0.1 - 1.0 M NaCl (pH 6) solutions show a narrower region of the lumen. Furthermore, this study response to voltage changes that is similar to that observed in demonstrated the passage of neutral flexible polymers across the negatively charged bilayers at 1 M NaCl. Here we present data on entire lumen of the channel. the influence of phloretin on the channel-forming activity of SRE in lipid bilayers. Addition of 100 mM phloretin to the membrane 602-Pos Board # B458 bathing solution results in uniformity of the voltage sensitivity of DYNAMICS OF A NEUTRAL FLEXIBLE POLYMER IN SRE channels: they are opened with cis-positive applied voltages THE LUMEN OF A TRANSMEMBRANE PROTEIN PORE. and closed with negative ones, independently of the membrane Liviu Movileanu, S. Howorka, S. Cheley, 0. Braha, H. Bayley; surface charge and the NaCl concentration. At the same time, Med. Biochem. & Gen., The Texas A&M University System phloretin strongly increases the cluster formation of SRE channels. Health Science Center, College Station, TX 77843-1114 The results are discussed the of considering charge asymmetry We present here two distinct approaches to study the behavior of SRE channels, as well as the well-known fact that phloretin decreases the dipole potential of lipid membranes. (Supported in neutral flexible polymers in the lumen of the a-hemolysin pore part by the Russian Fund for Basic Research, grant # 00-04- (acHL). First, a polyethylene glycol chain (3.4 kDa) was tethered at 49386). a defined site in the large vestibule [1]. The free end of the polymer was covalently attached to a biotin molecule. The biotinyl group moves across the lumen, as detected by reversible captures 600-Pos Board # B456 with a mutant streptavidin. Our results show how engineered POINT MUTATIONS AFFECTING THE ELECTRICAL protein pores can be used, at the single-molecule level, as PROPERTIES OF STAPHYLOCOCCAL F-HEMOLYSIN stochastic sensor elements for protein analytes at nanomolar CHANNELS concentrations [2]. Secondly, cysteine mutagenesis and polymeric Mauro Dalla Serra', Massimiliano Comai', Manuela Coraiola', sulfhydryl reagents were used to study the partition of flexible Sandra Werner2, Didier Colin2, Gilles Prevost2, Gianfranco polymers into acHL when present at millimolar concentrations. We Menestrina'; 'ITC-CNR Centre for the Physics of the Aggregated found a good agreement between experiment and the scaling States, via Sommarive 18, Povo, Trento 38050 Italy, Univ. L. theory for a flexible chain confined in a tube. Pasteur, 3, rue Koebertd, Strasbourg, F-67000 France [1] S. Howorka, L. Movileanu, X. Lu, M. Magnon, S. Cheley, 0. S. aureus y-hemolysins are bi-component toxins forming a family Braha and H. Bayley. 2000. J. Am. Chem. Soc., 122(11), 2411- with leucocidins and a-toxin. Two active toxins are formed 2416; combining either HlgA or HlgC with HlgB. The conductance of y- is [2] L. Movileanu, S. Howorka, 0. Braha and H. Bayley. 2000. Nat. hemolysin pores slightly larger than that of a-toxin and they are In cation- instead of anion-selective. Based on the 3D structure of the Biotechnol., 18(10) press. HlgB monomer and the a-toxin pore, and on the alignment of all sequences, a highly conserved region of 4 charges near the pore 603-Pos Board # B459 entrance was found. Such charges are all positive in a-toxin, PORE FORMATION BY HAGFISH INTESTINAL whereas one is negative in HlgA and HlgC, and two are negative in ANTIMICROBIAL PEPTIDES HlgB. To investigate their role, the following 4 mutants were Gorka Basafiezl, Ann E. Shinnar2, Joshua Zimmerberg'; 'NIH, 10 produced and tested: HlgAD44K, HlgBD48K, HIgBD50K and Center Drive, Bethesda, MD 20892, 2Barnard College, New York, HlgBD48K/D50K. In AB pores, the selectivity changes from NY 10027 slightly cationic to slightly anionic in the order: AB, AB50, Hagfish intestinal antimicrobial peptides (HFIAP) constitute a A44B50, AB48, A44B48, AB48/50, A44B48/50. The last couple, family of linear, cationic peptides showing potent, broad-spectrum with all D changed to K, has practically the same selectivity, antibacterial activity but very low antifungal or hemolytic activity. conductance and I-V curve of a-toxin. This confirms that the In an attempt to unravel the mechanism of action of HFIAP, we cluster of charges at the pore entrance has an important function as have begun to study their effect on model lipid membranes. electrostatic selectivity filter. CB pores are more cation selective, HFIAP-1 (37 amino acids) and HFIAP-3 (30 amino acids) caused and the coupling with HlgB mutants has a less dramatic effects fast and extensive release of ANTS in liposomes mimicking E. coli than with AB, although a small decrease in conductance was also membranes, but had a much less pronounced effect in liposomes observed. The role of other charged groups is under investigation. mimicking C. albicans or erythrocyte membranes. HFLAP-induced liposome leakage occurred concomitantly with liposome aggregation, followed by lipid mixing. Addition of PEG-ylated

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lipids to these membranes inhibited vesicle aggregation and lipid titrated against melittin in dimethyl sulfoxide, methanol, aqueous mixing without inhibiting liposome leakage, demonstrating that buffer, and dodecylphosphocholine micelles. The change in vesicle aggregation is not a pre-requisite for HFIAP-induced chemical shift of Trp19 resonances and the formation of a membrane permeabilization. Interestingly, HFIAP-1 but not precipitate at 1:1 mole ratio indicated binding. Solid-state NMR HFIAP-3, formed pores that allowed passage of high molecular spectra showed a change in chemical shift of two specifically weight dextrans, and potently decreased planar bilayer membrane labelled carbons near Pro14 of melittin and a change in 1H Tl lifetime. Altogether, these results are consistent with a model relaxation times when complexed with inhibitor. Rotational where HFIAP exert their host-defense activity by breaching the resonance experiments of melittin labelled in the proline region perneability barrier of bacterial membranes. indicated a change in conformation when complexed with inhibitor. This observation was also supported by CD data 604-Pos Board # B460 indicating a reduction in alpha helicity as inhibitor bound to AMPHOTERICIN B ACTION ON ERYTHROCYTES AND melittin. Details of the binding site in the melittin-inhibitor LECITHIN MEMBRANES. A TEMPERATURE WINDOW complex are being extracted using lanthanide ions which quench OF ACTIVITY. NMR resonances from molecular sites that are in close proximity Berenice Venegas', Heliodoro Celis2, Ivan Ortega-Blake'; 'Centro to the ion. Preliminary data have indicated that the inhibitor is de Ciencias Fisicas-UNAM, Av. Universidad s/n, Cuernavaca, bound to melittin at Ala15. It is proposed that the inhibitor binds in Morelos 62210 Mexico, 2lnstituto de Celular-UNAM the vicinity of Alal5 to Trpl9 and prevents insertion of melittin Fisiologia into cell membranes. The amphotericin B (AmB) is a widely used antibiotic though the side effects that it produces. The antibiotic action is thought to be due by channels formed in the membrane ofthe cell and its activity 607-Pos Board # B463 is greater in membranes containing sterols. To have a better insight CHANNEL FORMATION BY MAGAININ 2 IN VARIOUS on the AmB toxicity we made bilayers of erythrocyte lipids and BLACK LIPID MEMBRANES lecithin at the tip of a micropipette and compared the single Gallucci Enrico, Meleleo Daniela, Micelli Silvia, Picciarelli channel recording of both systems. The channel activity was Vittorio; University, V. E. Orabona 4, Bari, Italy 70126 Italy measured as a function of the temperature. In the erythrocytes we Magainin 2, a channel-forming peptide with antibiotic and found that there is a maximum in activity at around 37°C (± 30). tumoricidal activity, has been widely studied for its potential In order to know if this result was a particularity of the therapeutic application [1].This action has been attributed to its erythrocytes membranes we performed experiments with lecithin ability to form channels that dissipate the ionic gradient in the containing cholesterol (10, 20, 30 and 50 mol%) and without it. presence of membrane potential.The permeabilizing activity has The same result is seen only when the bilayer has 30 and 50 mol% also been demonstrated in liposomes in the absence of potential of cholesterol. This is hard to explain since apparently there is not [2].To evaluate the role played by lipid polar heads in the an important change in the physical properties of neither the incorporation process, membranes of Phosphatidylcholin, membrane nor the channels itself. We can think in a combined Phosphatidylcholin-phosphatidylglycerol(85:15%), Monoolein effect between membrane structure provided by cholesterol and the were used. AC method was used to monitor macroscopic thermal energy acquire by AmB allowing it for an easier diffusion incorporation [3].Single-channel experiments were performed in in the bilayer and so, a better formation of the aggregates; after the usual way. No steady state membrane conductance was increasing more the temperature, this thermal energy disrupt the detected during macroscopic Magainin 2 incorporation, unlike structure of the channel. peptides such as gramicidin A or calcitonin, indicating a different mechanism of interaction. In Monoolein membrane single-channel 605-Pos Board # B461 experiments, Magainin 2 acted with a periodic pattern of STRUCTURE OF MELITTIN PORES IN LIPID BILAYERS paroxysms of channel activity interrupted by silent periods; this Lin Yang, Thomas M Weiss, Lai Ding, Huey W Huang; Rice may indicate reduced peptide density facing the membrane after University, 6100 Main St., MS-61, Houston, TX 77251 the "burst". With polar membranes, no paroxysms were observed, We present evidence that indicates that melittin forms toroidal and the paucity of the channels' appearance made single-channel in similar to those induced analysis difficult.[1]Cruciani et al. 1991,PNAS; [2]Matsuzaki et al. pores lipid bilayers, by magainins. 1995, et Melittin is a cell Biochemistry 34:6521;[3] Micelli S. al. well-known lysing agent. Its structure and 2000,Bioelectrochemistry 52:63-75 function are similar to antimicrobial peptides. Many ofits activities depend on its ability to form transmembrane pores. Although it is commonly assumed that these pores are of the barrel-stave type 608-Pos Board # B464 like alamethicin pores, their structure has not been elucidated. MEMBRANE TOPOGRAPHY OF THE HYDROPHOBIC With oriented circular dichroism and x-ray/neutron scattering, we HELICES OF DIPHTHERIA TOXIN T DOMAIN studied melinttin in aligned lipid multilayers, varying temperature Mchael P. Rosconi, Gang Zhao, Tsu-Fan Cheng, Erwin London; and water content. We found the geometric parameters of melittin State University of New York at Stony Brook, Stony Brook, NY pores incompatible with the barrel-stave model. Further, in contrast 11794-5215 to alamethicin pores, these pores can crystalize, like magainin In the low pH-induced membrane inserted state we found the pores. hydrophobic helices 6-9 of the T domain of diphtheria toxin can adopt two distinct conformations, one close to the membrane 606-Pos Board # B462 surface and a second that is more deeply inserted. To further SPECTROSCOPIC INVESTIGATION OF MELITTIN - investigate membrane topography, single Cys were introduced in INHHBITOR BINDING SITES hydrophobic helix 5 and flanking residues, and then labeled with Frances Separovic, Yuen-Han Lam; University of Melbourne, fluorescent probes. After membrane insertion, apparent local School of Chemistry,Melbourne, VIC 3010 Australia polarity and accessibility to externally added antibodies was Melittin is a whose assessed by fluorescence. Under conditions giving shallow cytolytic peptide biological activity is lost upon insertion of to the with which it an helices 6-9, helix 5 is also shallowly inserted. It binding peptide, Ac-IVIFDC-NH2, forms to insert more under conditions insoluble complex. As a result, the structural determination of the appears deeply giving deep is difficult. Solid-state insertion of helices 6-9. However, residues in regions flanking complex NMR, however, allows insight into 5 the interaction between melittin and inhibitor. The conformation of helix are relatively exposed to external antibodies in both the complex was studied NMR and CD. Inhibitor conformations, suggesting that helix Sdoes not span the membrane using was in a stable fashion in either conformation. Combining these and

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previous results suggests that although helices 8 & 9 form a stable Yuri V. Griko', Stanislav D. Zakharov2, William A. Cramer2,; transmembrane hairpin under conditions of deep insertion, helices 'Johns Hopkins University, Baltimore, MD, 2Purdue University, 5-7 do not span the membrane in a stable fashion. An unstable West Lafayette, IN transmembrane state cannot be ruled out. To further explore DSC and CD were used to determine the number of structurally topography, a Cys-less construct containing the A chain and T independent domains, and the interdomain interactions necessary domain of the toxin has been expressed. It exhibits a low pH for the import of colicin El into E.coli. Analysis of the induced conformational change similar to that of the native A-T denaturation of the 522 residue colicin, and fragments of 342 and fragment. Introduction and fluorescent labeling of Cys residues in 178 residues containing domain subsets, showed the three major this construct may allow a more precise definition of toxin functional domains that differ in thermal stability: the COOH- topography in membranes. terminal channel-forming (C) domain was most stable, followed the BtuB receptor binding (R) domain, and the N-terminal 609-Pos Board # B465 translocation (T) domain with the smallest stabilization enthalpy DEUTERIUM NMR INVESTIGATIONS OF GATED and thermal stability. Interdomain interactions involve T-R HOMODIMERIC GRAMICIDIN CHANNELS interactions stabilizing R, and T-C and R-C interactions stabilizing Sigrid E. Schmutzerl, Eric M. Miller', Marvin C. Leister', Olaf S. R and T, but destabilizing C. Interacting extended helices of the R- Andersen2, Denise V. Greathouse', Roger E. Koeppe, 111; domain, possibly a coiled-coil, were implied by a high (>90%) a- 'University of Arkansas, Fayetteville, AR 72701, 2Cormell Medical helical content, cooperative decreases in a-helical content near the College, New York, NY 10021 Ttr of thermal denaturation, and a large denaturation enthalpy, Gramicidin A (gA) channels usually are not voltage dependent, but implying extensive H- voltage-dependent gating is observed in a few channels formed by ThermDdynamicParamters ofDenaturaion bond and van der These channels transitions between low- ofColicin El Domains at pH 8.0, I=0.1 M Waals interactions. It gA analogues. display Domain Tt, C AH, kI mor was inferred that the and high-conducting states, which vary as a function of the applied potential. Previously characterized voltage-dependent channels T 51.9 302.0 R-domain has a R 52.5 565.2 dominant role in were heterodimers, however, making structural analysis difficult. C 66.5 406.2 determining the A common feature of voltage-dependent gA channels is that they have a strain at the subunit interface, and the gating transitions conformation of other seem to be induced by stress on the peptide backbone. Based on domains, and that cellular import starts with the R domain binding this idea we designed a gramicidin that exhibits voltage-dependent to the BtuB receptor, and subsequent unfolding of the R-domain gating as a homodimer. The analogue, des-Vall-[Ala ]gA, has the coiled-coil and thereby of the T-domain, which then interacts with sequence formyl-A2A3LA_VVWLWLWLW-ethanolamine (D- the ToIC receptor (supported by NIH GM18457 and GM48036- residues are underlined). The chirality mismatch is due to L-Ala 07). rather than D-Ala at position 2. By varying the side chain and chirality at this position, we examine the structural basis behind the 612-Pos Board # B468 gating. We synthesized four gA analogues with a deuterium ASSOCIATION OF A FLUORESCENT SQUALAMINE labeled L-Ala at position 3, and L-Ala, D-Ala, Gly or Aib residues ANALOG WITH PHOSPHOLIPID VESICLES AS at position 2. Solid-state deuterium NMR spectra of these MEASURED BY FLUORESCENCE ANISOTROPY. analogues in oriented bilayers indicate multiple conformations for Lawrence Decker', Barry S Selinsky2, ; 'Augustiana College, the LAla and Aib analogues, whereas the spectra for the other two Rock Island, IL 61201, 2Villanova University, Villanova, PA analogues are consistent with a single conformation. 19085 A BODIPY-labeled analog of the aminosterol antibiotic 610-Pos Board # B466 squalamine (B-Sq) has been synthesized and characterized by 2H SOLID-STATE NMR STRUCTURAL AND DYNAMIC fluorescence spectroscopy. B-Sq is a highly fluorescent molecule STUDIES OF BIOTINYLATED GRAMICIDINS whose quantum yield increases in the presence of a hydrophobic Aphrodite Anastasiadis', Frances Separovic', Patrick CA van der environment. B-Sq forms pores in large unilamellar phospholipid Wel2, Roger E Koeppe 12; 'University of Melbourme, School of vesicles of a type similar to squalamine. The partitioning of Chemistry, Melbourne, VIC 3010 Australia, 2University of BODIPY-Squalamine into small unilamellar phospholipid vesicles Arkansas, Fayetteville, AR 72701 °BEcODPY-Squak|-Wn 0U,- (SUVs) was measured by Biotinylated gramicidins are an important component of the "ion NH4> ! _ fluorescence anisotropy. channel switch" biosensor. These gramicidin A (gA) analogues -<8F > ( > The measured partition have a biotin attached to the C-terminus of gA via an aminocaproyl --*.-..-e< 04 coefficients ranged linker. The conformation and dynamics of these linker groups are between 2100-4400, being investigated using 2H solid-state NMR. Deuterated where the partition coefficient increased with increasing aminocaproyl linkers have been synthesised and then coupled to percentages of anionic lipid in the SUVs. Future studies will gA to produce: gAXDBoc, gAXDXB, where XD indicates the examine the orientation and aggregation of fluorescent squalamine deuterated aminocaproyl groups. Biotinylated gA derivatives have analogs in lipid bilayers, with the hope of understanding the been incorporated into oriented bilayers in order to analyse the mechanism of pore formation by aminosterol antibiotics. order and dynamics of the aminocaproyl linker. The dynamics of the linker are being studied using deuterium T1 and T2 relaxation 613-Pos Board # B469 measurements. No differences are seen between the two deuterated CONTROL OF THE INTERACTION OF THE gA analogues. The small 2H splittings observed in each spectrum ALZHEIMER'S ABPS, AND HOMOLOGOUS suggest that the aminocaproyl linker is undergoing fast rotation in ANALOGUES, WITH LIPID MEMBRANES. both derivatives. The synthesis and incorporation of d4-gAXXB, Nelson Jose Arispe; Uniformed Seervices University of the health where the ethanolamine has been deuterated, and gA4XXDB into Sciences, 4301 Jones Bridge Rd, Bethesda, Maryland 20814-4799 oriented bilayers are in progress and will be compared to the A diverse group of proteins such as Alzheimer's APP (1-40) and conformation and dynamics of the gA analogues mentioned earlier. APP (1-42), and the toxic human amylin peptide and prion protein (106-126) incorporate into lipid membranes to form ion channels. 611-Pos Board # B467 They all have in common domains which are potentially able to STRUCTURAL STABILITY AND DOMAIN ORGANIZATION OF COLICIN El

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adopt I-pleated sheet conformations, and to promote self-assembly N796 and D800 contribute to site 2. All of these residues reside and aggregation. A liposome aggregation assay demonstrates that within the M4, M5, M6 and M8 transmembrane domains that are these proteins additionally interact with phospholipids to induce a clustered to approximate side chains for calcium coordination. concentration dependent liposome aggregation. We present here a Mutational analysis of S4 segment and M6/M7 loop indicates their comparative evaluation of the rate and the amplitude of these importance in providing a linkage between the Ca2+ binding and liposome aggregations. Aggregations were also evaluated under the phosphorylation domains. (Supported by U.S. NIH Program different phosphatidyl serine (PS) to phosphatidyl ethanolamine Project HL27867). membrane ratio and cholesterol and ganglioside (Gml) membrane content. The induction of liposome aggregation by the peptides 616-Pos Board # B472 required a minimum presence of PS, and was reduced by the PKA-DEPENDENT CA2+-INDUCED CA2+ RELEASE (CICR) presence of cholesterol into the liposome membrane. No liposome TRIGGERS EXOCYTOSIS AND ENDOCYTOSIS IN aggregation was observed at high cholesterol concentrations. The PANCREATIC B-CELLS. addition of the f-pleated sheet promoter GM, into the liposome Guoxin G membranes enhanced peptide-induced aggregation. Our results Kang, Oleg Chepurny, George G Holz; New York University School of Medicine, 550 First Ave, New York, NY signal the importance of the protein A-pleated sheet conformation 10016 and of the membrane lipid content to control the degree of the protein-lipid interaction. CICR in pancreatic fl-cells is facilitated by the insulinotropic hormone glucagon-like peptide-I (GLP-1) which sensitizes Ca2+ Endoplasmic Reticulum & Protein Trafficking release channels (ryanodine receptors) to stimulatory effects of Ca2+ entering via VDCCs. We now demonstrate that the action of GLP-1 is: 1) mediated by cAMP and protein kinase A (PKA), 2) is 614-Pos Board # B470 specific for caffeine and thapsigargin-sensitive Ca2+ stores, and 3) HOW DOES CALCIUM MEDIATE results in insulin exocytosis and endocytosis. The GLP-I NEUROTRANSMISSION? MECHANISM(S) OF CALCIUM receptor ACTIVATION OF SYNAPTOTAGMIN. agonist Exendin-4 proved to be an initiator of CICR in human f- cells and rat INS-1 cells, producing a spike-like increase of [Ca2+],, Hilary Arnold Godwin, Ricardo A Garcia, Cameron E Forde, the kinetics of which exhibited rapid onset and fast recovery. CICR Ryan Andersen; Northwestern University, 2145 Sheridan Road, was also triggered by forskolin or caffeine, and both substances Evanston, IL 60208-3113 acted in a synergistic fashion when applied together. Pretreatment Synaptotagmin I is a critical component of the synaptic machinery with the cAMP antagonist 8-Br-Rp-cAMPS or PKA antagonist H- that senses calcium influx and triggers fusion and neurotransmitter 89 inhibited CICR in response to Exendin-4 or forskolin, whereas exocytosis, but the mechanism by which calcium triggers the pretreatment with thapsigargin blocked the action of caffeine. fusogenic activity of synaptotagmin is not well understood. Here CICR induced by caffeine was accompanied by exocytosis, we report fluorescence resonance energy transfer studies that detected with carbon fiber electrodes as a burst of multiple demonstrate that calcium concentrations required for fusion induce amperometric events. Exocytosis and accompanying endocytosis a conformational change (EC50 - 3 mM) that brings the two were also detected by measuring whole cell capacitance in f-cells calcium-binding C2 domains in synaptotagmin I closer together. voltage clamped at -50 mV. The time course of this secretory The metal ion dependence for this conformational change mirrors process matched closely the fast transient increase of [Ca2+], the metal dependence of synaptotagmin-syntaxin interactions and accompanying CICR. In conclusion, these findings document a exocytosis. Analytical ultracentrifugation studies reveal that role for CICR in stimulus-secretion coupling, a process sensitized synaptotagmin I is monomeric even at high calcium by GLP-1, cAMP, and PKA in B-cells. Supported by NIH R01- concentrations, indicating that the calcium-triggered association DK52166 (GGH). between the C2 domains in synaptotagmin I is intramolecular, rather than intermolecular. By contrast, synaptotagmin II self- associates in the presence of divalent cations. These studies 617-Pos Board # B473 provide important insights into the molecular mechanism of CA INDEPENDENT BUT VOLTAGE DEPENDENT calcium mediated neurotransmission and clues into the different SECRETION IN DRG NEURONS activities of synaptotagmin family members. C. Zhang, L.C. Wang, Zhuan Zhou; Institute of Neuroscience, Chinese of 615-Pos Board # B471 Academy Sciences, Shanghai 200031, China MECHANISM OF SARCOPLASMIC RETICULUM Dorsal root ganglion (DRG) neurons are primary sensor cells, ATPASE (SERCA) ACTIVATION BY CA2+. which secrete neuropeptides from dense-core vesicles. In patch- clamped DRG neurons depolarization induces membrane Zhongsen Zhang, David Lewis, Lilin Zhong, Hailun Ma, capacitance (Cm) increase in Ca containing bath (2 mM), which is Giuseppe Inesi; University of Maryland, Baltimore, 108 N Greene interpreted as signal of Ca dependent exocytosis from soma St, Baltimore, Maryland 21201 (Huang & Neher, 1996). Using combined Cm and fluorescence Ca Occupation of two Ca2+ binding sites is required for catalytic assays, we found a novel depolarization-induced Cm increase in activation of the ATPase. The residues involved in Ca2+ binding, Ca free bath in rat DRG neurons. 100 ms depolarization-induced signal transmission and phosphorylation are characterized in detail Cm is larger in 2 mM Ca bath (600 ff) than that in Ca free bath in our laboratory by the use of recombinant SERCA expressed at (300 fF). In addition, depolarization evokes transient intracellular high level in Cos-l cells infected with adenovirus vectors Ca spikes up to 300 nM in Ca free bath, which is blocked by containing wild type and mutants under the control of the CMV thapsigargin, indicating existence of voltage dependent Ca release promoter. Direct measurements of Ca2+ binding and studies of (VDCR) from a unknown ER store. However, depolarization- various enzyme functions in the presence of ATP or Pi clarify the induced Cm remains even when VDCR is blocked, indicating cooperative mechanism of Ca2e binding and catalytic activation of existence of a Ca-independent and voltage-dependent Cm SERCA. Single mutations of E771, T799, D800 and E908 inhibit (CIVDC). CIVDC is further confirmed by the fact that, in Ca free total Ca2+ binding, whereas mutations of E309 and N796 inhibit bath, the voltage dependence (Cm is constant for Vm> -10 mV) is binding of one and the same Ca2+. The functional characterization very different from VDCR, which is similar as L-type Ca channel and high-resolution crystal structure of ATPase suggest in both kinetics and steady-state I-V curve. CIVDC can be cooperative and sequential Ca2+ binding in which side chains of blocked by intracellular dialysis of tripsin, but it is resistant to E771, T799, D800 and E908 contribute to site 1, while E309,

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external nifedipine, internal high EGTA and internal pH 6.2. 620-Pos Board # B476 Supported by grants from CAS, CDST and NSFC. ENERGETICS OF CHOLETEROL-INDUCED MEMBRANE PROTEIN SORTING. 618-Pos Board # B474 Jens A. Lundbiek1, Olaf S. Andersen2, Claus Nielsen3; 'St. Hans IDENTIFICATION OF THE INTRACELLULAR Hospital, Roskilde, Denmark, 2Cornell Univ. Weill Medical LOCALIZATION SIGNAL OF RYANODINE RECEPTOR. College, 1300 York Avenue, New York, NY 10021, 3Univ. Manjunatha B Bhat, Jianjie Ma; Case Western Reserve Copenhagen, Copenhagen, Denmark University, 10900 Euclid Avenue, Cleveland, OH 44106 Membrane protein-bilayer hydrophobic coupling means that the Ryanodine receptor (RyR) functions as a Ca release channel in the bilayer hydrophobic thickness will tend to match the hydrophobic membranes of sarcoplasmic/endoplasmic reticulum (S/ER). We length of inserted proteins. When there is mismatch between had previously reported that RyR expressed in CHO cells is bilayer thickness and protein length, the bilayer deformation incurs localized in the ER membranes suggesting that the sequence an energetic cost that varies with the bilayer thickness and material responsible for ER retention is present within the primary amino properties. Bretscher and Munro (Science, 1993) suggested that acid sequence of RyR. Confocal microscopic imaging of cells targeting of membrane proteins could result from such bilayer- expressing various segments of RyR as GFP fusion constructs mediated sorting, and that the uneven distribution of cholesterol indicated that the ER retention signal is present in the carboxyl among the membranes of eukaryotic cells constitutes a sorting (C)-terminal hydrophobic region of the protein, which also mechanism, as cholesterol thickens the plasma membrane which contains transmembrane segments forming the pore of the Ca favours the insertion of plasma membrane proteins that have release channel. To further identify the minimum domain(s) longer hydrophobic stretches than Golgi membrane proteins. We responsible for ER retention of RyR, we expressed fusion proteins evaluated the feasibility of cholesterol-induced protein sorting containing various fragments of RyR, GFP, and intercellular using a theory of elastic bilayer deformations and experimental adhesion molecule (ICAM-1), a glycoprotein that is normally bilayer material moduli (Nielsen and Andersen, Biophys. J. 2000). targeted to the plasma membrane. ICAM-GFP fusion protein was Longer ca-helices (20 AA vs. 15 AA) preferentially partition into targeted to the plasma membrane, whereas fusion proteins thicker bilayers (31 A vs. 25 A); but the major effect of cholesterol containing the C-terminal region of RyR were retained in the ER, is due to changes in membrane stiffness (material moduli) rather and those with the sequence preceding the transmembrane than thickness. In any case, there is sufficient energy to support a segments were targeted to the plasma membrane. Using bilayer-mediated sorting mechanism. glycosylation as a marker for plasma membrane targeting, it was 15 AA 20 AA determined that region(s) within the transmembrane segments 3 chol) e 25 kT 10 kT and 4 (a.a. 4830-4943) contain the sequence for ER retention of AAG2s(.. 31(no chol) RyR. AAG3I(no chol) . 31 (chol) 69 kT I kT

619-Pos Board # B475 THE CYTOSOLIC ENVIRONMENT INFLUENCES THE 621-Pos Board # B477 SOLUTE PERMEABILITY OF THE ENDOPLASMIC DIFFUSION IN THE ENDOPLASMIC RETICLUM OF AN RETICULUM AQUAPORIN-2 MUTANT CAUSING HUMAN William Frederick Wonderlin; West Virginia University, Robert NEPHROGENIC DIABETES INSIPIDUS C. Byrd Health Sciences Ctr., Morgantown, WV 26506 Marc Levin, Peter M. Haggie, L. Vetrivel, A. S. Verkman; UCSF The permeability of the ER membrane to small, uncharged Mutations of the AQP2 water channel cause the renal disease NDI. molecules is poorly characterized. We have examined the Recessive NDI is produced by retention of AQP2 mutants (eg. permeability of the ER to a small (MW 338) solute, 4- TI26M) at the ER. To determine whether the organellar retention methylumbelliferyl-alpha-D-glucopyranoside (4MG), that becomes of AQP2-T126M is due to its relative immobilization, we fluorescent when cleaved by neutral alpha glucosidases present in measured by FRAP the mobility of GFP-chimeras containing wt the ER. CHO cells grown in suspension were broken open by N2 and AQP2-T126M. In transfected LLC-PK1 cells, GFP-labeled cavitation in a buffer containing 140 K-glutamate, 2.5 MgCl2, 10 AQP2-T126M was localized to the ER and wt AQP2 to HEPES, pH 7.3, and the entry of 4MG into the ER was monitored endosomes/plasma membrane. The chimeras were localized to the by activation of the fluorophore. The permeability of the intact ER ER after brefeldin A (BFA) treatment. Photobleaching with image to 4MG was high, with a rate of fluorophore activation about 67% detection indicated that the GFP-AQP2 chimeras were mobile in of the rate observed after non-specific permeabilization with Na- the ER. Spot photobleaching revealed a diffusion-dependent deoxycholate. The permeability of the intact ER was increased by irreversible process that was dependent on spot size and abolished 10-15% by treatment with puromycin, which purges nascent by fixation. A novel reversible fluorescence recovery (t2n -2 s) peptides from the translocon pore. Both the puromycin- was characterized whose recovery was independent of spot size independent and puromycin-dependent entry of 4MG were and not affected by fixation. GFP-AQP2 diffusion in the ER was affected by substitution of other anions for glutamate according to not slowed by the TI 26M mutation; diffusion coefficients were (in the series: glutamate=gluconate>acetate>chloride=sulfate. cm2/s x 10-') 2.6 + 0.8 (wt/+BFA), 3.0 ± 0.6 (T126M/+BFA) and Decreasing the pH inhibited both routes of entry of 4-MG, with 25- 3.2 ± 0.5 (T126M/-BFA). ER diffusion of AQP2-T126M was not 40% inhibition at pH 6.2. The entry of 4MG was also inhibited by significantly affected by upregulation of chaperones, cAMP 1 mM spermine, but not 1 mM spermidine. We conclude that the activation, calcium elevation or actin disruption. These results ER membrane is highly permeable to a small glucose-dye indicate that the ER retention of AQP2-T126M does not result conjugate, and this permeability is sensitive to the cytosolic from restricted or slowed mobility, and suggest that the majority of environment. The puromycin-dependent entry of dye is probably AQP2-T126M is not aggregated or bound to slow-moving mediated by the translocon (sec61) pore, whereas the route for membrane proteins. puromycin-independent entry remains unidentified.

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622-Pos Board # B478 the readily releasable pool (RRP), is available for instant INHIBITORS OF VESICULAR PROTON GRADIENTS exocytosis, but it is not clear how RRP relates to granules that are SELECTIVELY AFFECT HIGH-FREQUENCY physically docked at the plasma membrane. In order to visually TRANSMISSION AT STRIATAL GABAERGIC SYNAPSES study exocytotic processes, we have labelled secretory granules in IN VITRO. the insulinoma cell-line Ins-I by transient transfection with an islet Jan Christoph Behrends, Eva Rumpel; University of Munich, amyloid precurser protein (IAPP)-enhanced green fluorescent Pettenkoferstr. 12, Bavaria 80799 protein (EGFP) fusion construct. Labelled granules with varying MtUnchen, Germany degrees of mobility were seen throughout the cell interior, while vesicles are loaded with transmitter Synaptic by transport proteins immobile granules were primarily found at the plasma membrane. of the vesicular membrane which use a H+-electrochemical During voltage-clamp depolarizations, single exocytotic events a vacuolar gradient (made by H+-ATPase) to pump transmitter could be detected by time-resolved confocal microscopy as sudden from the cytosol into the vesicular lumen. At present, little is disappearance of the membrane-associated known about the of this mechanism for immobile, plasma importance synaptic granule fluorescence. Analysis of a large number of such events strength in the CNS. In our preparation, the protonophore FCCP (5 combined with concurrend recordings of cell capacitance transmission on the 2nd suggests AM) selectively suppressed synaptic to 4t that 1) RRP is a subset of the docked pool of granules and 2) cargo impulse of a 33 Hz train of 5 presynaptic impulses, changing the release from individual granules is delayed relative to the fusion of depression index from 0.32+/-0.02 in control to 0.23+/-0.01 in the the granule with the plasma membrane. first 3 trains after drug application (n=6, p<0.005). Preincubation with the Ca2+-chelator BAPTA-AM (50 AM) and lowering extracellular Ca2+ to 0.5 or 0.8 mM had a protective action against 625-Pos Board # B481 FCCP-induced depression, while EGTA-AM or loading the A C-TERMINAL LEUCINE ZIPPER IS CRITICAL IN presynaptic cell with 20 mM ATP were without effect. MEMBRANE TRAFFICKING OF HIK1. Furthermore, FCCP (5 AM)application significantly and Colin A Syme, Aaron C Gerlach, LeeAnn Giltinan, Simon C progressively reduced the mean amplitude of mIPSCs and Watkins, Daniel C Devor; University of Pittsburgh decreased their time constant of decay. Similar results were We previously demonstrated that C-terminal truncations of the 427 obtained with a blocker of the vacuolar H+-ATPase, amino acid channel, hIK1, results in compromised surface BafilomycinAl. We hypothezise that slower refilling of synaptic expression. To delineate the structural motifs within the C- vesicles may selectively affect rapidly cycling synaptic vesicles at terminal tail responsible for cell surface localization we utilized high frequency of transmission. (Supportedby the DFG, BeJ 739-2) immunofluoresence and whole-cell recording techniques. Truncation at amino acid K402 resulted in a complete loss of 623-Pos Board # B479 surface membrane expression, whereas truncation at L414 had no PHYSIOLOGICAL STIMULATION LEADS TO TWO effect. Located in this region is a di-leucine motif (L409-410) and DISTINCT MODES OF ENDOCYTOSIS IN BOVINE a leucine heptad repeat ending at L406. Mutation of the di-leucine ADRENAL CHROMAFFIN CELLS. motif resulted in a small decrease in surface expression, whereas Shyue-An Chan, Corey Smith; Medical College of Georgia, mutation of the two terminal leucines (L399P/LA06F; Augusta, Georgia 30912-3000 L399A/LA06A) within the leucine zipper abrogated membrane localization of hIKi. Additional mutations within the Exocytosis-coupled endocytosis has recently been the subject of heptad intense repeat (L385A/L392A) or of the a positions (1396A/L403A) study. Membrane retrieval has been reported to occur by resulted in a near different at different and complete loss of membrane localized channel. In paths, rates, magnitudes and under contrast, mutating individual leucines did not compromise channel different molecular control mechanisms. With such a varied range of trafficking or function. Co-localization studies revealed that endocytotic modes reported, we wondered which would be L399A/L406A found to occur electrical in the failed to escape the ER, although membrane following physiological stimulation localization could be restored by incubation at 27 °C. In neuroendocrine adrenal chromaffin cell. Here we present data under conclusion, these findings demonstrate that the C-terminal leucine demonstrating that, physiological stimulation, the zipper is critical to facilitate oorrect folding and membrane neuroendocrine chromaffin cell utilizes two plasma pharmacologically and trafficking of hIKl. (Support by NIH DK54941-02). kinetically separable modes of endocytosis to internalize cell membrane. By comparing differences in cell capacitance and integrated amperometric currents we found that following a single 626-Pos Board #B482 stimulus, cells exhibited a robust retrieval mechanism. Increasing RELEASE RATES AND MEPSC AMPLITUDES OF A GLUTAMATERGIC cell firing (0.02 - 6 Hz) steadily decreased endocytotic efficiency. SYNAPSE ESTIMATED FROM Further increases in activity (6 - 16 Hz) resulted in a recovery of POSTSYNAPTIC CURRENT FLUCTUATIONS: the endocytotic efficiency. The calcineurin inhibitor cyclosporin A VARIANCE, SKEWNESS AND KURTOSIS (4TH selectively blocked the recovery of endocytosis observed above 6 CUMULANT). Hz firing. Based on the findings reported here, we propose that in Erwin Neher, Takeshi Sakaba; Max Planck Institut biophys. response to a range of physiological activity, bovine adrenal Chem., Am Fassberg, Goettingen, 37120 Germany chromaffin cells alternate between at least two activity-modulated of Postsynaptic currents at the Calyx of Held synapse fluctuate forms endocytosis. strongly during episodes of longlasting stimulation. These fluctuations contain information about the release process, such as 624-Pos Board # B480 on release rate and mEPSC amplitude. Unfortunately, 'residual VISUALISING THE DYNAMICS OF SINGLE INSULIN- glutamate' in the synaptic cleft prevents a straightforward analysis, CONTAINIG GRANULES UNDERGOING EXOCTOSIS due to ,residual current'. We show that highpass filtering (Segal et Sebastian Barg, Charlotta Olofsson, Jenny Schriever-Abeln, Anna al., 1985 Biophysical J. Vol 47. pp. 183 -202) effectively Wendt, Erik Renstrom, Patrik Rorsman, Samuel Gebre-Medhin; eliminates the adverse effects of residual current and that Lund University, Solvegatan 19, Lund, SE-22354 evaluation of the three noise parameters variance, skewness and Release of neuropeptides and hormones is preceded by the kurtosis (the 4th cumulant) allows to solve for release rate, mEPS recruitment, docking at the plasma membrane, and priming of amplitude, and an effective single channel amplitude of glutamate secretory granules for release. This is reflected by the kinetics of channels. We show that mEPSC amplitude decreases during exocytosis, which suggest that granules exist in functionally continued stimulation due to receptor desensitization and that this distinct pools. Only a fraction of the cell's granules, referred to as effect is removed by cyclothiazid (CTZ). We find that release rates

138a Sundae POSTERS 626-627.05 drop to a low value of approximately 10 events/msec during strong 627.03-Pos Board # B485 stimulation, which depletes the readily-releasable pool of vesicles. A CALCIUM CLAMP FOR STUDYING EXOCYTOSIS AND This rate is as expected for a pool recovery time constant of about ENDOCYTOSIS 300 msec, assuming that the steady state release represents James Dunning, Kevin D Gillis; University of Missouri - vesicles, which are newly recruited to the pool and immediately Columbia, Research Park, Columbia, MO 65211 released. Photorelease of Ca from caged compounds has been widely used to study Ca-activated cellular processes such as exocytosis and Exocytosis & Endocytosis endocytosis. However, photolysis of caged compounds is usually done in an "open loop" configuration without tight control of the 627.01-Pos Board # B483 amplitude and time course of the resulting free Ca concentration MODELING OF CALCIUM DEPENDENT PRESYNAPTIC ([Ca]). We have implemented a feedback control system using a RELEASE SYSTEMS. monochromator both to photolyze the cage and excite a fluorescent Najl V. Valeyev; Kazan State University, Kremlevskaja - 18, dye to report [Ca]. We have been able to elevate [Ca] from -150 Kazan, Tatarstan Republic 420008 Russian Federation nM to 1 jM in about 200 ms and maintain [Ca] within -+1-5% of Calcium signaling proteins display a large variability concerning the desired level indefinitely. Complex [Ca] stimulus patterns are their affinity, specificity, and kinetics. The question is how the possible. In another configuration, [Ca] can be elevated within - 1 necessary selectivity of activation of diverse targets can be ms with a flash lamp and then maintained at a constant level using regulated by calcium concentration. We have developed the the monochromator. In our system, the Ca cage NPEGTA is mathematical model of nonequilibrium Ca2+ distribution in the maximally photolyzed at a wavelength of -360 nm. In order to presynaptic terminal. The model has been created in analogy with minimize photorelease while measuring [Ca], we excite the Ca model previously developed by Markram H et. al. 1998 for indicator dye bisfura2 at 330 and 410 nr. The system could be dendritic like compartment. To explore the possible implications of further improved if a high-affinity, ratiometric Ca indicator excited nonequilibrium calcium dynamics in presynaptic terminal, we at long wavelengths could be found. We believe that the Ca clamp simulated calcium influx, buffering and binding by proteins crucial will be particularly useful for studying both the priming and for neurotransmitter release. The results suggest that the triggering of exocytosis at [Ca] levels of - 1 jiM where the rate of presynaptic calcium signaling system may activate different exocytosis is slower than the response time of the Ca clamp. release systems selectively by different calcium accumulating in buffers and binding by proteins far from equilibrium. 627.04-Pos Board # B486 CALCIUM ELEVATION IS REQUIRED FOR VESICLE 627.02-Pos Board # B484 POOL MOBILIZATION AT HIPPOCAMPAL SYNAPSES. SECRETION CAN BE EVOKED BY ACTIVATION OF Timothy Aidan Ryan, Thomas Fernandez-Alfonso; Weill EXOGENOUS CALCIUM CHANNELS IN A MOUSE Medical College Cornell, 1300 York Ave, New York, NY 10021 PHEOCHROMOCYTOMA CELL LINE. Using multiple optical tracing approaches of synaptic vesicle Amy B. Harkins', Anne L. Cahill', Arthur S. Tischler2, Aaron P. traffic (FM 1-43 and synaptopHluorins) we show that prolonged Fox'; 'University of Chicago, 947 E. 58th Street, Chicago, IL and repeated action potential stimulation in minimal external 60637, 2Tufts University School of Medicine, Boston, MA 02111 calcium ([Ca2k],,) leads to turnover of only - 10-15 % of the total We have characterized a recently-developed immortalized mouse recycling pool. Stimulation in intermediate levels of [Ca2i]. leads pheochromocytoma (MPC) cell line to determine whether these to larger but stil incomplete pool turnover compared to that which cells can be used as a model cell for secretion. MPC cells contain occurs in elevated [Ca2'].3. Our data indicate that in addition to the small synaptic-like vesicles as determined by electron microscopy. calcium sensor for exocytosis, a calcium-sensitive switch controls Additionally, MPC cells contain both syntaxin I and whether vesicles in the reserve pool make transit to the readily- synaptotagmin I as determined by immunocytochemistry. releasable pool to participate in vesicle recycling. Finally our data However, when single, untreated MPC cells were patch-clamped in indicate that vesicles that undergo exocytosis during minimal the whole-cell recording configuration, the cells expressed a Na+ calcium elevation become functionally mixed within the reserve current (n=88) and little or no endogenous Ca2+ current. Of the 88 pool upon recycling. cells, only 19 had a small endogenous Ca2' current which averaged 78 ± 8 pA (mean ± SEM). No measurable secretion was observed 627.05-Pos Board # B487 in MPC cells (n=27). Cells treated with either NGF (n=6) or EGF A TECHNIQUE FOR MEASURING MEMBRANE (n=8) did not exhibit an increase in either Ca2+ current amplitude CAPACITANCE DURING A DEPOLARIZING STIMULUS or secretion. MPC cells were transiently transfected with Ca2+ Peng Chen, Kevin D Gillis; University of Missouri - Columbia, channel subunits (a,o, 2, and a28). Stimulation with a train of 5 Research Park, Columbia, MO 65211 depolarizations from a holding potential of -80 mV to +20 mV Depolarization-evoked exocytosis is often inferred from the (200 ms test pulse, 50 ms interpulse) produced a Ca2+-influx of 880 change in membrane capacitance (Cm) measured at hyperpolarized x 106 ions (+ 96 x 106 ions, n=15), and peak secretion of 376 fF (± intervals before and after a depolarizing pulse. Cm measurements 75 fF, n=15). o-Cgtx GVIA (1 pM) completely blocked the are usually not attempted in excitable cells during the depolarized transfected a,B Ca2+ current (n=5), as well as secretion (n=3). pulse because the activation of nonlinear time-dependent Thus, previously unavailable MPC cell lines can serve as surrogate conductances invalidates the simple 3 component equivalent models of secretion, complementing existing cell lines such as circuit of the cell. This necessitates complex pulse protocols to PC12 cells. Furthermore, the MPC cells are readily transfected indirectly infer the time course of exocytosis during a and may be particularly useful for studies in which a mouse cell depolarization-evoked influx of Ca. We have found that the line would be advantageous. Supported by grants to APF addition of one additional time constant (2 parameters) to the (NS33826-04) and AST (NS37685). equivalent circuit is able to model both the nonlinear, time- dependent Ca conductance and the current due to the motion of "gating" charges of voltage-dependent ion channels. We have used a broadband "pseudo-random binary sequence" voltage stimulus and a nonlinear least-squares algorithm to estimate the 5 model parameters during membrane depolarization and find that

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