SUNDAY SODIUM-CALCIUM EXCHANGE 303-309

303 - Pos 305 - Pos Ca ACTIVATION OF THE Na/Ca EXCHANGER IN MOUSE MYOCYTES BETA-BLOCKERS REDUCE HYDROXYL RADICAL INDUCED REVERSE SODIUM- OVEREXPRESSING CANINE NCX1. CALCIUM EXCHANGE IN CARDIAC MYOCYTES. Chrls R. Weber', Kenneth S. Ginsburg', Kenneth D. Philipson2, Donald M. Bers', 'Loyola Mohammed S Qayyum, Joshua I Goldhaber, UCLA School of Medicine University Chicago, 2UCLA School of Medicine, Stimulation of sodium-calcium exchange (NaCaX) by oxygen free radicals (OFR) may be an We have shown that outward and inward exchange currents (INx) increase with increasing [Ca]i in important cause of Ca overload and cellular injury during ischemia-reperfusion in cardiac ferret myocytes (BJ., 76, A299, 1999). We concluded that the increase inINX(..) reflects allosteric myocytes. We determined whether Carvedilol, a 1-blocker with antioxidant properties, could Ca; activation of NCXI. Note that f[Ca]i reduces thedriving force forINX(O,,) and favors greater prevent hydroxyl radical (.OH) mediated increases in reverse NaCaX. We measured INwC,X in INX(ti). Here we measure [Ca]j, INX (Ni-sensitive), and contraction during alternating 100 ms singlerabbit ventricular myocytes using whole cell patch clamp. The Na-Tyrodes bath contained voltage clamp pulses (-100 mV to +100 mV), with SR Ca release and all other currents blocked. (mM): 1.8 CaCl2, 0 K, and 0.01 TTX. Thapsigargin and ryanodine were included to deplete SR INX(,1) (at +100mV) in mouse myocytes overexpressing canine NCX1 increases with T[Ca]j (as in Ca. The Cs-aspartate pipette solution contained 20 mM Cl and substrates for endogenous ATP ferret). However, this[Ca]i-dependent activation was not seen in myocytes from either WT mice synthesis. IN.C.X was measured as the Ni-sensitive current at the end of a3 second depolarization or those overexpressing mutant canine NCXI (A680-685; lacking a Ca regulatory domain). INX(i.) from aVH of-40 to +80 mV. When ImM H202 and 100 AM FeSO4 were added to the bath to (at -100 mV) still increased with generate .OH there was a >2-fold increase in IN.C.x. from (pA, mean±SE) 70±13 to 184±35 (n=5, buttheeffectwaslessthanin 6- T[Ca]j, - TG-Dog p

0 1 2 3 time (a)

Pos 307- Pos FULL-LENGTH, ACTIVE CARDIAC SODIUM-CALCIUM MOLECULAR CLONING AND CHARACTERIZATION OF THE HUMAN RETINAL PROTEIN IN THRICHOPLUSIA NI LARVAE MEMBRANE VESICLES. CONE SODIUM-CALCIUM+POTASSIUM EXCHANGER Calvin Hale, Zimmerschied, Chananada K. Hill, Elmer M. Price, Julie Bossuyt, Clemens F Prinsen, Robert T Szerencsei, Paul P Schnetkamp, University of Calgary, 3330 University Missouri, Dalton Cardio. Research Center, Research Park, Columbia, MO 65211 Hospital Drive NW, Calgary, Alberta T2N 4N1 Canada sodium-calcium exchanger (NCXI) has been expressed in insect larvae The plasma membrane of retinal rod and cone photoreceptors contains a Na-Ca exchanger which using different baculovirus vectors. One derived from the transfer vector Bac300O, is removes calcium that enters rods and cones in darkness via the light-sensitive channels. The rod baculovirus-encoded protease gene is eliminated. Bac3000 was chosen since Na-Ca exchanger has previously been shown to be a sodium-calcium exchanger that requires and NCXI protein expressed from the traditional viral vector appears as a 70 kDa form when transports potassium and its cDNA was the first member of the NCKX gene family identified. In analyzed immunoblots. The mature form of the exchanger is 120 kDa. This suggests that the this study we have determined the identity ofthe retinal cone exchanger by cloning cDNAs coding protein is proteolytically clipped during expression/purification. The NCXI for chicken and human cone NCKX; transcripts were localized to the cone photoreceptor inner expressed the Bac3000-derived virus also contains a His-tag at the carboxy terminus (NCX- segments ofchicken and human retina, respectively. The putative topology of cone NCKX is very His). was inhibited by exchange inhibitory peptide (ICso = < I uM) and the 120 and 70 similar to that of rod NCKX, but the two large hydrophilic loops are much shorter in cone NCKX NCX I were observed under nonreducing SDS-PAGE while only the 70 kDa protein and lack the large stretch dominated by acidic residues observed in mammalian rod NCKX. Two present under reducing conditions. Comparison of NCX1 specific activity between the 2 splice variant were cloned from both species, the shorter one missing 17 amino acids compared to a lower value for the protease-deleted version (1.7 vs. 0.045 nmol Ca/mg the full-length protein. We observed potassium-dependent Na-Ca exchange activity after prot./s). NCX-His was purified via chelated Ni affinity column chromatography, yielding the 120 heterologous expression ofboth splice variants of the human cone NCKX in cultured insect cells. forms. Affinity purified NCX-His activity in reconstituted proteoliposomes was 0.362 Activity of cone NCKX measured with 45Ca uptake was significantly higher when compared with Ca/mg prot./s (n=4), an 8-fold increase. Elimination of the viral protease facilitated rod NCKX, and allowed us to directly demonstrate and characterize potassium transport via cone expression length NCX1 but in lower amounts. The expressed 120 kDa protein is cleaved NCKX using "6Rb uptake. Supported by MRC ofCanada. I site and is held together by disulfide bridge interactions. (Supported by AHA- Consortium (CCH), NIH (CCH), COR-CVM (CCH), CF Foundation (EMP), and AHA- EI (EMP).

309- Pos EXCHANGER CURRENTS MEASURED IN INSECT CELLS TRANSFECTED FUNCTIONAL ANALYSIS OF A DISULFIDE BOND IN THE CARDIAC Na+-Ca'+ THE RETINAL CONE NCKX cDNA. EXCHANGER (NCX1.1) Jian-Zhong Sheng, C. F.M. Prinsen, R. B. Clark, W. R. Giles, Paul P.M. Schnetkamp, Michela Ottolia', Lucia Santacruz-Toloza2, Debora A Nicoll', Kenneth D. Philipson', 'UCLA, University Calgary Dept. of Physiology, Los Angeles, CA, 2Dept. of Molecular Physiology and Biophysics, Baylor darkness, cGMP-gated channels carry a significant calcium influx across the plasma membrane College of Medicine, Houston, Texas cone photoreceptors. This calcium influx is balanced by calcium efflux mediated Na+-Ca2+ exchanger activity increases after treatment with redox agents. A rearrangement of an by cone-specific Na-Ca+K exchangers. We have determined the molecular identity of the intramolecular disulfide bond has been proposed as the mechanism responsible for this stimulation Na-Ca+K exchanger, and, using 45Ca fluxes, have shown that it is a potassium- (Reeves et al.; JBC 261: 4948-4955, 1986). Using cysteine mutagenesis and biochemical analysis, dependent Na-Ca exchanger. Here, we have applied whole-cell patch clamp to examine we demonstrate the presence of an intramolecular disulfide bond between cysteine 792 and transmembrane currents in insect cells transfected with cDNA of the chicken and human retinal cysteine 14 or 20. We further investigate the effect of redox reagents on NCXI.I and various Na-Ca+K exchanger. Inward current was observed when the bath solution contained sodium mutants. NCX1. I was expressed in Xenopus oocytes and exchange currents were characterized solution contained pipette potassium together with micromolar free calcium concentration. using the giant patch technique. Upon application of internal Na+an outward current was elicited current was observed when the pipette solution contained sodium and EGTA, and the (8 mM CaW+ in the pipette). The exchange current peaked and than decayed with time (Na+- contained both potassium and calcium. No current changes were observed in dependent inactivation). Cytoplasmic application of 100 I&M FeSO4 and 3 mM DTT (Fe-DTT) cells. Similar membrane currents were observed in cells transfected with the bovine induced exchanger activation mainly by removing the Na+-dependent inactivation. Peak and heartNa-Ca exchanger, but in this case outward current required only the presence of calcium in steady state current increased by 2.2 ± 0.6 and 7.8 ± 2.2 fold (n=30) ([Ca2+]i= 1 pM), respectively. bath solutions. These results demonstrate that retinal cones an express electrogenic and FeSO4 or DTT by themselves were unable to evoke an effect. Activation by Fe-DTT was also potassium-dependent Na-Ca exchanger. A quantitative comparison will be presented between observed in a cys-lessexchanger with cysteines 14, 20, and 792 reintroduced and in the wild type transmembrane ionic currents generated by the rod and cone Na-Ca+K exchangers expressed in exchanger with cysteines 14, 20 or 792 mutated to alanine. The data indicate that cysteines insect cells. Supported by the Medical Research Council of Canada. residues are not involved in Fe-DTT stimulation.

53A 310-315...... SODIUM-CALCIUM EXCHANGE SUNDAY

- 310 - Pos 311 Pos FUNCTIONAL CHARACTERIZATION OF A "SPLIT" Na+-Ca5+ EXCHANGER A NOVEL EXCHANGER FROM E. COLI: SIMILAR IN STRUCTURE BUT Michela Ottolls, Zhiyong Qiu, Kenneth D. Philipson, UCLA, Dept. of Physiology, Los Angeles, DIFFERENT IN FUNCTION TO Na+/Ca5+ EXCHANGERS. CA Dan H. Schulze, Abdul M. Rukludin, University of Maryland, 655 W. Baltimore St., Baltimore, We have constructed two cDNA clones containing non-overlapping fragments of the full length MD 21201 exchanger. One clone codes for the N-terminal 5transmembrane helices and part of loop f and the In mammalian cells the Na+/Ca2+ exchanger is an important transport molecule involved in Ca2+ other clone consists of the remainder of loop f and the C terminal transmembrane helices. homeostasis. There are three mammalian genes, NCX1, NCX2 and NCX3, which produce Coexpression of the two clones in Xenopus oocytes or HEK cells leads to an active exchanger as Nae/Ca2+ exchanger proteins and these proteins function similarly. In invertebrates, the Nae/Ca2+ detected by Na+-dependent 45Ca2' uptake and electrophysiological measurements. Expression of exchanger has been cloned from Drosophila melanogaster and it also functions like mammalian either domain separately does not result in exchange activity. Assembly of the split exchanger Na+/Cae+ exchangers. By sequence homology several sequences from bacteria to mammals, viz. appears to require disulfide bond formation between Cys2O and Cys792. The biophysical gb/X66481, sp/P34322, dbj/D35735, gb/Z46259 and gblU18997, are suggested to belong to the properties of the split exchanger were analyzed using the giant-patch technique in the inside out superfamily of the Na+/Ca2+ exchangers. To study the function of the bacterial exchanger, we have configuration. Application of internal Na' elicited an outward current when 8 mM Ca2+ was cloned the E.coli gb/U18997 gene by PCR cloning method from E.coli genome. We subcloned the present in the pipette. The split exchanger displayed Nae dependent inactivation that decayed cDNA for E. coli exchanger into pSD64TF vector to facilitate expression in Xenopus laevis exponentially with a time constant of 2.4±0.2 sec reaching a steady state that was 60±5 % of peak oocytes. When the cRNA-injected oocytes were studied using 45Ca2+ method for the Na+/Ca2+ current (100 mM Nae, I pM CaW') (n=l I). The split exchanger showed partial Ca2' regulation. exchanger activity, we did not detect any significant Na+-dependent Ca2+ uptake suggesting that Upon removal of regulatory Ca2', a Ca2'-insensitive current component remained (60±3 % of this is not a Na+/Ca2+ exchanger. Further experiments are in progress in characterizing this steady state current, n=4). The split exchanger was chymotrypsin and XIP sensitive. Split transporter. exchangers with serial truncations of loop f were also constructed and found to be active.

312 - Pos 313 - Pos CIRCULAR DICHROISM SPECTROSCOPY OF THE Na+-Ca2+ EXCHANGER TEMPERATURE DEPENDENCE OF CARDIAC Na+-Ca5+ EXCHANGER: REVEALS -SHEET STRUCTURE IN THE Ca2+-BINDING CYTOPLASMIC LOOP COMPARISON OF CANINE (NCX1) AND SALMONID (NCX-TR1) ISOFORMS Shannon K Lewis', R. S. Nunn', D. A. Nicoll2, K. D. Philipson2, M. J. Yeagerl, 'The Scripps X H Xue', C L Elias2, A Omelchenko2, L V Hryshko2, Glen F Tibbits', 'Simon Fraser University, Research Institute, Department of Cell Biology, 10550 North Torrey Pines Road, La Jolla, 2University of Manitoba California 92037, 2UCLA School of Medicine, Departments of Physiology and Medicine Sarcolemmal vesicular studies have shown the trout, a cold water salmonid, NCX to have very The 938 residue Na+-Ca2+ exchanger is a polytopic membrane protein that maintains cellular Ca2' high activity at 7°C in comparison to that from mammals and this unique property is attributable to levels by exchanging three extracellular Nae ions for one intracellular Ca2+ ion. The exchanger is differences in protein structure. In this study, we describe the temperature dependencies of cloned regulated by a high affinity cytoplasmic Ca2+ binding site, which is separate from the Ca2+ wild type NCX-TRI, NCXI and chimera constructs of these cDNAs cRNA was injected into transport site. Residues 240-679 in the cytoplasmic loop were expressed in E. coli as a fusion Xenopus oocytes and after 3-5 days outward currents were measured using the giant excised patch protein [Levitsky et aL, J. Biol. Chem. 269, 22847-22852 (1994)]. Ca2e overlay blots and technique. After application of 100 mM Na+ and I AM Ca2+ to the cytoplasmic surface at 30'C, mobility shift assays showed that this fragment displayed Ca2+ binding activity. Based on the both NCXI and NCX-TRI exhibited a peak current (I,k) which decayed to a steady-state current analysis of N- and C-terminal truncation mutants, the high affinity Ca2+ binding domain was (I.) with r <8s. The magnitudes of Ik and I. were enhanced dramatically by increasing [Ca2+], localized within residues 371-525. Secondary structure prediction algorithms suggest that this from 0 tol FM in both wild types. Although reducing the temperature from 30 to 7C, reduced Ipk region folds with a high content of f-pleated sheet. To test this prediction, residues 355-540 and and 1. in both wild types, the Qls values were markedly different between the species. For the an N-terminal hexahistidine tag were cloned into the Qiagen E. coli expression plasmid pQE-32. mammalian isoform the Qis values were 2.5 for both lpk and I., while these values were 1.2 and The recombinant protein was purified to homogeneity using nickel-chelate chromatography. 1.5 for Ip, and I., respectively in the salmonid isoform. A chimera (DTT) was constructed in Circular dichroism (CD) spectroscopy was performed using an AVIV spectropolarimeter. At pH which the first 279 aa (corresponding to first 5 transmembrane segments and XIP site) of the 968 7.0 in 10mM NaH2PO4 buffer, the CD spectrum displayed a broad minimum at -215 nm and a aa in the trout cDNA were replaced with that of canine. DIT exhibited a mammalian-like sharper maximum at 198 nm. These features are characteristic for a P-sheet conformation. temperature dependence suggesting that the species difference exists in this segment of the Experiments are underway to evaluate whether Ca2+ binding induces a change in secondary molecule. structure.

314-Pos 315- Pos A UNIQUE FORM OF Ca2+rDEPENDENT REGULATION OF THE KIDNEY Na+-Ca2+ INHIBITION OF Na+-Ca2+ EXCHANGE CURRENTS BY KB-R7943. EXCHANGER, NCX1.3. Chadwick L. Elias', A. Omelchenko', G.J. Gross2, M. Hnatowich', L.V. Hryshko', 'Inst. Alexander Omelchenko, C.L. Elias, M. Hnatowich, L.V. Hryshko, Inst. Cardiovasw. Sci., St. Cardiovasc. Sci., St. Boniface Gen. Hosp. Res. Ctr., Univ. Manitoba, Winnipeg, Canada, R2H Boniface Gen. Hosp. Res. Ctr., Univ. Manitoba, Winnipeg, Canada, R2H 2A6 2A6, 2Cardiology Division, Hospital for Sick Children, Toronto, Ontario, Canada, M5G IX8 All Na+-Ca2+ exchangers examined to date are regulated by the transported ions, Nae and Ca2+. The effects of the novel isothiourea derivative, KB-R7943, were examined on the canine cardiac Furthernore, differences in these ionic regulatory processes have been found among altematively Nae-Ca2+ exchanger, NCXI .1, expressed in Xenopus laevis oocytes using the giant excised patch spliced Nae-Ca2+ exchanger isoforms. Here, we show a unique regulatory response for NCX1.3, technique. We examined inward, outward, and combined inward-outward Na+-Ca2+ exchange the exchanger commonly referred to as the kidney splice variant. Ca2+i-dependent regulation of currents before and after applying various concentrations of KB-R7943 to the cytoplasmic surface outward Na+-Ca2+ exchange currents was examined using the giant excised patch technique in X of the patch. Similar to previous reports, we observed a preferential inhibition of outward NaW- (cardiac splice variant) or With laevis oocytes expressing either NCXl.l NCX1.3. NCXl.l, Ca2+ exchange currents. The ICso for inhibition of pure outward currents was -2 pM. For pure inactivation, whereas regulatory Ca2+, could completely alleviate Naei-dependent (II) I, inward currents, applying 20 FM KB-R7943 resulted in 20% inhibition and an IC50 value was we a unique response of inactivation of NCX1.3 was barely affected. In addition, observed not determined. This preferential inhibition of the outward transport mode was maintained in prior NCXI.3 with respect to the influence of pre-incubating patches with regulatory Ca2` to patches where combined inward-outward currents were examined. Deregulation of exchange was observed regulatory Ca2' was current activation. For NCXI.I, little difference whether current with a-chymotrypsin led to a small reduction in the extent of inhibition. The effects of with In contrast, NCXI.3 demonstrated a applied prior to or during current activation Na+. KB-R79343 on outward Na+-Ca2+ exchange currents were examined at different concentrations of marked inhibition of peak current with regulatory Ca2' pre-incubation, exhibiting 100% larger Ca2+0 and Na+, and the inhibitory potency and IV characteristics will be described. currents when Nae and Ca2+ were applied simultaneously. Our results indicate that regulatory Ca2+ exhibits both stimulatory and inhibitory effects on outward Na+-Ca2+ exchange currents for NCXI.3.

54A SUNDAY SODIUM-CALCIUM EXCHANGE 316-321

316- Pos 317- Pos FREQUENCY-DEPENDENT ACTIVITY OF ALTERNATIVELY SPLICED Na+-Ca2+ THE CHANGE OF THE FREE ENERGY OF Na+/Ca2'+-EXCHANGER DURING EXCHANGERS METABOLIC INHIBITION. Larry V. Hryshko, C.L. Elias, S. Shurraw, M. Trac, A. Omelchenko, M. Hnatowich, Inst. Antonius Baartscheer, Cees A. Schumacher, Joris R. de Groot, Jan W.T. Fiolet, Academic Cardiovasc. Sci., St. Boniface Gen. Hosp. Res. Ctr., Univ. Manitoba, Winnipeg, Canada, R2H Medical Center, Meibergdreef 9, Amsterdam, 1105 AZ Netherlands 2A6 Calcium homeostasis depends on the kinetic properties of the Na+/Ca2'-exchanger, in which its Ionic regulation by the transport ions, Na+ and Ca2+, modulates the activity of all members of the free energy (AG,,,,) plays a key role. Na`-Ca2+ exchanger superfamily. Na'-dependent inactivation describes a process whereby the During metabolic inhibition (2 mmol/l cyanide) cytoplasmic [Ca2+]i, [Na'],, action potential and introduction of cytoplasmic Na' which activates exchange current also leads to inactivation. Ca2+- SR calcium content were measured in ventricular myocytes (2 Hz, 37°C). activation describes the where recruits out of an dependent process cytoplasmic Ca2+ exchangers After 5 min of metabolic inhibition diastolic [Ca2']i decreased from 150 to 40 nmol/l and systolic state. In this we examined the activity of three inactive study, frequency-dependent altematively [Ca2+], with 75%. After 14min transients were disappeared and diastolic [Ca2+], started to increase spliced Na+-Ca2+ exchangers, NCXl.l, NCXI.3, and NCXI.4, expressed in X. laevis oocytes. to 700 nmol/l. During the first 10 min [Na'], remained constant where after it increased to 29 Outward Na+-Ca2+ exchange currents were examined using the giant excised patch technique. mmnol/l. The APD90 decreased 30% in the first5 min and remained constant for5 min where after Considerable differences in exchanger activity were observed between splice variants in response it largely decreased until the myocytes became unexcitable after 14 min. The SR calcium content to different solutions frequencies (up to 4 Hz) and durations of activity/recovery switching decreased similar compared to systolic The average increased from 6.2 to 7.6 (analogous to alterations in action potential duration). In all cases, the extent of exchanger [Ca2']i. AG,,ch kJ/mol in the first four minutes of metabolic inhibition. After 15 min was decreased to 0 inactivation was reduced solution application compared to continuous application. AG,,,c, by pulsatile kJ/mole where after it remained constant. Following deregulation of the exchangers by treatment with a-chymotrypsin, all frequency- dependent responses were lost. Our results demonstrate that altemative splicing may represent a Conclusion: Metabolic inhibition caused a temporally increase of the AG,,,h caused by a decrease means of fine tuning exchanger recruitment pattems to be appropriate for their different cellular of the APD. This resulted in a decrease of diastolic [Ca2']i and an enhancement of the decrease of environments. systolic [Ca2+]i and SR calcium content. Diastolic [Ca2+], started to increase when AG,,,,h reached equilibrium.

318-Pos 319 Pos INCREASED Na/Ca EXCHANGE & REDUCED IKI FACILITATE TRIGGERED SR Ca RELEASE TRIGGERS ACTION POTENTIALS (AP) THROUGH Na/Ca ACTION POTENTIALS IN AN RABBIT MODEL OF HEART FAILURE. EXCHANGE CURRENT IN MYOCYTES FROM FAILING AND NON-FAILING Jose L. Puglisi, S. M. Pogwizd, W. Yuan, D. M. Bers, Loyola University Chicago; Maywood, IL RABBIT AND HUMAN 60153; University of Illinois at Chicago IL 60612 Klaus OA Schlotthauer', Steven M Pogwizd2, Donald M Bers', 'Loyola University Chicago, Rabbits with heart failure (HF) show spontaneous ventricular tachycardias (VT) of non-reentrant 2160 South First Ave., Maywood, Illinois 60153, 2University of Illinois origin (e.g. delayed afterdepolarizations, DADs). HF myocytes exhibit 117% increase in Na/Ca Caffein"activated DADs. APs In non-ischemic heart failure (HF) delayed after- exchange current (INcx), 49% and 36% decrease of IKI and It,, and unaltered Ic. and SERCA2 in a rabbit myocyta depolarizations (DADs) are likely candidates for non- levels. These changes may underlie aftercontractions in these myocytes. We developed a computer a2H reentrant tachyarrhythmia. DADs are associated with model (LABHEART 4.0) to mimic our measured currents and twitch and caffeine-induced A[Ca]i 5R 20- 9/ .sH spontaneous SR Ca release, activating Ca induced inward in control (Ctl) and HF myocytes. Spontaneous SR Ca release activated Its and DADs which could J § >SE0.2Ht currents such as IN./C., aCI(C) and INS(C.), which depolarize the trigger APs in Ctl. For a given SR Ca cDADs myocyte. Application of 10 mM Caffeine at 37C caused SR \42Y t Ca release caffeine-activated release, the 117% increased INCX 0g and DADs (cDADs) in isolated lowered the 1reshold for triggered 40 t555 is.,10meCafre myocytes of HF human, HF rabbit and control rabbit. The APs to 360 .50 ([Ca],h 680 nM). Similarly E Nickel blocks cDAD cDADs amplitude increased with elevated SR Ca load (by decreasing IKI by 49% increased . I l .C enhanced pacing) and eventually reached threshold to trigger DADs and reduced threshold for 9 HF 'INCX !'IKI an AP in all three cell types. Blocking IN./C. with 5 mM Ni triggered APs (to 380 nM). Changing -60- IK1 CalM,,.AD - for 2 sec prior and during Caffeine prevented cDADs and both INCX and IKI as in HF myocytes 0 tINCRI E triggered APs, whereas 30 ,uM niflumate (to block Icl(c,)) did greatly lowered the amount of Ca 5 .70 Contro 'a notblock cDADs nor triggered APs. We conclude that SR Ca release required to trigger a .s release evokes DADs in an SR Ca load dependent manner via spontaneous AP (to 170 nM). Changes Caftine '° §inward IN./C. and can trigger APs. In heart failure this in It. had no effect. We conclude that M -80 Threshold E405iNI,,klI arrhythmogenic potency could be increased by the 2-fold the triggered APs that contribute to forAP 7 0 Il 5 - enhanced INVC. and the -50% reduction of IKI, which nonreentrant VT in HF are due to . stabilizes E, near EK. changes in both INCX and IKI 0 100 200 360 400 500 660 760 Spontaneous A[Ca]J (nM)

320- Pos 321 Pos SODIUM-CALCIUM EXCHANGE, POTASSIUM AND EC COUPLING IN VOLTAGE- IN SQUID AXONS MgATP MODULATION OF Na/Ca EXCHANGE INVOLVES CLAMPED GUINEA PIG VENTRICULAR MYOCYTES. RECOVERY FROM INTRACELLULAR H+ AND Na+ INHIBITION. Neal Shepherd, Holly B. McDonough, VA Medical Center, Research Service, 508 Fulton Street, R. DiPolo', L. Beaug62, 'IVIC-Caracas-Venezuela; M.y.M. Ferreyra C6rdoba-Argentina, 'Marine Durham, NC 27705 Biological Lab, Woods Hole, MA Replacement of K+ with Cs+ in physiologic solutions is reported to interfere with the role of An increase of intracellular Na+ and H+ concentration inhibit the Na/Ca exchanger. On the other sodium-calcium exchange (NCX) in e-c coupling (Wasserstrom and Vites, J. Physiol., hand, ATP regulates exchange activity by altering critical kinetic parameters including Ca2+,, Na+, 493:559,1996). To see if K' directly affects NCX under physiologic conditions, we studied the and Na+, affinities. We examined if ATP regulation includes modification of H+, inhibition. We effects of K+ on changes in INCx due to rapid changes of extracellular [Na]+ At a holding did that by exploring the relationship between cytoplasmic H+ Na+ and ATP in internally of-45 = potential mV, with [K +] =130 mM and [K +] . 5.4 mM, rapid replacement of 25-100% of dialyzed squid axons. Our results show that: 1-in the absence of internal ATP inhibition by Na' Na+, by Li+ -100 ms prior to depolarization to 0 mV caused a large, fast outward current change is strongly dependent on H+ The K, for Na+ were 10, 25 and 90 mM at pH: 6.9, 7.5 and 8.8 during depolarization (At), and increased twitch tension (AT). 10 ,sM nifedipine blocked At, respectively. 2- In the presence of ATP, inhibition by Na+' is practically insensitive to pH, with a leaving a small steady-state outward shift. 200 pM DIDS, 5 mM anthracene, I mM Ba+, 10 mM K, close to 100 mM both at pH 6.9 and 8.8. 3-At pH (6.9) and in the absence of internal Na', Cs+, 20 mM TEA or 50 nM charybdotoxin had no effect on the waveform or magnitude of Al. 200 ATP induces a large increase in the Na,-dependent Ca efflux. The present results indicate that pM ouabain did not alter the waveform of Al but increased its magnitude, presumably reflecting ATP brings about a relief of H+ inhibition on the exchanger and also decreases the affinity for an increase in At internal Na+ inhibition. These findings are predicted by a model in which increasing [Na+]i The dependence of on V., [Ca,+] , [Na'], and [Na']. and its resistance H+ to channel blockers, is consistent with its being a pure outward shift of sodium-calcium exchange concentration reduces the "off' rate of Na+' bound to the carrier and the rate of Ca efflux, and these are counteracted An experimental not current (IN.C.). Replacing intracellular K+ with Cs+ had little or no effect on Al or AT. However, where effects by ATP. finding, predicted by the replacing extracellular K+ with Cs+ appeared to shift Al markedly inward, whether intracellular K+ model, is that in the absence of AT?, the affinity of the Na/Ca exchanger for external Na+ is markedly increased by rising intracellular H'. The apparent K,,, for external Na+ goes from about was present or not, without changing AT significantly. Thus, the effect of K', on At is most likely 80 mM at to due to a K+ channel, buta modulating effect of K+ on IN.-c. cannot yet be excluded. pH 8.8, 10 mM at pH 6.9; this suggests that intracellular H+ might have "trans" effects on the exchanger-cations interactions. The reliefof H+ and Na+ inhibition induced by ATP could be important in cardiac isquemia in which a combination of acidosis and rise in [Na+], occurs. (Supported by CONICIT SI-2000-Venezuela, CONICET 4904/97, CONICOR 4021/97 and FONCYT PICT-97 05-00000-01092, Argentina and USA-NSF IBN-963 1107).

55A 322-323 SODIUM-CALCIUM EXCHANGE SUNDAY

322 - Pos 323 - Pos FEEDBACK INHIBITON OF SODIUM/CALCIUM EXCHANGE ACTIVITY BY NA+/CA2+ EXCHANGER (NCX1) REDUCES CYTOTOXICITY CAUSED BY CALCIUM MITOCHONDRIAL CALCIUM ACCUMULATION. OVERLOAD Kwabena Opunl, John P. Reeves, UMDNJ-Grad. Sch. Biomed. Sci., Dept. Pharmacology & Yihong Wang, Robert A. Colvin, Ohio University, Athens, OH 45701 Physiology, 185 S. Orange Ave., Newark, NJ 07103 In order to address the role ofNa+/Ca2+ exchange in neuronal calcium homeostasis, we transfected Reduction of extracellular Na in Chinese hamster ovary cells expressing the bovine cardiac Na/Ca PC12 cells with an NCX1 isoform. 45Ca2+ uptake was rapid initially in overexpressing cells, bus exchanger results in an increase in both cytosolic and mitochondrial Ca, measured using the Ca- within 2 minutes there was no net Ca2+ transport. A Vmax of 0.4nmol/U5cells/2min and Km of sensitive dyes fura-2 and rhod-2 respectively. Fura-2 loaded cells were subjected to two 15uM were obtained. Next, cells were allowed to accumulate 45Cs2+ for 2 minutes and then sequential intervals of low extracellular Na (20 mM) separated by a 5 min recovery period in 140 switched to 137 mM sodium. After two minutes, 45Ca2+ remaining in the cells was reduced by mM Na; all solutions contained 1 mM CaC12. The change in cytosolic Ca during the second 50%. 5mM nickel completely abolished NCX activity in overexpressing cells. KB-R7943 showed interval in low Na, compared to that during the first, was 20% under control conditions, 100% in a dose dependent inhibition of NCX activity in NCXI overexpressing cells with half maximum the presence of the uncoupler Cl-CCP (2 pM), and 5% in the presence of diltiazem (100 pM), an inhibition at 3OuM. Concentration dependency of A23187 and ionomycin cytotoxicity was tested inhibitor of the Na-dependent mitochondrial Ca efflux pathway. A minimal first interval duration on vector and NCXI transfected cell lines using LDH and MTT assay. The overexpressing cells of3 min was required to induce inhibition of Ca uptake during the second interval. Nocodazole (5 showed less cell death. To test whether the ionophore induced cytotoxicity was calcium pg/mI), which depolymerizes microtubules, and the protein kinase inhibitors K252a or dependent, we used 3uM ionomycin in a medium with 1.1mM Ca2+. The LDH reading of the staurosporine (1 pM) equalized Ca uptakes during the first and second interval. We conclude that vector transfected cell line was twice the reading of the overexpressing cell line. But when treated the mitochondrial Ca accumulation during the first interval inhibits Ca influx by Na/Ca exchange with 3uM ionomycin and 1. 1mM EGTA, the two groups showed LDH readings similar to control during the second. The mechanism is unknown, but the effect of nocodazole suggests that (no ionomycin treatment). The results show that PC12 cells overexpressing Na+/Ca2+ exchanger mitochondrial location with respect to the plasma membrane and/or intracellular Ca stores is a are afforded some protection from Ca2+ dependent cell death induced by ionophore treatment. critical factor in this interaction.

324-327 MEMBRANE FUSION SUNDAY

324 - Pos 325- Pos EFFECT OF SUPPORTS AND COSURFACTANTS ON THE FORCES AND FUSION OF HYDROPHOBIC SURFACTANT PROTEINS ALTER THE THERMODYNAMICS OF BILAYERS INITIAL ADSORPTION TO AN AIR / WATER INTERFACE Chad K Park, Jacob N Israelachvili, University of California, Santa Barbara, Chemical Vinceat Schram, Stephen B. Hall, Oregon Health Sciences University, 3181 S.W. Sam Jackson Engineering, Santa Barbara, CA 93117 Park Road Mail code NRC-3, Portland, OR 97201-3098 The surface forces apparatus (SFA) was used to observe the fusion of apposed, solid and soft supported, phospholipid in time. bilayers real Previous work has involved investigation ofeffects We determined the influence of the two hydrophobic surfactant proteins, SP-B and SP-C, on the like packing or deposition density, lipid shape and lipid phase state. Recently, we have supported thermodynamic barrier to adsorption of surfactant at the air / water interface. The initial rate of bilayers on thin, swellable polyelectrolyte layers. This allows for a more mobile and fluid adsorption, followed by measuring surface tension with a Wilhelmy plate, was compared for Calf membrane, while still allowing manipulation of the bilayers' position. Current work focuses on Lung Surfactant Extract and for the set of surfactant Neutral and the effects of additives of (CLSE) complete Phospholipids dimethylsulfoxide (DMSO) and ethanol as positive and negative (N&PL) depleted of the proteins at a series of temperatures and lipid concentrations. When curvature promoting cosurfactants. present, SP-B and SP-C 1) increased the initial adsorption rate for CLSE approximately 10 fold relative to N&PL, 2) reduced the activation energy from 59.3 kJ/IK/mole (N&PL) to 43.9 kJ/°K/mole (CLSE), and 3) decreased the Gibbs free energy of activation (AGo*) from 74.6 kJ/lK/mole to 64.9 kJ/°K/mole. The proteins reduced AGo* by producing a favorable decrease in the activation enthalpy AHo* from 56.7 kJ/mole to 41.4 kU/mole that more than compensated for an unfavorable lowering ofthe activation entropy ASon from -58.4 J/K/mole to -77.3 J/PK/mole. These results suggest a model in which the major barrier to adsorption is a decrease in interactions among acyl groups in a tightly curved intermediate. SP-B and SP-C may then facilitate adsorption by lowering the unfavorable enthalpy barrier presented by this tightly curved intermediate.

326- Pos 327- Pos MEMBRANE TENSION FACILITATES VOLTAGE DEPENDENT SURFACE ELECTROSTATICS, SURFACE HYDROPHOBICITY, LEAKAGE, AND PERMEABILIZATION AND FUSION FUSION OF MIXED LIPID MEMBRANES CONTAINING PHOSPHOINOSITIDES. Justda A TEISSIE, Corinne RAMOS, CNRS, IPBS (UPR 9062), Route de narbonne, Toulouse, Matthias MIllfer', Olaf Zsch6mig', Klaus Amold', Shinpei Ohki2, 'University of Leipzig, 31062 France Liebigstr. 27, D-04103 Leipzig, Germany, 2State University ofNew York at Buffalo Cell Electropulsation induces a position dependent membrane potential difference modulation. Phosphoinositides constitute only a small part of cellular membrane phospholipids, however have This can bring a local transient membrane This a permeabilization. perturbation induces locally an enormous biological importance. Our studies are focused on the influence of headgroup fusogenic state of the membrane. In vivo such perturbations are proposed to be involved in structure and the negative charge of differently phosphorylated phosphoinositides on the surface exocytosis. 10 years ago liposome electropermeabilmization was shown to be facilitated by a properties and fusion of membranes containing phosphoinositides at their physiologically relevant bilayer tension increase. A new method was designed to increase the membrane tension in a low concentrations. We investigated different steps ofcation-induced fusion of vesicles population of mammalian cells. A consisting biocompatible filter is used. This does not affect the cell of 5 mol% PI, PIP, PIP2, and PIP3 in PC or PE and compared them to phosphoinositides enriched viability. Contacts are established between cells. Electric pulses are easily applied to the cells. lipid mixtures from bovine brain. Surface potential and zeta potential in the absence and When the cell membrane is under the tension, permeabilization is observed under lower voltage presence of cations are related to the spatial charge distribution caused by phosphoinositides. conditions. This permeabilized state always supports fusion. An increase in membrane tension Cation effects on membrane surface hydrophobicity and membrane stability for non- therefore acts in synergy with the a transmembrane voltage to create permeabilized and fusogenic phosphorylated as well as for phosphorylated phosphoinositides, increase with the state. The higher the mechanical stress is, the lower the electrical contribution has to be. Homo as phosphorylation level and the number of negative charges in the membrane. While non- well as heterofusion can be obtained with a very high yield. This property of cell membrane has phosphorylated PI inhibits fusion (phospholipid mixing), the presence of even small amounts of to be taken into account in exocytosis associated fusion, where are mechanical stresses due to the PIP, PIP2, or PIP3 promotes fusion depending on phosphorylation state. The behavior of complex protein complex acting to bring vesicles in contact with the membrane. lipid mixtures with phosphoinositides is described as a balance of steric and electrostatic effects. (Supports from the ARC and the region Midi Pyrenees are acknowledged) This work was supported by DFG, SFB 197.

56A SUNDAY MEMBRANE FUSION 328-333

328 - Pos 329 - Pos CURVATURE AND MECHANICAL STRESS IN PEG-INDUCED VESICLE FUSION. "NATURE'S OWN" FUSOGENIC LIPID BILAYER Malinll, B R Lentz, University of North Carolina, Dept. of Biochemistry, MEJB M E Haquel, T McIntosh2, B R Lentz', 'University ofNorth Carolina, Dept. of Biochem. & Prog. CB#7260, Chapel Hill, NC 27599 in MolecJCell Biophys, Chapel Hill, North Carolina 27599, 2Duke University PEG in solution affects lipid vesicles by 3 possible ways: it brings vesicles together into close We have examined polyethylene glycol (PEG) mediated fusion of highly curved (SUV) and contact due to "depletion forces"; it creates mechanical stress in membranes and induces shrinkage uncurved (LUV) membrane vesicles composed of four lipids in different ratios in order to identify (and changing of membrane curvature) due to osmotic stress. To test the role of these factors, we an optimally fusogenic membrane. We have found that vesicles composed of DOPC/ monitored contents mixing (CM) and lipid mixing (LM) between different sized vesicles under a DOPE/Sphingomyelin(SM)/Cholesterol (CH) fuse very well at a 35/30/15/20 molar ratio. This variety of osmotic conditions. CM between highly curved vesicles (SUV, 25 nm diameter) was up composition is very close to natural synaptic vesicle composition. Each lipid seems to have a times greater than between nearly uncurved vesicles (LUV, 120 nm diameter) at a low PEG specific role in membrane fusion. PE disrupts bilayer packing (TMA-DPH lifetime, C6NBD-PC concentration (< 10 %), while LM was the same. A negative osmotic gradient (- 5 atm, hypertonic partitioning and DPH anisotropy) without significantly altering inter-bilayer approach (X-ray outside) increased CM (but not LM) 2 - 3 times for both types of vesicles. We propose that this diffraction), and thus significantly enhances fusion. An optimal ratio of PC/PE is critical to the effect of negative osmotic stress is due to mechanical stress for SUV and shrinkage for LUV. A balance between fusion and rupture. CH and SM, when present at an optimal ratio in vesicles positive gradient (+ 5 atm, hypertonic inside) nearly eliminated CM but had no effect on LM. We containing the optimal PC/PE ratio, reduce rupture without significantly reducing fusion. This conclude that contacting vesicle membranes easily form an intermediate accompanied by outer optimal CH/SM ratio also enhances outer leaflet packing, indicating that fusion does not correlate leaflet mixing as soon as they come into close contact. However, formation of the final fusion only with decreased outer leaflet packing - the inner leaflet must play a significant role. CH alone product depends critically on membrane curvature and mechanical stress. Supported by USPHS enhances rupture relative to fusion, while SM alone reduces both rupture and fusion. We conclude grant GM32707. that the synaptic vesicle lipid composition is optimized with respect to fusogenicity. Supported by USPHS grant GM32707.

Pos 331- Pos MECHANISTIC AND KINETIC STUDIES OF SAPOSIN C INDUCED VESICLE FUSION A SYSTEM FOR T1HE STUDY OF VESICLE-BILAYER INTERACTIONS BY ATOMIC Xiaoyang Qi G. A. Grabowski, Children's Hospital Research Foundation and University of FORCE MICROSCOPY College of Medicine, Cincinnati, Ohio 45229 Sanjay Kumar, Jan H. Hoh, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Saposin C is a small (80 amino acids, no Trp), multifunctional lysosomal protein. Saposin C Baltimore, MD 21205 comprises four amphipathic a-helices (Hl, 2, 3, and 4) linked with three disulfide bridges. In We describe an in vitro system for studying vesicle-bilayer interactions based on direct addition to activation of acid I-glucosidase and promotion of neurite outgrowth, saposin C induces visualization by atomic force microscopy (AFM) of vesicles bound to a supported lipid phospholipid lamellar vesicles at acidic pH. The mechanism and kinetics of saposin C's membrane. Vesicle-bilayer complexes were formed using the vesicle adsorption technique while fusogenic activity were evaluated by fluorescence spectroscopy, including maximum emission retaining excess vesicles. Pure dipalmitoylphosphatidylcholine (DPPC) vesicles appear to form shifts, quenching and dequenching, resonance energy transfer, and stopped-flow analysis. Trp was patchy lamellae on top of the bilayer, sometimes forming small dome- and saucer-like structures introduced into the selected sites of saposin C as a fluorescence reporter. The size changes of instead. The use of mixed DPPC-cholesterol vesicles dramatically increases the fraction of these liposomes were monitored by EM and N4+submicron particle sizer. These analyses indicated that latter two structures. Two lines ofevidence suggest that these structures are bound vesicles. First, protein-membrane and protein-protein interactions are involved in the fusion. During relative elasticity measurements show that they are far more compliant than the underlying bilayer. protein-lipid association, HI at the amino- and H4 at the carboxyl- termini anchored rapidly to Second, adjacent structures appear to come into flat interfacial contact, similar to vesicle-vesicle negatively charged membrane surface through ionic binding. This was followed by a slower complexes studied previously by cryo-electron microscopy. The observation that cholesterol embedding phase into the phospholipid bilayer through hydrophobic interaction. The entire stabilizes these vesicular structures may be due to cholesterol's well-documented ability to fortify association process was completed in 3-4 seconds. Upon binding to lipid membrane, a phospholipid bilayers. Evidence for this is provided by mechanical abrasion experiments on the conformational change of saposin C was determined by circular dichroism. The alteration opened supported membrane at varying cholesterol concentrations. High concentrations of cholesterol H3 regions to expose hydrophobic patches for saposin Cs on other liposomes to binding. appear to confer resistance to probe-induced bilayer defects and alter the rate at which those on these results, we proposed a clip-on model, i.e., liposome-bound saposin Cs clip one to defects subsequently heal. through hydrophobic interaction, and induce liposome fusion.

333 - Pos INTERACTION OF WHOLE INFLUENZA VIRIONS WITH PLANAR BILAYER LIPID LIPID-ANCHORED ECTODOMAIN OF HEMAGGLUTININ (GPI-HA) CAN INDUCE MEMBRANE. FUSION PORES. Ekaterlna Yurievna Simonenko, A. A. Butylin, Lomonosov Moscow State University, R. M. Markosyan, F. S. Cohen, G. B. Melikyan, Physiology, Rush Medical College, Chicago IL. Vorobievy Gory, Moscow, 1 19899 Russian Federation It is known that GPI-HA can induce hemifusion without formation of fusion pores. We now Influenza virus penetration into the host-cell is not well understood. In model systems (bilayer report that under more optimal conditions than used previously, GPI-HA can also induce bonafide lipid membranes, virus BLM) only purified components are usually used (hemagglutinin, HA, fusion pores. GPI-HA (or wt HA) cells were bound to red blood cells (RBCs) and fusion was Ml, M2-proteins). We have studied the fusion of the whole virions with BLM. Whole influenza monitored in the whole-cell patch clamp mode using admittance measurements. Lowering pH to virion particles (A/Aichi; 0,08pg/ml) were added to asolectin ganglioside-bearing BLM, formed 4.8at 30"C yielded pores between GPI-HA cells and unlabeled RBCs in 1/3 of the experiments. by method of Mueller in buffer solution (Hepes, 0,1 M KCI). Only when pH was changed GPI-HA induced pore formation was steeply dependent on pH and temperature, as expected of an 7,0 to 5,0 (HCI) channel look like increases of conductance were observed in the range 40 pS HA-mediated process. Appreciably more fusion occurred for RBCs labeled with amounts of pS(+60 mV). The channel's life time varied from 130 ms(single) to 5 s(in group). At high fluorescent lipid dyes (RI8, DiI, or rho-PE) routinely used to monitor membrane continuity by dye virus concentration (up to 8 pg/ml) conductance increased to more than 1500 pS and was spread. Pores always formed prior to lipid dye spread (determined by fluorescence microscopy) dramatically reduced to the noise level in the presence of 70xI04M of amantadine. Influenza M2 even though the dyes passed freely from RBCs to GPI-HA cells. The lipid dyes moved less well protein is known to form ion channels into BLM. The being incorporated parameters of channels through pores of the same size formed by wt HA. Pores formed by GPI-HA did not enlarge as are the same as has. It seems we have M2-protein managed to model in vitro the well as those induced by wt HA - only.a small percentage of the GPI-HA pores permitted small and fusion of whole influenza virion with cell surface. aqueous dyes to transfer. This study directly demonstrates that the transmembrane (TM) domain of HA, absent in GPI-HA, is not essential for fusion pore formation. But the presence of the TM domain facilitates pore formation and is vital for pore enlargement. The existence of GPI-HA pores strongly suggests that lipids of formerly separate membranes have merged by the time the initial pore forms.

57A SUNDAY 334-339 MEMBRANEMEMBRANE FUSIONFUSION SUNDAY~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

334 - Pos 335- Pos A SINGLE POINT MUTATION IN THE TRANSMEMBRANE (TM) DOMAIN OF EFFECTS OF SIALYTION OF CELLS EXPRESSING INFLUENZA VIRUS INFLUENZA HEMAGGLUTININ (HA) ARRESTS FUSION AT ROOM TEMPERATURE HEMAGGLUTININ (HA) AND LIPID COMPOSITION OF TARGET MEMBRANES ON AT A STAGE PRIOR TO PORE FORMATION. MEMBRANE FUSION. G. B. Melikyan', R. M. Markosyan', M. G. Roth2, F. S. Cohen', 'Rush Medical College, Chicago, A. V. Samsonovl, V. I. Razinkov2, V. A. Frolov', F. S. Cohen2, 'Frumkin Inst. of IL, 2University of Texas at Dallas. Electrochemistry, RAS, Moscow, Russia, 2Physiology, Rush Medical College, Chicago, IL. Mutation of the semiconserved Gly 520 to Leu (G520L) of the TM domain of Japan/305/57 HA Cells constitutively expressing HA (HAb2 cells) were bound to voltage-clamped planar lipid conferred a temperature-sensitive phenotype of fusion. For as long as one hour after lowering pH bilayer membranes and fusion triggered by lowering pH. Pore formation was measured by followed by reneutralization at room temperature, neither lipid mixing, nor fusion pore formation electrical admittance and lipid continuity was determined from spread of the lipophilic dye rho-PE were detected between red blood cells (RBCs) and G520L-expressing cells. A subsequent from the planar to cell membrane. For cells treated with neuraminidase (as is conventional), increase in temperature to 37C at neutral pH resulted in fusion. We are characterizing the stage at flickering pores followed by full pore enlargement were observed after lowering of pH for which this room temperature-arrested intermediate (TAI) is blocked. TAI is at or downstream of diphytanoylPC (DPhPC) + 5mol% Gdl planar membranes. In contrast, for cells not treated with hemifusion because adding lyso-lipids (known to block hemifusion, but not the subsequent stages neuraminidase, gradual, irreversible, and equal increases in the in-phase (Y.) and DC (Yoc) of fusion) to the TAI did not inhibit fusion when temperature was elevated. In fact, TAI could be admittance components appeared, accompanied by lipid dye spread. This shows that hemifusion a state of local hemifusion: adding chlorpromazine (a membrane-permeable drug that induces occurred, that the hemifusion diaphragn was tight, but that the planar membrane became leaky. fusion ofhemifused cells) at room temperature to the TAI of RBC-G520L cell complexes resulted But Y. eventually became greater than YDc, indicating that the hemifusion diaphragm developed in efficient fusion. Separate and independent experiments suggest that the ectodomain alone leaks. In contrast, including 30 mol% DPhPE in the planar membrane led to pores that flickered induces local hemifusion, whereas the TM domain helps destabilize the hemifusion diaphragm to before a pore fully enlarged even if the cells were not treated with neuraminadase. It is well induce pore formation. The G520L mutation could thus affect the structure of the TM domain known that PE supports fusion better than PC and that fusion is prevented before hemifusion, as and/or ectodomain or their ability to undergo critical structural changes, downstream of local fusion conditions are made less optimal. Our data shows that the presence of sialate on the cell hemifusion, that are required for fusion. surfaces hinders fusion and provides evidence that sialate on HA itselfreduces the ability of HA to induce fusion. Supported by NIH FIRCA R03 TW0715.

336- Pos 337- Pos MUTANTS WITHIN THE FUSION PEPTIDE AND TRANSMEMBRANE (TM) DOMAIN INFLUENZA HEMAGGLUTININ: MEMBRANE-BOUND STRUCTURE OF LINKER OF INFLUENZA VIRUS HEMAGGLUTININ (HA) DO NOT COMPLEMENT EACH REGION STUDIED BY ELECTRON PARAMAGNETIC RESONANCE OTHER. Daniks Lyane LeDuc, Yeon-Kyun Shin, University of Califomia, Berkeley, c/o Shin Lab, Emma Borrego-Diaz, M. E. Peeples, G. B. Melikyan, F. S. Cohen, Depts of Physiology and Berkeley, Califomia 94720 Immunology/ Microbiology, Rush Medical College, Chicago, IL. Influenza hemagglutinin (HA) is an amphipathic membrane protein responsible for the WT HA induces fusion at room temperature (RT) and 37°C; the mutation Gly520Leu within the binding (subunit HAI) and fusion of the virus to the host cell (HA2). The fusion mechanism has TM domain of HA inhibits lipid and aqueous dye mixing at RT, but fusion results when not been fully determined. The dominant paradigm of HA-mediated fusion is the "spring-loaded" temperature is subsequently raised to 37C (Melikyan et al., this meeting); the mutation ofthe first model in which a dramatic pH-induced conformational change provides part, if not all, of the residue ofthe fusion peptide from Gly to Val (GlylVal) inhibits fusion at both temperatures (Qiao energy to overcome the thermodynamic barriers to membrane fusion. We have recently et al., 1999) without preventing cleavage. For mutants of HA coexpressed from the same strain, demonstrated that a shortened construct of HA2, FHA2-127 (a.a.1-127), induces lipid mixing in trimers assemble with individual monomers selected atrandom (Bouley et al., 1988). Mutant HA lipid vesicles. According to the "spring-loaded" model, this protein should be fusion-inactive, and wt HA of the Japan/305/57 strain were cotransfected (two forms at a time) into HEK293T because it does not undergo an energy-producing conformational change. In an effort to develop a cells and COS-7 cells. RBC ghosts were colabeled with a membrane (R18) and an aqueous (CF) new mechanistic model, we are determining the structure of the linker region (a.a. 20-40) in the fluorescent dye and bound to the transfected cells. Fusion was monitored after a 2-min low pH context ofthe lipid membrane, as it may be critically involved in the fusion process. Spin-labeling pulse followed by neutralization, all done at RT. Coexpressing equal amounts of wt HA and electron paragmagnetic resonance results from the study of FHA2-127 reconstituted in synthetic GlylVal HA yielded only somewhat less fusion than expressing wt HA alone. In contrast, lipid vesicles will be used to determine the structure ofthis region by analyzing the pattemofspin coexpressing Gly520Leu and GlylVal yielded limited lipid and aqueous dye transfer even after label mobility at sequential positions. raising the temperature to 37°C. These preliminary data indicate that each monomer within a trimer need not be fully functional for fusion to occur, but two different monomers, each nonfunctional, within a trimer do not complement each other to cause fusion.

- 338 Pos 339 - Pos FHA2: A MINIMAL HEMIFUSION MACHINERY IN HEMAGGLUTININ-MEDIATED MOLECULAR DYNAMICS OF THE INFLUENZA HEMAGGLUTININ FUSION? Guoliang Qian, Themis Lazaridis, City College of CUNY Danika L. Eugenia Leikina', LeDuc2, Jed C. Macosko2, Raquel F. Epand3, Richard M. Epand3, Molecular dynamics simulations of the influenza hemagglutinin were performed using the Yeon-Kyun Shin2, Leonid V. Chernomordik', 'LCMB, NICHD, 2UC,Berkeley, Berkeley, EEFI Ontario energy function (CHARMM19 with an implicit solvation potential). One simulation was carried 3McMaster Univ., Hamilton, out at 300 K to evaluate the stability of the high-pH form of the trimer in solution. The energetics Fusion mediated by the low pH confonnation of the influenza hemagglutinin (HA) is a prototype fusion of assembly of the trimer were subsequently studied and the contributions of the various subunit reaction. Initially the HA2 subunit is locked in ametastable state by interaction with HAl, another subunit interactions to the binding energy were determined. Targeted Molecular Dynamics simulations of HA. Low pH unlocks HA2, allows it to reach its low energy conformation, and thus, triggers fusion. were performed to study the mechanism and pathway of the conformational change from the high- HA-mediated fusion apparently involves an earlier hemifusion intermediate: merger of only the outer pH (inactive) state to the fusogenic state and to provide a model for the whole molecule membrsne monolayers, with the aqueous contents remaining distinct. To the functional units in (HAI+HA2) in the state. Studies of the this identify fusogenic energetics of burial of the fusion peptide into hemifusion/fusion machine, we compared the fusion activity of a construct comprising the 127 amino the trimer core and how it is influenced by pH are in progress. These studies will hopefully shed acid ectodomain fragment of HA2, FHA2, with thatof the whole HA. The conformation of FHA2 at both light on the mechanism of hemagglutinin-mediated membrane fusion. neutral and low pH is similar to the lowest energy conformation achieved after treating HA with low pH. To test fusion activity of FHA2 on cells bound by HAI-receptor connections, characteristic of HA- mediated fusion, labeled erythrocytes wereadded to cells expressing HA in the fusion-incompetent HAO form. For this system and for different HA-free cells we observed lipid mixing between bound cells only upon FHA2 application followed by a low pH. Both the pH- and the membrane concentration of FHA2 required for lipid mixing (-1000 trimers of FHA2/jsm2, estimated by Westerm blotting) were close to those required in HA-mediated fusion. As for HA, FHA2-induced lipid mixing was reversibly blocked by LPC, by low temperature (4tC), and inhibited by thermolysin. In contrast to HA-mediated fusion, FHA2- mediated lipid mixing was accompanied by neither measurable content nor formation. Mutated mixing syncytia forms of FHA2 with single amino acid substitution in the fusion peptide or in the kink region and the truncated formlacking the entire kink region of FHA2 had almost no fusion activity. These results, along with earlier reports on FHA2-mediated aggregation and fusion of liposomes, are consistent with the hypothesis that FHA2 serves as a minimal functional unit responsible for HA-mediated hemifusion. If the so, mechanism of this reaction for FHA2, presumably corresponding to the "discharged"lowest energy conformation ofHA2 and in addition lacking the transmembrane domain, is very intriguing.

58A SUNDAY MEMBRANE FUSION 340-345

340- Pos 341 Pos COOPERATIVE ACTIVATION OF INFLUENZA VIRUS HEMAGGLUTININ DURING A HOST-GUEST FUSION PEPTIDE SYSTEM TO STUDY THE THERMODYNAMICS VIRAL ENTRY OF PEPTIDE INSERTION INTO LIPID BILAYERS Ingrid Markovic, Eugenia Leikina, Leonid V. Chernomordik, LCMB, NICHD, Bldg. 10, Rm. Xing Han, Lukas K Tamm, Department of Mol. Physiol. & Biol. Phys. University of Virginia 10D03, Bethesda, MD 20892 Health Sciences Center, 1300 Jefferson Park Avenue, Charlottesville, VA 22908 Influenza virus entry into the target cell requires major structural rearrangement of the fusion Previous studies of the thermodynamics and structure of viral fusion peptides were limited protein, hemagglutinin (HA), which drives fusion of the viral envelope with the endosomal because of the extremely low solubility of these peptides in aqueous buffers. To overcome this membrane. Such major structural refolding is initiated by low pH-triggered release of the fusion problem, we have designed a host-guest peptide system in which guest fusion peptides of various peptide from hydrophobic groove and proceeds to a fusion-competent conformation through a lengths and sequences are coupled to a highly water-soluble host peptide via a flexible linker number of poorly understood intermediate structures. While the actual fusion event apparently (sequence: GCGKKKK). In a first application of this new concept, we have used as guests N- involvesconcerted action of multiple HA trimers, early activation has been thought to develop at terminal peptides of increasing lengths (n=8, 13, 16, 20) of the transmembrane subunit of the level of individual trimers. In the present work we studied the effects of the surface density of influenza hemagglutinin (HA2). The secondary structures of these peptides were measured in HA molecules on the percentage of low pH activated HA. Conformational change in HA from solution and after binding to lipid bilayers composed of POPC and POPG (4:1). In solution, they initial to an activated form, was assayed by susceptibility to protease (e.g., thermolysin) digestion were mostly random coil at low salt, but aggregated into more ordered structures at higher salt and disulfide reduction. The rate of HA activation was also tested by functional assays using concentrations. When bound to lipid bilayers, the shorter peptides still did not exhibit regular fluorescence microscopy. HA-expressing cells were treated with a first low pH pulse in the secondary structures, but the longer peptides became increasingly more helical as their absence of the target membrane. Such pretreatment resulted in activation and then inactivation of hydrophobic lengths were increased. Detailed binding studies allowed us to measure incremental HA molecules. The extent of this activation/inactivation under different conditions was evaluated free energies of insertion as the peptides were "grown" into the lipid bilayers. by subsequent addition of human erythrocytes followed by the second low pH pulse. Generally, the greater HA activation during the first pulse, the fewer HA was available for fusion and thus the lower the extent of fusion. Using biochemical and functional approach we found that HA activation of two different strains of influenza virus, i.e. Japan and X:31, considerably increases with the increase in HA surface density. Such finding supports the notion that HA activation is a cooperative event. This cooperativity can reflect either direct trimer-trimer interaction (e.g., via fusion peptides of adjacent trimers) or trimer interactions with the membrane they are anchored to.

342 - Pos 343 Pos THE EFFECTS OF LIPOSOME COMPOSITION ON CELL-LIPOSOME FUSION AND DP178 INHIBITS HIV-1 ENTRY BY BLOCKING THE FOLDING OF GP41 INTO ITS PROTEIN INACTIVATION IN MEMBRANE FUSION MEDIATED BY INFLUENZA FUSOGENIC CONFORMATION HEMAGGLUTININ YossefKliger, Yechiel Shai, The Weizmann Institute of Science, Biological Chemistry, Rehovot, Michael Zhukovsky, Austin Bailey, Eugenia Leikina, Leonid Chemomordik, LCMB, NICHD 76100 Israel Low pH triggers major conformational changes in influenza virus hemagglutinin (HA). Depending HIV entry into cells is modulated by its transmembrane envelope glycoprotein (gp4I). The core of on conditions, this refolding either enables HA to mediate membrane fusion or yields an inactivated the activated conformation of gp4l consists of a heterotrimeric coiled-coil comprising a protein conformation. Here we studied how these processes depend on the lipid composition of the leucine/isoleucine zipper sequence (represented here by the synthetic peptide N36 or by the longer target membrane. CVI fibroblasts, expressing HA, were incubated with extruded liposomes at 4°C. N51 peptide) and a C-terminal highly conserved region (represented here by C34). A correlation After removal of unbound liposomes, cells with bound liposomes were exposed to low pH medium. was found between the action of DP178, which is a potent inhibitor of HIV entry into its host cell, Then, we simultaneously raised the pH to neutral and the temperature to 37°C and followed lipid and its ability to interact with the leucine/isoleucine zipper sequence. This correlation was fusrther mixing as the dequenching of a fluorescent lipid probe upon its dilution. We estimated that most of tested and confimed by CD spectroscopy-We found that whereas DP178 perturbs the partial a- the cell surface was covered by liposomes and up to 50% of these liposomes fused with cells. helix nature of peptides corresponding to the leucine/isoleucine zipper sequence (N36 or N51), it Addition of non-bilayer lipids (i.e., lysophosphatidylcholine (LPC) and cardiolipin in presence of Ca cannot perturb the coiled-coil conformation, modeled by the complex of N36 or N51 with C34. ions) to the different monolayers of the liposomal membrane changed lipid mixing rates and extents Therefore, we suggest that the already formed heterotrimeric coiled-coil is not the target of in ways consistent with the hypothesis that membrane fusion involves subsequent bending of outer inhibition by DP178. Our results are consistent with a model in which DP178 acquires its and inner monolayers of the membrane in opposite directions. Liposome composition was also inhibitory activity by binding to an earlier intermediate of gp4l, in which the N and C peptide found to affect the rate of fusion inactivation. For liposomes of the mixture of distearoyl regions are not yet associated, thus allowing DP178 to bind to the leucine/isoleucine zipper phosphatydilcholine (DSPC) and cholesterol with or without gangliosides, serving as HA receptors, sequence and consequently to inhibit the transition to the fusion-active conformation. the extent of liposome-cell fusion decreased with increasing duration of low pH treatment at 40C. This fusion inactivation with time at low pH reached a plateau at times longer than 15 min. In contrast, such inactivation was very small or was not observed, if the liposome composition was supplemented with 5 mol% cardiolipin or for liposomes of dioleoyl phosphatydilcholine (DOPC)- cholesterol mixture, correspondingly. In experiments with human erythrocytes as the target membrane as well as for DSPC-cholesterol liposomes exposed to low pH at 37°C, the extent of fusion increased when the duration of low pH application changed from 15 sec to 10 min. This dependence of the fusion inactivation on the target membrane composition can be interpreted by the effects of this membrane either on HA refolding into an inactivated state at low pH and at 4°C or on fusion upon raising the temperature.

344 - Pos 345 Pos ARE "RAFTS" INVOLVED IN HIV-1 ENTRY? THE POLAR REGION CONSECUTIVE TO THE HIV -1 FUSION PEPTIDE Mathlas Viard, Sherimay Ablan, Han-Ming Joseph Lin, Peter Hug, Anu Puri, Robert PARTICIPATES IN MEMBRANE FUSION Blumenthal, NCI/NIH Sergio Gerardo Peisajovich', Raquel F. Epand2, Moshe Pritsker', Yechiel Shai', Richard M. HIV-1 gains entry into susceptible cells by fusion of the viral membrane with the cell plasma Epand2, 'Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, membrane. This process is mediated by the interaction of the HIV-1 envelope glycoprotein with Israel, 2McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5 Canada CD4 on the host cell surface and requires additional co-receptors, such as CXCR4 or CCR5, that The fusion peptide of HIV-I gp41 is formed by the 16 N-terminal residues of the protein. The 16 determine the tropism of different HIV-1 isolates. After binding to the cell surface, several viral amino acid peptide, as several viral fission peptides, caused a reduction in the bilayer to hexagonal envelope glycoprotein oligomers assemble into a viral fusion machine, forming a molecular phase transition temperature (TH), indicating its ability to promote negative curvature in scaffold that the viral and cell membranes into close apposition and precipitates the brings target membranes. Surprisingly, an elongated peptide corresponding to the N-terminal 33 amino acids actual fusion event. We show that inhibitors ofglycosphingolipid biosynthesis affect HIV-I fusion raised TH, although it was more potent than the 16 amino acid fusion peptide in inducing lipid and infection, and that fusion can be recovered following addition of certain purified activity mixing with large unilamellar liposomes. The 17 acid glycosphingolipids to the impaired cells. Since glycosphingolipid and cholesterol-rich domains amino C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anchored to a membrane by palmitoylation of the exist as phase-separated "rafts" in the membrane, which may serve as sites for HIV-1 entry, we amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the examined what effect cholesterol depletion of appropriate target cells might have on HIV-I fusion potency of the FTIR us activity. of human osteosarcoma cells, expressing fusogenic peptide. spectroscopy lead to postulate a structural model in Although methyl-beta-cyclodextrin treatment which the CD4 and CXCR4, did not alter cell-surface expression of these receptors, it did significantly amino-terminus ofthe fusion peptide is inserted into the membrane, as a P-sheet and the 17 reduce the susceptibility of these cells to HIV-1 envelope glycoprotein-mediated fusion. These C-terminal asnino acids lie on the surface, adopting an a-helical conformation. Thus, the 16 amino acid HIV as data indicate that glycosphingolipid-enriched domains may be involved in the assembly and peptide, other viral fusion peptides, lowers the bilayer to hexagonal phase function of the HIV- I fusion machine. transition temperature. Other segments of the viral fusion protein also facilitate membrane fusion by mechanisms unrelated to changes in intrinsic membrane curvature.

59A 346-351 MEMBRANE FUSION SUNDAY

346 - Pos 347 - Pos OPPOSITE PROPERTIES OF TWO PEPTIDE HOMOLOGS OF THE EFFECT OF SURFACE DENSITY OF VIRAL RECEPTOR MOLECULES ON VIRAL ANTIMICROBIAL PEPTIDE TRICHOGIN: LYTIC AND FUSOGENIC ACTIVITIES FUSION AND INTERMEMBRANE DISTANCE BETWEEN VIRAL ENVELOPE AND Raquel F. Epand', Richard M. Epand', Fernando Formaggio2, Marco Crisma2, Claudio Toniolo2, TARGET MEMBRANES 'McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5 Canada, 2University Shinpel OhkIl, Thomas D. Flanagan', Richard M. Epand2, 'State University of New York at of Padova, Padova, Italy Buffalo, 224 Cary Hall, Buffalo, New York 14214, McMaster University, Hamilton, Ontario, Two analogs of the antimicrobial decapeptide trichogin were synthesized. In one of these analogs Canada each ofthe four Gly resides was substituted for Ser to give the peptide: Fusion of Sendai virus with target membranes bearing the Sendai receptor, glycophorin, was n-Octanoyl-Aib-Ser-Leu-Aib-Ser-Ser-Leu-Aib-Ser-Ile-Leu-OMe (Ser-Trichogin). studied with respect to the surface density of the receptor molecule. The distance between the This anolog has an increased hydrophobic moment compared with the native peptide. A synthetic viral envelope and target membranes was measured with the same membrane system by use of precursor of Ser-Trichogin has the hydroxyl function of Ser blocked with the tert-butyl group: fluorescence resonance energy transfer. An optimum surface density of the receptor molecule on Ser(tBu)-Trichogin. This peptide has only hydrophobic amino acids with the bulkier amino acids target lipid membranes was found at which the maximum extent of viral fusion and the shortest being on one face of the helix, suggesting that it may enter a membrsne at an oblique angle. intermembrane distance were obtained. The intermembrane distance correlated well with the Ser(tBu)-Trichogin lowers the bilayer to hexagonal phase transition temperature (TH) of extent of viral fusion. Incorporation of lysolipid into the target membranes inhibited the viral dipalmitoleoyl phosphatidylethanolamine (DiPoPE), indicating that this peptide promotes negative fusion but did not appreciably affect the intermembrane distance from that for the control case curvature. In contrast, Ser-Trichogin raises TH of DiPoPE indicating that this peptide promotes (without lysolipid incorporation). With the trypsin treated HN-virus, on the other hand, no positive curvature and therefore likely lies on the membrane surface. The action of these peptides appreciable fusion of the virus with the target membranes occurred and the intermembrane on vesicle leakage and on lipid mixing between vesicles using large unilamellar vesicles with lipid distance was greater than that obtained for the intact virus over various surface densities of the compositions having varying ratios of dioleoyl phosphatidylcholine (DOPC) and dioleoyl receptor molecule. These results suggest that the accessibility of the ectodomain of the receptor phosphatidylethanolamine (DOPE) from pure DOPC to DOPC:DOPE 4:6 were measured. None molecules is important to induce viral fusion and this factor along with membrane binding by the of these vesicles produced leakage with Ser(tBu)-Trichogin but Ser-Trichogin induced rapid F-protein determines both the juxtaposition of the viral membrane and the rate of subsequent leakage of aqueous contents with the amount of leakage increasing with vesicles having a higher membrane fusion. mol fraction of DOPC. Ser(tBu)-Trichogin promoted significant lipid mixing, but only with vesicles rich in DOPE; there was also a measurable amount of aqueous contents mixing in these systems. In contrast, the lipid mixing promoted by Ser-Trichogin was smaller and qualitatively different. Thus, these two homologous peptides destabilize bilayers in ways which are mechanistically very different.

348 - Pos 349 - Pos NEUTRON DIFFRACTION STUDIES OF VIRAL FUSION PEPTIDES PROBING THE MECHANISM OF FUSION IN TWO-DIMENSIONAL COMPUTER Jerem!y P. Bradshaw', Malcolm J.M. Darkes', Thad A. Harroun', John KatsaraS2, Richard M. SIMULATION. Epand, 'University of Edinburgh, R(D)SVS, Summerhall, Edinburgh, EH9 IQH, 2Steacie Alexandr Chanturlya, Puthupparampil Scaria, Martin Woodle, Genetic Therapy Inc., 19 Institute for Materials Science, 3McMaster University Firstfield Rd, Gaithersburg, Maryland 20878 Membrane fusion plays a vital role in a large and diverse number ofessential biological processes. Two-dimensional analogs of liposomes and planar membranes were shown to undergo hemifusion Despite this fact, the precise molecular events that occur during fusion are still not known. We are and complete fusion in response to the increase of lateral tension in outer monolayer of both currently engaged on a study of membrane fusion as mediated by viral fusion peptides. These structures. This simulation study was extended further to investigate possibility of protein peptides are the N-terminal regions of certain viral envelope proteins that mediate the process of participation in the tension-driven mechanism of fusion. First, we tested if fusion can be observed fusion between the viral envelope and the membranes of the host cell during the infection process. in the model with lateral tension in only one oftwo contacting bilayers. It was found that breaking As part of this study, we have carried out neutron diffraction measurements at the ILL, BeNSC of the bilayer and formation of hemifusion-like structure is possible under this condition but and Chalk River, on a range of viral fusion peptides. Here we report on lamellar diffraction occurred only when attraction forces in the area of contact were reduced in both monolayers. measurements of simian immunodeficiency virus fusion peptide incorporated into stacked Effect of lysolipids was modeled as an insertion of a number ofextra molecules into contacting or phospholipid bilayers. Using peptides specifically deutersted at one of three different locations, distal monolayers of two interacting bilayers at different stages of simulation. It was found that we have been able to test models of the peptide in terms of peptide conformation, location and insertion into contacting monolayer inhibit fusion while insertion into distal monolayer promote orientation relative to the bilayer. fusion. In the next series of simulations one of two interacting structures contained a number of "protein molecules". Condensation of these molecules was sufficient to break monolayers in the area of contact. Our results support the idea that fusion can be regulated by lateral tension in membranes and this may be the universal mechanism for both purely lipidic and biological fusion.

- 350 Pos 351 - Pos MOLECULAR DYNAMICS OF HYDROPHOBIC DEFECTS IN MEMBRANE FUSION TILTED PEPTIDES: STRUCTURAL MOTIVES INVOLVED IN PROTEIN FUNCTION Joe Bentz', D. Peter Tieleman2, 'Drexel University, 32nd and Chestnut Sts., Philadelphia, PA L. Lins', A. Thomas', R. Brasseur2, 'INSERM UIO, Paris, France, 2CBMN, Gembloux, Belgium 19104, 2Univ. of Groningen, The Netherlands A molecular model proposed for the low-pH induced membrane fusion by influenza Tilted peptides are short sequence fragments (10-20 residues long) which possess an asymmetric hemagglutinin (HA) suggests that the initial defect is formed because the conformational change hydrophobicity gradient along their sequence when they are helical (1). Due to this asymmetric to the extended coiled coil of2 or 3 HAs in the center of a close packed aggregate of HAs extracts gradient, they adopt a tilted orientation towards any interface separating a hydrophobic phase from the fusion peptide from the viral envelope and creates a hydrophobic defect in the outer monolayer a hydrophilic one. This oblicity allows them to destabilize organized molecular systems presenting of the virion of the order of 10-20 nr2 (Bentz, Biophys. J., 2000, in press). This defect is such an interface, shifting them from one structural state to another. The asymmetric proposed to be stabilized by the closely packed HAs, which are inhibited from diffusing away hydrophobicity gradient appears to be crucial for the destabilizing activity of tilted peptides, since from the site to admit lateral lipid flow, since that would initially increase the surface area of mutants loosing this gradient are experimentally non-destabilizing. Tilted peptides were detected hydrophobic exposure. The other obvious pathway to heal this hydrophobic defect is recruitment by molecular modelling and, when possible, experimentally evidenced in many proteins, notably of lipids from the outer monolayer of the apposed target membrane, i.e. fu.sion. A first step to in viral fusion proteins (e.g. SIV gp32) (2), in signal peptides (3) and in membrane proteins (4). understanding the behavior of such a hydrophobic defect is a molecular dynamics simulation of a They were also found in lipases and in neurotoxic proteins (for instance in A( peptide and PrP patch of phospholipids with a fixed perimeter of boundary lipids, wherein several phospholipids protein) (5,6). have been extracted to mimic the effect of removal of the fusion peptides from a site of restricted We propose that tilted peptides represent a widespread structural motif involved in dynamical lipid flow. The simulations focus on how the remaining lipids reorient to minimize the free functional processes ofvarious proteins. energy of this hydrophobic defect in the bilayer. (I)Brasseur, R, Pillot, T, Lins, L, Vandekerckhove, J, Rosseneu, M, 1997, TIBS,22,128-131 (2) Voneche, V, Portetelle, D, Kettmann, R, Willems, L, Limbach, K, Paoletti, E, Ruysschaert, JM, Bumy, A, Brasseur, R, 1992, Proc. Natl. Acad. Sci. USA, 89,3810-3814 (3)Talmud, P, Lins, L, Brasseur, 1996, R Prot. Eng., 9, 317-321 (4) Rahman, M, Lins, L, Thomas-Soumarmon, A, Brasseur, R, 1997, J. Mol. Model., 3, 203-215 (5)Pillot, T, Goethals, M, Vanloo, B, Talussot, C, Brasseur, R, Vandekerckhove, J, Rosseneu, M, Lins, L, 1996, J. Biol. Chem., 271, 28757-28765 167-171 (6)Pillot, T, Lins, L, Goethals, M, Vanloo, B, Baert, J, Vandekerckhove, J, Rosseneu, M, Brasseur, R, 1997a, J. Mol. Biol., 274, 381-393

60A SUNDAY MEMBRANE FUSION 352-353

352 - Pos 353 - Pos DIFFERENT MODES OF ANNEXIN IV BINDING TO PHOSPHOLIPID VESICLES IN BILAYER/NONBILAYER PHASE TRANSITIONS OF N-MONO METHYLATED THE ABSENCE AND PRESENCE OF CALCIUM DIOLEOYLPHOSPHATIDYLETHANOLAMINE. Olaf Zschlrnig, Frank Opitz, Guido Kohler, Klaus Arnold, University of Leipzig, Liebigstr.27, Vadim G Cherezov', David Paul Siegel', W. Shaw2, S. L. Burgess2, M. Caffrey', 'The Ohio State Leipzig, D-04103 Germany University, 100 W. 18th Ave, Columbus, OH 43210, 2Avanti Polar Lipids We studied the temperature and kinetics of nonbilayer phase formation in N-monomethylated Annexin IV belongs to a class of Ca2'-binding proteins for which an involvement in exocytosis dioleoylphosphatidylethanolamine (DOPE-Me), using DSC and time-resolved x-ray diffraction. and in the coagulation processes are discussed. All these functions are related to the ability of DOPE-Me is often used to study the effects of peptides, e.g., viral fusion peptides, on inverted annexins to bind to acidic phospholipids. Annexin IV strongly binds to phosphatidylserine (PS) phase formation and membrane fusion. Using slow temperature-scan and temperature-jump and phosphatidic acid (PA) large unilamellar vesicles (LUV) at acidic pH, at neutral pH only weak experiments, we found that the bilayer/nonbilayer transitions between 59° and 67° C are complex. binding to PA and no binding to PS occurs. Addition of 40 AM Ca?' leads to a strong binding to In general, inverted cubic (Ql,) phase forms at lower temperatures than inverted hexagonal (H,,) the lipids also at neutral pH. Binding ofannexin IV induces dehydration ofthe vesicle surface and phase, and forms very slowly (hours). Formation of Q,, phase precursors lengthens the time a decrease ofthe lateral diffusion within the bilayer. Both effects are much greater at pH 4 than at required for Hi, phase to nucleate from the lamellar phase (minutes to hours). Once nucleated, H, pH 7.4. Annexin IV promotes the Ca2+-induced fusion of PA LUV by diminishing the Ca2' phase forms rapidly, although it is not the equilibrium phase, and it eventually disappeara in favor threshold concentration at pH 7.4, whereas fusion of PS LUV is not affected. At pH 4 annexin IV of Ql, phase. The first Q', lattices to form have large lattice constants (> 30 nm), but these shrink induces fusion of either vesicles without Ca2+.It is concluded, that 1. annexin IV penetrates into with increasing time at constant temperature. Under some circumstances, the first Q,l lattice the bilayer at pH 4 contrary to the known location at the vesicle surface at neutral pH., 2 observed has Im3m symmetry, and later coexists with another Q,, lattice (Pn3m). These annexin IV seems to bind two PA vesicles simultaneously however only one PS vesicle at the observations agree with predictions of a recent theory of the phase transition mechanism. same time at pH 7.4. Supported by DFG SFB 197/ A 10. Different lots of DOPE-Me from the supplier showed significant differences in phase behavior, but more highly purified preparations reproduced the behavior described earlier for DOPE-Me using similarly-purified material (Biochem. 29:5975 [1990]).

SUNDAY MEMBRANE STRUCTURE 354-357------

354 - Pos 355- Pos MEMBRANE MECHANICS AND PEARLING INSTABILITY IN TUBULAR VESICLES. ELECTROPHORETIC BEHAVIOR OF HUMAN RED BLOOD CELLS COATED WITH Sa§a Svetina, B. Bozic, B. Zek§, Institute of Biophysics, Faculty of Medicine, Ljubljana, J. Stefan POLY(ETHYLENE) GLYCOL Inst., 1000 Ljubljana, Slovenia Bjoern Neu, Jonathan K. Armstrong, Timothy C. Fisher, Herbert J. Meiselman, Univ. Southern When a long tubular fluid membrane fixed at both sides is locally exposed to the laser beam of California optical tweezers, above a certain threshold value of the laser intensity, the membrane exhibits a We have recently developed (e.g., Biorheology 36:53, 1999) a technique to covalently bind non- sinusoidal instability (Bar-Ziv and Moses, Phys.Rev.Lett. 73: 1392, 1994). This, often called ionic polymers such as PEG to the surface of human red blood cells (RBC); this process "masks" pearling instability propagates outward from the tweezing point in both directions with a constant membrane antigenic sites and inhibits RBC aggregation. The effects of such coating on the velocity and a characteristic can wavelength. The phenomenon be explained by the tendency of regions near the RBC glycocalyx were explored using a new cell electrophoresis molecules to move phospholipid into the laser trap, causing the tube to shorten. Thus the lateral system (HaSoTec). Electrophoretic mobilities were measured, in polymer-free buffer, as functions tension is induced in the membrane having its magnitude proportional to the laser intensity. The of PEG MW (3.35, 18.5, 40 kDa), structure (linear or 8-arm branched) and concentration; dynamical aspects of the pearling instability were interpreted (Goldstein et al., J.Phys.II France mobilities were measured at various Debye-Huckel lengths. Our results indicate marked decreases 6: 767, 1996) by considering it to be analogous to the Rayleigh-type instabilities. In more recent of mobility (up to 85%) which were affected by polymer MW (18.5>3.35) and structure experiments (Bar-Ziv et al., Biophys. J. 75: 294, 1998) a similar sinusoidal instability was induced (branched>linear). Since PEG is neutral and binds only to positive amino groups, such decreases also by increasing membrane tension mechanically. In view of this it can be argued that the basis of mobility likely reflect structural changes of the RBC surface penetrable layer. To analyze for the formation of peristaltic shapes may reside in the equilibrium properties of the system. The these results, we have extended "hairy sphere" models to consider friction and thickness of two idea is supported by possible solutions of the shape equation of the axisymmetrical bilayer separate layers: charged glycocalyx +PEG, uncharged PEG. Calculated length (8) and penetration membrane obtained for shapes which do not deviate significantly from a cylinder (BoziC et al., in depth values (I/a) from this model are both realistic (e.g., 40 kDa: 6 = 7.6 nm, I/a > 2.26 nm) and preparation). Here we present the conditions at which the a vesicle shape is periodic sinusoidal consistent increases These results an function. The predicted (6 with MW). provide approach to determining the amount dependence of the wavenumber on the lateral membrane tension is in and dimensions of PEG coatings and suggest the usefulness of electrophoresis for studies of accord with the related experimental data. polymer surface adsorption.

356- Pos 357 - Pos EFFECT OF DISACCHARIDES AND PHLORETIN ON THE HYDRATION AND A COMPARATIVE STUDY OF THE ACTION OF AMPHOTERICIN B CHANNELS ON DIPOLE POTENTIAL OF LIPID BILAYERS. LIPID BILAYERS FROM BACTERIA AND ERYTHROCITES Edgardo Anibal Disalvo', Flavia Amalfa', Sonia Diaz2, Maria C. Luzardo3, Ana M. Nunez', Berenice Venegas', Heliodoro Celia2, Jorge Ramirez2, Ivan Ortega-Blake', 'Centro de Ciencias Angela C Biondi de Lopez2, 'Farmacia y Bioquimica, Universidad de Buenos Aires Argentina, Fisicas-UNAM, Av. Universidad s/n, Cuernavaca, Morelos 62210 Mexico, 2Instituto de Fisiologia junin 956, 2P, Buenos Aires, CP 1 13 Argentina, 2U. N. Tucuman, 3U. La Habana, Cuba Celular-UNAM The effect ofphloretin, sucrose and trehalose on on the dipole potential and the vibration mode of Amphotericin B (AmB) is a very effective antimicotic but produces severe side effects carbonyls of in the phosphatidylcholines has been measured in monolayers and in bilayers with Fourier patient, therefore it has been studied to understand its action and to reduce its toxicity. Several Transform Infrared Spectroscopy (FTIR). Lipid on an monolayers spread air/water interface experiments have shown a direct correlation between channel formation and sterol content in the showed a decrease from 480 mV in pure water to 250 mV in 0.25M trehalose. Results obtained membrane, leading to the proposal that sterols form an integral part of the channel. Nevertheless, FTIR denoted that trehalose binds to the carbonyl groups at the sn, and the sn2 positions. Sucrose AmB channels can be formed in sterol-free DMPC bilayers and in the presence of it we have does not change the dipole seen potential and does not affect the C=O vibrations at the sn, or sn2 a cholesterol dependence in the dynamical properties but not in channel conductivity. Analysis of positions; phloretin decreases the dipole potential by 110 mV but does not affect the carbonyl these results suggest that cholesterol changes the partition coefficient, facilitating the incorporation vibration frequencies. From titrations a of DMPC solution in chloroform and measurement of ofthe AmB to the lipid phase by changing the structure ofthe bilayer. water activity it is concluded that 7 out of 13 water molecules from the hydration shell of the In this work we used the patch-clamp technique, making lipid bilayers at the tip ofa phospholipids are replaced by trehalose. Two ofthem are linked to the carbonyls and contribute to micropipiette and recording single channel currents. We compare the formation and the dynamical the dipole potential of the membrane. In contrast, sucrose displaces three water molecules and properties (frequency, probability of open channel, average of open time) of the AmB channel in bilayers phloretin none. The different action of the sugars are related to the number of OH groups in equatorial position and its properties ofhydration. made of lipid extractions of E. coli and erythrocites, as an atempt to understand the differences in toxicity in each cell at the molecular level. For each bilayer we used two temperatures and two colesterol concentrations as membrane structuring agents.

61A 358-363 MEEMBRANEMEMBRANE STRUCITURESTRUCTURE SUNDAY

358 - Pos 359- Pos SPECTROSCOPIC CHARACTERIZATION OF NANOERYTHROSOMES IN THE OPTIMIZATION OF KEY PARAMETERS FOR GENE TRANSFER: TOWARDS A ABSENCE AND PRESENCE OF POLYETHYLENEGLYCOLS: A FTIR AND 31P NMR UNIVERSAL DESCRIPTION OF TRANSFECTION EFFICIENCY STUDY Nelle L. Slack, A. Ahmad, Heather M. Evans, Alison J. Lin, Uwe Schulze, Ana H. Martin, C. X. Roxane Pouliot', Audrey Saint-Laureee, Ren6 C.-Gaudreaulte, Camille Chypre4, Michele Auger2, George, C. E. Samuel, C. R. Safinya, UC Santa Barbara, MRL, Santa Barbara, CA 93106 'Universite Laval/DiagnoCure Inc./CERSIM, Pavillon Vachon/Cite Universitaire, Quebec, 7P4 de Recherche Quebec GIK Canada, 2Universite Laval/CERSIM, 3DiagnoCure Inc./Centre de Previous work on cationic lipid:DNA (CL:DNA) non-viral carriers for gene therapy has shown l'Hopital St-Francois d'Assise, 4DiagnoCure Inc. that in systems containing univalent cationic lipids mixed with neutral lipids the overall structure of the carrier and the membrane charge density may be important variables for designing an We have recently developed from red blood cells a new delivery system so called efficient carrier'. Using x-ray diffraction and biological assays, we show key parameters for nanoerythrosomes. These particles offer a high degree of versatility for the encapsulation of optimizing gene transfer that are mediated by properties of the lipid. Results from multivalent CL biological or non-biological compounds and for the binding oftargeting agents. Their membranes complexes show that transfection efficiency dependence on membrane charge density and are composed of both phospholipids, proteins and cholesterol with a respective weight ratio of geometric shape may be universal in vitro. We examine the effects of addition of PEG lipid (PL) 2/1/1, with the protein molecules significantly protruding the phospholipid bilayer. to these complexes for applications in vivo. The presence of PL introduces enhanced stability of Polyethyleneglycols can be coupled to the amino acid residues present on the different proteins as complexes, but is expected to inhibit transfection efficiency. Multivalent cationic PL were masking component to overcome immunogenicity and rapid clearance from circulation. In the synthesized and are found to enhance transfection efficiency under some conditions in vitro. present work, we have investigated the effect of temperature on nanoerythrosomes in the absence Supported by NIH ROI-GM59288-01 and R37-A12520-24, UCBiotechnology Research and and presence of polyethyleneglycols. More specifically, polyethyleneglycols having various Education Program (97-02), NSF-DMR-9972246. molecular and and different structural were weights (2000 5000) characteristics covalently linked J. Raedler, I. Koltover, T. C. R. Science 810 T. J. to and 31P NMR and FTIR were used to evaluate the Salditt, Safinya, 275, (1997), Koltover, Salditt, nanoerythrosomes spectroscopies degree of Raedler, C. R. Safinya, Science 281, 78-81 (1998), A. J. Lin, N. L. Slack, A. I aggregation, the lipid order and dynamics and the hydration ofthe nanoerythrosomes. The results Ahmad, Koltover, indicate that the nanoerythrosome lipid chain order is not significantly affected by heating the C. X. George, C. E. Samuel, C. R Safinya, Joumal ofDrug Targeting (to appear) nanoerythrosomes at temperatures up to 500C. They also indicate that the nanoerythrosomes aggregate at temperatures above 37°C, this effect being less important in the presence of polyethyleneglycols.

360- Pos 361 - Pos WHY DO ELECTROPHORETICALLY-DETERMINED HYDRODYNAMIC EXTRUSION STUDIES OF LIPID VESICLES. THICKNESSES OF NEUTRAL POLYMER COATS DEPEND ON IONIC STRENGTH? Philipus Josepus Patty, Barbara J. Frisken, Simon Fraser University, 8888 University Drive, Joel A. Cohen, Univ. ofthe Pacific, San Francisco, CA 94115 Burnaby, BC V5A IS6 Canada Liposomes decorated with surface-grafted neutral polymers, typically PEG, are pharmaceutically Lysis tension of POPC (1-palmitoyl-2-oleoyl-sn-phosphatidycholine), POPC: Cholesterol and useful due to their low immunogenicity. The hydrodynamic thickness ofthe polymer coat can be POPC: Ceramide mixtures was measured by extrusion method [1]. In this method, solutions studied by microelectrophoresis. The zeta potential gives the location ofthe shear surface relative containing large multilamellar vesicles are forced or extruded through small pores using to the membrane charge surface if the electrostatic-potential profile is known. The hydrodynamic pressurized gas. Extruding the solutions at least 10 times (10 passes) through polycarbonate filters coat thickness then is defined as the distance between the shear and membrane surfaces. Since of 100 em pore size, we measured the flow rate ofthe solutions for each pass. We find for the first PEG is strictly neutral, the coat thickness is expected to be nearly independent of ionic strength. pass, flow rates are proportional to applied pressure. There is a minimum pressure, associated with However, when the zeta potentials of PEG-grafted liposomes are measured at different ionic the lysis tension, below which no vesicles rupture. To support our picture ofthe extrusion process, strengths and the hydrodynamic thicknesses calculated, a strong ionic-strength dependence is we also have checked the effects of flow rate, pressure, and concentration on the size and found, the coat thickness decreasing as ionic strength increases. As previously indicated by Cohen polydispersity ofvesicles as measured by dynamic light seattering and field flow fractionation. Stuart et al. (Colloid & Polymer Sci 1984 262, 423), this effect is largely an artifact associated with application of the Smoluchowski equation to electrophoretic particles having partially- draining surface polymer layers. The Navier-Stokes equation including surface-polymer frictional [1] D.G. Hunter and B.J. Frisken, BiophysJ. 74, 2996 (1998). flow shows that Smoluchowski gives the correct steric coat thickness only for very low ionic strengths and/or high hydrodynamic friction in the polymer layer. As ionic strength increases and/or friction decreases, Smoluchowski yields an apparent hydrodynamic thickness that is smaller than the steric thickness and decreases toward zero. Analytic solutions and criteria for valid electrophoretic determinations ofcoat thickness are presented.

362 - Pos 363 - Pos FRUCTANS STRONGLY INTERACT WITH LIPIDS IN MODEL MEMBRANES DOXORUBICIN BINDING TO CARDIOLIPIN CONTAINING MAGNETICALLY Ingrld J. Vereyken, R. A. Demel, J. C.M. Smeekens, B. de Kruijff, Utrecht University, ORIENTED PHOSPHOLIPID BILAYERS Padualaan 8,Utrecht,3584 CH Netherlands Kathleen P. Howard, Margaret Parker, Swarthmore College, Swarthmore, PA 19081 One of the strategies plants have developed to protect themselves from the adverse effects of Doxorubicin is a potent anthracycline cancer drug whoseprimary mode of action is intercalation drought is the biosynthesis of fructans (fructose polymers). Recent studies showed that transgenic into DNA, but whose interaction with anionic membrane phospholipids, such as cardiolipin, is tobacco plants, able to synthesize microbial-typefructans, are more lrought tolerant than the wild- also thought to contribute to its cytotoxicity. We are studying the membrane interaction of type plant. Inour group the hypothesis was put forward that fructans increase drought tolerance by doxorubicin with a cardiolipin-containing model membrane system. The model membrane system protecting the membrane. Fructan-membrane interactions were analyzed using monomolecular and is composed of a bilayer forming phospholipid and a detergent that breaks the extended bilayers lipid layers bilayer systems to further extend an earlier study by Demel et al.(BBA (1998), into disc-shaped micelles (bicelles). Since these phospholipid bilayer arrays orient with respect to 1375, 36-42). Bacterial type fructans (polymerisation grade higher than 15) increased the surface the magnetic field, anisotropic NMR parameters such as chemical shift and dipolar and pressure of thelipid monolayer, thereby showing penetration into this layer. The maximal effect for in quadrupolar coupling are measured which can be translated into high resolution information on fructans this system was much larger than the effectsobtained using small carbohydrates conformation and dynamics. We are using 2H NMR of headgroup and acyl chain deuterated like sucrose and trehalose or the polysaccharide-dextran under the sameconditions. In addition, DMPC and 31P NMR of the phospholipid headgroups to observe the changes that occur as fructan binding to lipid vesicles resulted in turbidity changes due to either vesicle or fusion. aggregation doxorubicin is added to thesystem. Changes in NMR anisotropic parameters upon drug binding Furthermore, 31P-NMR demonstrated that fructans retarded the L-OH,, transition areinterpreted in terms on the topology of interaction of the drug with the bilayer. We are also indicating a stabilization of the La-phase under hydrated conditions. From this it can be concluded starting to synthetically deuterate doxorubicinso we can directly measure how the drug is oriented that fructans strongly interact with lipid membranes. with respect to the lipid bilayer.

62A SUNDAY MEMBRANE STRUCTURE 364-367 SUNDAY 364-367~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 364 - Pos 365- Pos THE INFLUENCE OF SALTS ON THE MORPHOLOGY AND ORIENTATION OF CHANGES IN CELL MECHANOCHEMICAL PROPERTIES AFFECT NEUTROPHIL BICELLES IN A MAGNETIC FIELD: A SOLID STATE NMR STUDY TETHERING Alexandre Arnold, Reiko Oda, Erick Joel Dufourc, Institut Europeen de Chimie et Biologie- Warren D. Marcus, Robert M Hochmuth, Duke University, Mechanical Engineering, Box 90300, Ecole Polytechnique, Avenue Pey Berland, Talence, BP 108 33402 France Durham, NC 27708 We have investigated the influence of salts (KCI, NaCI, CaC12 and MgCl2) on the size of The separation of the lipid membrane from the cytoskeleton is performed by forming membrane DMPC/DHPC bicelles along with the quality of their orientation by 31P and 'H nuclear magnetic tethers from human neutrophils. A study of this separation process is fundamental to our resonance. It was found that the thickness of the central bilayer, determined by 2H NMR, is understanding of the association between membrane and cytoskeleton. Specifically, analysis of approximately of 40 A and that the nature of the studied salt solutions has practically no effect on the force on a tether allows for the calculation of the adhesion energy between the lipid membrane the thickness. We have determined the bicelle diameters by 31P NMR using a model which assigns and cytoskeleton and the identification ofthe dissipation that occurs when a tether is extracted at a the two peaks ofthe NMR spectra to two geometrical environments ofthe bicelle (bilayer or rim). finite rate. In our initial experiments, the neutrophil surface area is mechanically dilated by The results show that the calculated diameters vary between 350 and 450 A when KCI or NaCI are partially aspirating the cells into a micropipette. Another suction pipette, containing a latex bead present in the lipid solution, and between 450 to 700 A when CaC12 and MgCI2 are added. We coated by antibodies to human CDI b, is used to determine the membrane tether velocities of have measured the quality of the bicelles orientation (mosaicity) from the shape of the 31P NMR neutrophils over a range ofsuction forces. The results show an increase in the initial force to pull spectra. It was observed that the mosaicity is of 6° in a salt free solution and that this value can a tether once the area has been expanded. This expanded stretching of the elastic cell membrane decrease to 1° in salt solutions. This result is indicative of the high cooperative alignment of alters the association of the membrane with the cytoskeleton. Further studies using cytochalasin- bicelles under certain conditions. In conclusion, this study showed that it is possible to control the D and osmotic shrinking/swelling of cells are also underway to determine their effects on the size of bicelles and their orientation by adjusting the salt concentration of the sample. It is forces required to form a tether. This work is supported by NIH grant HL23728. interesting to note that the studied cations are physiologically relevant. Preliminary results using electron microscopy bear out sizes found by NMR.

366- Pos 367- Pos CHLORHEXIDINE DIGLUCONATE ALTERS STRUCTURAL PARAMETERS OF PHYSICAL PROPERTIES OF MEMBRANE FRACTIONS ISOLATED FROM HUMAN OUTER MEMBRANES ISOLATED FROM CULTURED PORPHYROMONAS PLATELETS: IMPLICATION FOR CHILLING INDUCED PLATELET ACTIVATION GINGIVALIS Nelly M. Tsvetkova, John H. Crowe, Naomi J. Walker, Lois M. Crowe, Fern Tablin, University Il Yun', C.D. Kim', I.K. Chung', C.H. Choi', I.S. Kim', S.H. Kim', G.J. Cho', J.S. Kim', H.O. ofCalifornia Davis, I Shields Avenue, Davis, CA 95616 Jang2, 'Pusan National University, 1-10, Ami-Dong, Seo-Gu, Pusan, 602-739 Korea, Republic of, Human blood are 2Dong-Eui Univerisity platelets extremely sensitive to chilling. If they are cooled below 20 "C, they markedly change shape, exhibit an increase in the cytosolic calcium and actin polymerization, and Intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py-3-Py) and fluorescence increased aggregation. Such platelets, said to be 'activated', lose hemostatic function. In previous polarization of 1,6-diphenyl- 1,3,5-hexatriene (DPH) were used to evaluate effects ofchlorhexidine studies we suggested that chilling induced activation of the platelets is related to the membrane digluconate on lateral and rotational mobility of bulk bilayer structures of outer membranes lipid phase transition from liquid crystalline to gel phase. In this study we developed a novel (PGOM) isolated from cultured Porphyromonas gingivalis (PG). In a concentration-dependent method for separation of dense tubular system and plasma membranes. This method was used to manner, chlorhexidine digluconate increased the lateral and rotational mobility of the bulk demonstrate that the thermal phase transition seen in intact platelets is also seen in the two structures of PGOM. Selective quenching of Py-3-Py by trinitrophenyl groups was used to membrane fractions and it can be to the membrane The examine the assigned phospholipids. thermotropic transbilayer asymmetric effect of chlorhexidine digluconate on the rate and range of phase transitions ofthe two fractions, determined DSC and FTIR, were found to occur at about the lateral of the by mobility PGOM. The transbilayer asymmetric effect of the drug on the range of 15 °C, similarly to the T, of intact platelets. Lipid analysis showed a high abundance of the rotational mobility of the PGOM was also determined by cationic TMA-DPH and anionic phosphatidylcholine and in the two whereas cholesterol and PRO-DPH probes. phosphatidylethanolamine fractions, Chlorhexidine digluconate had a greater increasing effect on the lateral and sphingomyelin were predominantly located in the plasma membranes. These results are discussed rotational mobility of the inner monolayer as compared to the outer monolayer of the PGOM. It in relation to the role of the DTS and plasma membranes in the cold induced activation of human has been proven that the drug exhibits a selective rather than nonselective fluidizing effect within platelets. the transbilayer domains of the PGOM. Fluorescence quenching methods were used to evaluate This work was a R01HL57810-01 from the National effects of the drug on annular lipid fluidity, protein distribution and thickness of bulk bilayer supported by grant Institutes ofHealth. structures of the PGOM. Chlorhexidine digluconate alters annular lipid fluidity, protein distribution and thickness ofthe membrane lipid bilayer PGOM.

SUNDAY CELL SIGNALING 368-369YUCF-.YU7

368 - Pos 369- Pos A MALDI-TOF STUDY ON LIPID SECOND MESSENGERS IN NEUTROPHILIC PITP'S UNFILLED HEADGROUP SITE MAY PLAY A REGULATORY ROLE IN ITS GRANULOCYTES FUNCTION: EFFECTS OF GPC AND GPI. Klaus Arnold, Stefan Benard, Jftirgen Schiller, Sabine Reichl, Jtrgen Arnhold, University of Hiroaki Komatsue, Jan Westerman2, Karel W. A. Wirtz2, Theodore F. Taraschi', Nathan Janes, Leipzig, Liebigstr. 27, D-04103 Leipzig, Germany 'Thomas Jefferson University, Philadelphia, PA 19107, 2Utrecht University Phosphatidylinositol transfer protein (PITP) transfers PI to membrane sites in exchange for PC. MALDI-TOF (matrix-assisted laser desorption and ionization time-of-flight) mass spectrometry is The single lipid binding site contains a shared acyl-chain binding motif and distinct headgroup a rapidly developing method. Astonishingly, there are only few reports on lipid analysis, although binding motifs, suggesting that the unused headgroup site is either alternately open-and-empty the or sensitivity, the excellent mass accuracy and the simple experimental setup are considerable alternately closed. Glycerophosphocholine (GPC) and glycerophosphoinositol (GPI) are advantages of lipid MALDI-TOF. Additionally, lipids can be analyzed in a single solvent since the lipid metabolites that are known to vary with cell growth and with chronic ethanol exposure. We as well as the matrix are readily soluble in organic solvents. This warrants high reproducibility due investigated whether these polar metabolites might bind to the empty headgroup motif and alter to the formation of even matrix/analyte cocrystals. The activation of the respiratory-burst-oxidase, the activity ofPm and possibly provide a regulatory role in PITP function. the key enzyme for the generation ofreactive oxygen species (ROS) in neutrophils is mediated by The assay for PMa (recombinant mouse) mediated PI transfer employed large unilamellar donor lipids: Arachidonic acid, phosphatidic acids and diacylglycerols are important lipid second- vesicles prepared by extrusion (200 nm pore) and sonicated acceptor vesicles messengers that favour the ROS formation. It is paper (Donor, shown in this that MALDI-TOF enables the PI:PC:PS:PE:LacCer, 2.5/2.5/2.5/82.5/10; Acceptor, PC:PA, 98/2). At its physiological analysis of complex lipid mixtures without a previous separation of the individual lipids and concentration in rat liver 1.4 GPC inhibited PI without any (ca. mM), transfer by 50%. GPI had an opposite, derivatization. Also a quantification of lipids by using internal standards can be but effect on PI transfer. It performed, whereby the detection weaker, enhanced PI transfer by 50% at 7 mM. Thus, the unused limit is in the picomolar range. Additionally, changes in the headgroup site appears to be open and to lipid composition of neutrophils induced by accessible small polar lipid metabolites. Their levels the modulation of the cell response by specific drugs may play an important regulatory role in PITP function. (e.g. enzyme inhibitors) are also detectable by MALDI-TOF: Due to the known lipid composition ofneutrophils most peaks can be easily assigned.

63A 370-375 CELL SIGNALING SUNDAY

370 - Pos 371 - Pos INABILITY OF PITP TO TRANSFER PHOSPHATIDYLETHANOL. FUNCTIONAL CONSEQUENCES OF MEMBRANE TOLERANCE: CHRONIC Hiroaki Komatsu', Karel W. A. Wirtz2, Theodore F. Taraschit, NathanJanes', 'Thomas Jefferson ETHANOL EXPOSURE INDUCES MEMBRANE RESISTANCE TO THE ABILITY OF University, Philadelphia, PA 19107,2Utrecht University ETHANOL TO ENHANCE PHOSPHATIDYLINOSITOL TRANSFER PROTEIN'S Phosphatidylethanol (PEt) is an ethanol metabolite of phospholipase D that possesses a small ACTIVITY. nonpolar ethyl headgroup terminus that endows it with properties that are unusual for native Hiroaki Komatsun, Nhan N. Nguyen', Barend Bouma2, Karel W. A. Wirtz2, Theodore F. mammalian phospholipids. Phosphatidylinositol transfer protein (PITP) is influenced by acute Taraschil, Nathan Janes', 'Thomas Jefferson University, Philadelphia, PA 19107, 2Utrecht and chronic ethanol exposure. We tested the possibility that PITP couldtransfer PEt. University A transfer assay was developed that allowed for the observation of transfer efficacies for a number The physical status of biomembranes is critical to membrane function. Environmental changes of phospholipids under identical conditions. The assay employed donor large unilamellar vesicles often induce an adaptive remodeling to maintain homeostasis. For chronic ethanol exposure, this prepared by extrusion (200 nm pore) and acceptor sonicated vesicles. The assay used dilute levels is termed "membrane tolerance". Whereas many functional consequences of adaptation are of transferable phospholipids in a nontransferable phosphatidylethanolamine donor matrix. The known, no functional consequences of membrane tolerance have been identified. donor membranes were separated from the acceptor membranes by agglutination/centrifugation. Phosphatidylinositol transfer protein (PITP) transfers phosphatidylinositol (PI) from its sites of Donor vesicles contained PC/PV/PEt/PE/LacCer, 2.5/2.5/2.5/82.5/10 mol%. Acceptor vesicles synthesis to its sites of utilization. The assay for recombinant mouse PITP-a activity measured contained PC/PA 98/2 mol%. the transfer of PI from donor rat liver microsomes to acceptor sonicated liposomes. PITP-mediated PEt transfer was very small. It amounted to 1% of PI transfer and 3% of PC Ethanol and chloroform enhanced PI transfer. Kinetic analyses indicated that ethanol increased transfer. These are innate transfer rates obtained at equimolar membrane concentrations. In cells, the flux of PITP at membrane surfaces. PITP's activity was greatly enhanced activity at highly PI and PC are much more abundant than PEt so that the difference in the effective transfer rates is curved surfaces. Ethanol enhanced PI transfer to all curvatures, but its effect was modest by likely to be considerably larger. We conclude that while PITP may play a direct role in the acute comparison, as expected given the relative magnitudes of the membrane perturbations. and chronic effects of ethanol, it does not show appreciable activity toward the phospholipid The effects of membrane tolerance were investigated with donor microsomes from ethanol-fed metabolite of ethanol, PEt. rats. In the absence of ethanol, the rates of PI transfer from the ethanol-fed and control animals were indistinguishable. In the presence of ethanol, PI transfer from the tolerant membranes was diminished by three-fold. Ethanol feeding induced an adaptive response toward functional homeostasis. This is the first demonstration that membrane tolerance has functional consequences.

372 - Pos 373 Pos INTERACTION PARTNERS OF THE HUMAN PI4K230 SYNERGISTIC AMPLITUDE AND FREQUENCY MODULATION OF METABOLIC Huelya Guelkan, L. M. G. Heilmeyer, Ruhr University Bochum, Universitaetsstr. 150 MA 2/35, SIGNALS IN CELL ACTIVATION. Bochum, 44801 Germany Andrei L Kindzelskii', Y Adachi2, T Yadomae2, N Ohno2, H R Petty','Wayne State University, Polyphosphosphoinositides cover a wide range of functions other than just being or producing Detroit, Michigan, 2Tokyo University of Pharmacy and Life Sciences second-messengers. Phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is a critical plasma- Intracellular chemical oscillations may encode information in their amplitudes and frequencies. membrane precursor of several intracellular messengers that mediate the action of many We test the hypothesis that synergistic responses to dual cytokine exposure correlate with cross- hormones, transmitters and growth factors. It has been also implicated in other cellular functions, talk between metabolic signaling pathways. Polarized macrophages exhibited NAD(P)H including regulation of activity of certain actin-binding proteins, of clathrin assembly and of autofluorescence oscillation periods of-20 sec. IFN-y tripled the NAD(P)H amplitude. Although receptor mediated endocytosis or exocytosis. Hormone sensitive and -insensitive pools of IL-6 had no effect, treatment with IFN-y and IL-6 increased amplitude and frequency. Parallel inositolphospholipids have been described, as well as a nuclear inositolphospholipid system that changes were noted with IFN-y and IL-2. IL-11 and TNF-a provided negative controls. To appears to be regulated independently of receptor-activated inositolphospholipid tumover at the determine if frequency doubling required IFN-y signaling or just NAD(P)H amplitude modulation, plasma membrane. Phosphatidylinositol 4-kinase (P14-kinase) phosphorylates phosphatidylinositol an electric field was applied to cells at NAD(P)H troughs, which enhances NAD(P)H amplitudes. (PtdIns) at the D4 position of the inositol ring and catalyzes the formation of phosphatidylinositol Electric fields led to frequency doubling with IL-6 or IL-2 alone. Since NADPH donates electrons 4-phosphate (PtdlnsP). Several cDNAs have been cloned which code isoforms of P14-kinase and to NO, we tested NO production. Although IL-6 and IL-2 had no effect, IFN-y plus IL-6 or IL-2 which can be divided into wortmannin-sensitive and wortmannin-insensitive P14-kinases. In enhanced NO release. NO release increased incrementally with the same phase and period as human the cDNA sequences of two wortmannin-sensitive isoforms (P14K230 and PI4K92) are NAD(P)H. Amplitude and frequency modulation of NAD(P)H oscillations may contribute to known whereas a wortmannin-insensitive PI4-kinase (Pl4K55) is only enzymatically cytokine synergy and NO production. characterized. Recent studies have been implicated an involvement of P14-kinases in endocytosis, exocytosis, neurotransmitter release and retrograde axonal transport. By performing a yeast two hybrid screen two interesting interaction partners have been isolated. Whether these interacting proteins verify an involvement of the human P14K230 in the above mentioned processes have to be discussed.

374 - Pos 375- Pos SIGNAL PROCESSING TIMES IN NEUTROPHIL ACTIVATION: DEPENDENCE ON REACTIVE OXYGEN SPECIES AND NITRIC OXIDE MEDIATE ACTIVITY- LIGAND CONCENTRATION AND RELATIVE METABOLIC PHASE DEPENDENT PLASTICITY OF NEURONAL CALCIUM SIGNALING. Eric A Albrecht, Andrei Kindzelskii, Howard R Petty, Wayne State University Olems Yermolaleval, N Broe, H Weissbach3, S H Heinemann4, T Hoshi', 'University of Iowa, Eric Albrecht, A.L. Kindzelskii, and H.R. Petty Bowen Science Building, Iowa City, Iowa 52242, 2Cornell University Medical Center, New York, Department of Biological Sciences, Wayne State University, Detroit MI 48202 NY, 3Florida Atlantic University, Boca Raton, FL, 4Friedrich Schiller University, Jena, Germany Reactive oxygen/nitrogen species (ROS/RNS) are often considered as tissue damaging agents involved in oxidative stress, aging and neurodegenerative diseases, especially in the presence of Intracellular NAD(P)H oscillations exhibited by polarized neutrophils display a20 sec. periods elevated cytosolic Ca2'. However, ROS and RNS may participate in neuronal signal transduction which are halved to sec. stimulation FNLPNTL. Utilizing l10 upon with the chemotactic peptide that could be involved in the regulation of neuronal function and plasticity. We found that in this change, we have measured accurately the time between stimulus and binary frequency young rat cortical brain slices and undifferentiated PCl2 cells, simultaneous application of metabolic A was to allow rapid delivery of frequency doubling. microscope chamber designed depolarization/agonist stimulation and oxidation induces long-lasting potentiation of subsequent FNLPNTL to adherent cells. Using a fluorescent FNLPNTL conjugate, we found delivery to be Cae+ signaling. This potentiation is dependent on nitric oxide (NO) production and involves and stable within -400 msec. were into the chamber at concentrations complete Peptides injected cellular ROS utilization as measured by the fluorescent dyes. The reducing agent DTT and from 106 to 10-9 M. Injections also varied with respect to the relative phase of the ranging hypoxia abolished the Ca2' signal potentiation. The NO scavenger hemoglobin as well as the NO NAD(P)H oscillations. The time interval between injection of 106 M ligand and frequency synthase inhibitor L-NAME inhibited the development of potentiation. Moreover, application of doubling was found to be 11±2 sec. when injections occurred at the NAD(P)H oscillation peak but NO donors and stimulation of NO synthase activity mimicked the effects of paired stimulation and was 22+2 when with a were found to be 26±7 and 32±6 coinciding trough. At 10-8 M, lag times induced the Ca2' signal potentiation. A sustained elevation of cytosolic Ca2' also appears to be an for and at M had no effect on injections at NAD(P)H peaks troughs, respectively. FNLPNTL 10-9 important element of potentiation. The ability to develop the Ca2' signal potentiation was metabolic lack of physiological frequency doubling, consistent with its previously described dependent on the developmental stage, decreasing markedly in adult rat cortical neurons and on at this dose. signal effects neutrophils These studies show that the kinetics of transmembrane differentiated PC12 cells. Our results suggest that ROS and NO may participate in the neuronal processing, in contrast to a simple transmembrane chemical reaction, can depend upon both ligand plasticity at the cellular level. Supported by NIH GM57654 and HL14388. dose and its temporal relationship with metabolic oscillations.

64A SUNDAY CELL SIGNALING 376-381

377 - Pos NITROGEN IN RAT PAROTID ACINAR CELLS AND ITS ROLE OF INTRAMITOCHONDRIAL NITRIC OXIDE DURING HYPERTENSION IN CA+ PROCESSES. RAT HEART AND KIDNEY MITOCHONDRIA Dissing, Tritsaris, Dagnia K Looms, Birgitte Nauntofte, University of Leopoldo Aguilera-Aguirre', Rafael Villalobos-Molina2, M6nica Clemente-Guerrero', Alfredo Copenhagen, Blegdamsvej 3, Copenhagen, 2200N Denmark Saavedra-Molina', 'Universidad Michoacana, 2CINVESTAV-IPN. Depto. de Farmacol y Toxicol. investigated signalling processes mediated by noradrenaline (NA) in rat parotid acinar rapid increase in nitrogen oxide (NO) production as measured by Nitric oxide (NO) is an important reactive molecule in many organisms. NO is the strongest use dye receptor activated formation of NO causes synthesis of endogenous vasodilator known to date. Recently, it has been described a mitochondrial nitric by process by ODQ, an inhibitor af soluble guanylate cyclases. By incubating oxide synthase (mtNOS). In view ofthe importance ofnitric oxide during hypertension, the aim of detergent CHAP, an enzyme that converts NAD+ into the Ca+ this work is to study the role of mitochondrial nitric oxide in some physiological mitochondrial mobilizing compound, cyclic ADP-ribose. This was accomplished by characterizing the enzymatic aspects. properties enzyme, using NGD+ as a substrate, which was converted into the fluorescent Results obtained show that intramitochondrial free calcium concentrations values are low in compound cGDPR. data show that this reaction is inhibited competetively by NAD+ with a kc- hypertensive rat (SHR) heart mitochondria (0.504 pM), compared with normotensive rat (WKY) FM. Our data are consistent with NAD+ being the true substrate, the heart mitochondria (1.178 AM); in kidney hypertensive rat mitochondria, free Ca2+ was 0.435 pM, product being cADPR, the enzyme ADP ribosyl cyclase. The NA induced rise in cGMP, while in normotensive 0.638 AM; p<0.01). The addition of L-arginine (NOS substrate) increased dependent G-kinase, that can be inhibited by the cGMP derivative Rp-8-pCPT- the mitochondrial permneability transition induced by calcium in heart and kidney mitochondrial of cGMPS, the ADP ribosyl cyclase. The synthesized cADPR was found to hypertensive rats (91±0.3% and 84±0.07%, respectively; p<0.01); but the addition of L-NAME Ca2+ release stores. our data are ryanodine-sensitive Ca2+ Thus, consistent with a (NOS competitive inhibitor) partially prevented the permeability transition induced by calcium in NO/cGMP/cADPR signalling mechanism participates in the control of the Ca2' kindey mitochondria of hypertensive and normotensive rats (89±0.1% and 91i 0. 1%, respectively; permeability the ER membrane. p<0.01). (Supported in part by: CONACYT 29609 N and CIC-UMSNH 1999).

Pos 379 - Pos MODEL SYSTEM STUDY WITH LIPID ANCHORED RGD PEPTIDE CONSTRUCTION AND CHARACTERIZATION OF A MODEL SYSTEM OF THE IMMOBILIZED INTEGRIN QUls CELL-TISSUE-INTERACTION Guttenberg, Lorz, Hu, E. Sackmann, Physik Department E22, Technische Universitat Barbara Lorz, Z. Guttenberg, B. Hu, E. Sackmann, Technical University of Munich, Biophysics Mtlnchen, Franck Strasse, 85748Garching, Germany Laboratory (E22) platelet transmembrane protein ztIlb[3 is a member of the integrin receptor family and For studying the physical basis of the cell-tissue-interaction a model system is established RGD sequence of extracellular-membrane proteins like fibrinogen or fibronectin. To consisting of DMPC giant vesicles modelling the cells and surface grafted integrins aisiSs. Lipid system for cell adhesion, we used a cyclic RGD-containing hexapeptide, anchored cyclic RGD-peptides are embedded as conjugate ligands. The glycocalix of the cell is synthesized lipid For the model system the lipid anchored peptides were embedded modelled by repeller molecules. The RGD-containing vesicles adhere to the integrin-coated giant vesicles, that also contained lipids with a polymer headgroup, to prevent surface and the adhesion ofthe vesicles is observed by reflexion interference contrast microscopy unspecific binding. experiments the solubilized integrin was directly absorbed to the (RICM). The specificity ofthe receptor-ligand-coupling and the influence ofconcentrations of the surface. vesicles was observed with an interferometric technique. the receptors, the ligands and the repeller molecules is systematically investigated. We found three peptide and lipo-polymer concentrations was investigated. A discrete two different states of adhesion: weak undulation dominated adhesion, strong adhesion which is phase strong and weak adhesion could be observed with increasing RGD-lipid dominated by specific lock and key interactions and coexistence of both states. These results can lipo-polymer content yielded a shift of the critical RGD concentration. be explained in terms of a theory of adhesion-induced lateral phase separationsimilar to the from experiments with other model system experiments using mobile wetting transition of fluids an solid supports. This wetting-like transition is also veryfied by ligands supported bilayers. We also observe a nonlinear adhesion dynamic. Both findings can evaluating the kinetics of adhesion whose area-time plots show a sigmoidal shape. An essential explained by theory R Bruinsma, considering undulation, steric repulsion and specific aspect ofbiological systems is the non-equilibrium state. For this purpose we use a flow chamber receptor-ligand binding. which allows us to expose our model cells to hydrodynamic flow fields and observe the reaction by RICM.

Pos 381 - Pos STRUCTURAL MOTIF FOR CARBOHYDRATES. COVALENT MODIFICATION REGULATES LIGAND BINDING TO RECEPTOR Eugene Stevens, Dept. Chemistry, SUNY, Binghamton, N.Y. 13902 COMPLEXES IN THE E. COLI CHEMOSENSORY SYSTEMI Carbohydrates no simple set of secondary structural motifs analogous to the a-helix and J- Robert M Wel, Guoyong Li, University ofMassachusetts, Chemistry, LGRT 701, Amherst, MA sheet proteins, but the gt conformation about the glycosidic bond can usefully serve as a 01003-4510 motif in carbohydrates even when linkages are not exclusively gt. Covalent modification of transmembrane receptors in the E. coli chemosensory system allows group oligosaccharides have all gt linkages' (are 100% ordered), but mannose cells to adapt to a wide range of ligand concentrations. Evidence is presented that covalent oligosaccharides display flexibility.2 Modeling of the (1-3)-linked a-D-mannose dimer34 modification strongly influences the binding affinity only when receptors are in ternary complexes indicates a gtpopulation of 30-60%. with the cellular signaling proteins, CheW, and the kinase CheA. Receptor-regulated steady-state Flexibility the mannose dimer is also indicated by its circular dichroism (CD).5 The far uv CD kinase activity was measured in a reconstituted system using serine receptors with defined levels carbohydrates correlates with a gtlinkage geometry and is regularly reduced upon disordering.6 of modification. From the lowest to the highest modification level, the serine concentration that conventional structure, conformations of carbohydrates may be partitionable into produced half-maximal kinase inhibition increased by 104, about ten-fold for each site modified. disordered components by CD or other means. (Unlike the a-helix, the gt linkage has Since previous ligand-binding measurements were conducted in the absence of CheA and CheW, which has and found little influence unique (p,p specification, worked against its visibility as astructural motif.] of covalent modification, it is concluded that the binding affinity is & strongly affected by covalent modification in the ternary As Cagas, Bush, C.A., Biopolymers 30, 1123-1138 (1990). only complex. this is the physiologically relevant form of the receptor, it seems that optimum receptor sensitivity is 2.Cumming, D.A. & Carver, J.P., Biochem. 26, 6664-6676 (1987). maintained in vivo by changes in binding affinity. Moreover, data analysis by an all-or-none model 3.Carver,J.,Mandel,D.,Michnick,S.,Imberty,A.&Brady,J.,in Comp. Model. Carbohydr. Mol., of cooperativity suggests that the kinase is regulated in a receptor cluster (- 7 receptor dimers for J.Brady,Eds.,266-280 (ACS,1990). the most highly modified form), and that the cluster size decreases as the level of covalent 4.Homans, W., Biochem. 29, 9110-9118 (1990). modification decreases. 5.Arndt, & Stevens,E.S., Biopolymers 38, 567-571 (1996). 6.Stevens,E.,in Circular Dichroism,G.Fasman,Ed.,501-530Plenum,1996).

65A 382-386 CELL SIGNALING SUNDAY

382 - Pos 383 - Pos SOLID-STATE NMR DISTANCE MEASUREMENTS PROBE MECHANISMS OF SPATIO-TEMPORAL PATTERN FORMATION OF GLYCOLYTIC ACTIVITY IN A TRANSMEMBRANE SIGNALING IN BACTERIAL CHEMOTAXIS RECEPTORS YEAST EXTRACT. Frank Kovacs, Owen J Murphy III, Binumol James, Yaci S Balazs, Jesse Charette, Erin Sicard, Thomas Mair, S C Mueller, University Magdeburg, Universitaetsplatz 2, 39106 Magdeburg, Lynmarle K Thompson, University ofMassachusetts Germany The binding of serine to its chemotaxis receptor in E. coli sends a signal across the bacterial inner The generation of dynamic as well as stationary patterns is a property of nonequilibrium systems membrane which ultimately controls the swimming direction of the cell. Efforts to determine the with nonlinear reaction dynamics. Necessary prerequisite for this kind of pattern formation is the mechanism of transmembrane signaling have been hampered by the difficulty of studying the 120 coupling of an autocatalytic (nonlinear) reaction with diffusion. The metabolic flux through kDa intact receptor dimer in a lipid bilayer environment. We are using site-directed solid-state glycolysis is greatly affected by autocatalytic reactions via allosteric enzymes that obey feed-back NMR distance measurements to map local structure and structural changes in the intact Ser activation/inhibition. Hence, the glycolytic flux can become nonlinear (e. g. oscillatory). We have receptor in a hydrated native lipid environment. REDOR measurements have detected a ligand- demonstrated, that glycolytic oscillations are associated with the formation of circular and spiral induced change in the distance between the axl and a4 helices of the 4-helix bundle periplasmic shaped traveling NADH- and proton waves [J. Biol. Chem. 271, 627-630, 1996.]. The allostericaly domain (p-'9F-Phel63 to '3CO-Cys56). The '9F-Phel63 to '3CO-Cyst64 distance determines the regulated enzyme phosphofructokinase (PFK) has an important impact for the initiation and chil torsion angle for Phel63, and is unchanged by ligand binding. These high-resolution distance control of the patterns, as demonstrated by the effects of its allosteric activators AMP and measurements in the intact receptor should determine whether one of the two conformational fructose-2,6-bisphosphate upon the propagation dynamics. Since the activity of the PFK is changes which have emerged from crystal structures of the receptor periplasmic fragsnent occurs regulated by adenine nucleotides, the energy charge affects the patterns as well. Increasing the in the native receptor. Rotational resonance measurements of 13C to 13C interhelical distances in ATPase activity (decrease of energy charge) leads to the formation of both, reaction/diffusion the transmembrane domain have thus far not detected the ligand-induced distance changes waves and phase waves. Transient stationary patterns can be observed, when the ATP-inhibition predicted by current models. This suggests that confonnational changes observed in the of PFK is partly released by the addition of ammonium ions. These results indicate, that the periplasmic domain crystal structures may not simply propagate through the membrane. different energetic states of glycolysis can be translated into different spatio-temporal patterns, opening new possibilities for biological intra- and intercellular communication.

384- Pos 385- Pos INTERCELLULAR COMMUNICATIONS IN GREEN PLANTS: EfFECTS OF ELECTRICAL SIGNALING IN GREEN PLANTS: EFFECTS OF 2,4-DINITROPHENOL UNCOUPLERS. ON RESTING AND ACTION POTENTIALS IN SOYBEAN. Alexander George Volkovl, Daniel J. Collins2, John Mwesigwa', 'Oakwood College, Huntsville, John Mwesigwa', Daniel J. Collins2, Alexander George Volkov', 'Oakwood College, Huntsville, Alabama 35896, 2Department ofSoil and Plant Sciences, Southern University, Baton Rouge, LA AL. 35896, 2Department ofSoil and Plant Sciences, Southern University, Baton Rouge, LA Action potentials in higher plants are the information carriers in intercellular and intracellular 2,4-Dinitrophenol induces fast action potentials and decreases the resting potential in a soybean. comsnunication in the case of environment changes. Action potentials take an active part in the The presence of pesticide 2,4-dinitrophenol in soil is one of the most serious environmental expedient character of response reactions of plants as a reply to external effects. These impulses problem and has an impact on agriculture and human health. The speed of the propagation of transfer a signal about the changes of conditions in a conducting bundle of a plant from the root action potentials in a soybean induced by 2,4-dinitrophenol is 2 m/s. The duration of single action system to the point of growth and conversely. The response reactions of plant tissues and organs potentials after treatment by 2,4-dinitrophenol was 0.02 s. Measured velocities for transmission of can be local or they can be transmitted from cell to cell over long distances via the plasmodesmata. action potentials in green plants, which have been reported by different authors, include: 0.2 m/s in The transfer of excitation has a complicated character, accompanied by an internal change in cells Dionaea flytrap, 0.044 n/s in Mimosa pudica leaf stalk, 0.005 m/s in Drosera, 0.018 m/s and tissues. The high sensitivity of protoplasm and all cell organelles to any natural and chemical following cold stimulation in Cucurbita pepo, 0.0005 - 0.0008 m/s in etiolated pea plants that effects is the basis for excitability. The most rapid methods of long distance communication were wounded, up to 0.1 m/s via sieve tubes in Cucurbita pepo, and 0.001-0.007 cm/s in response between plant tissues and organs are bioelectrochemical or electrophysiological signals. In the to wounding. Action potentials induced in soybean by 2,4-dinitrophenol are many times faster. present work we show that uncouplers induce fast action potentials and decreases the resting Adding 2,4-dinitrophenol to soil decreases resting potential to zero level. The automatic potential in soybeans. The speed of the propagation of action potentials in soybean induced by measurements of the electrical potential difference can be effectively used in environmental plant adding an aqueous solution ofpentachlorophenol to soil reaches 30 mI/s. This is the first attempt of electrophysiology, for the studying of molecular mechanisms of transport processes and the high-speed automatic measurements ofaction potentials in green plants. influence ofexternal stimuli on plants.

386- Pos CELL SURFACE AND INTERFACIAL TENSIONS IN EMBRYONIC MORPHOGENESIS Jennifer M. Folger', Gabor Forgacs', Stuart Newman2, 'Clarkson University, Box 5822, Potsdam, NY 13699, 2New York Medical College The spreading of one cell population over the surface of another is a common means of embryonic morphogenesis. This embryonic cell movement closely imitates the behavior of immiscible liquids. The differences in the surface and interfacial tensions ofthese liquids are the determinants of their immisciblity. According to Steinberg's "differential adhesion hypothesis"(DAH; Steinberg, 1998), the liquid-like behavior of cell populations is due to their possession of surface and interfacial tensions, generated by adhesive and cohesive interactions between the component subunits. Recent experimental results have demonstrated that (i) tissue surface tension is a well- defined intensive physical parameter that characterizes the equilibrium shape of certain multicellular aggregates (Foty et al., 1994) and (ii) the measured values of these tensions account for the liquid-like behavior ofthe corresponding tissues (Foty et al., 1996). An important event in the development of a chicken embryo is the protrusion of limb buds from the surrounding flank tissue. If these embryonic tissues indeed behave as immiscible liquids, with differing surface tensions, according to the DAH this would provide a driving force for the limb bud cell population to migrate from the flank. Using a specifically designed apparatus, we have measured the surface tensions of embryonic chicken limb bud and flank tissues and correlated their values with the morphogenetic changes ofearly limb development.

Steinberg, M.S. (1998). Goal-orientedness in embryonic development Integrative Biology 1, 49- 59.

66A SUNDAY MEMBRANE RECEPTORS & SIGNAL TRANSDUCTION 387-392

387 - Pos 388- Pos SOLUTION STRUCTURE OF CYTOPLASMIC DOMAINS OF THE cz1s-ADRENERGIC A FLUORESCENT ANTAGONIST OF THE MU OPIOID RECEPTOR THAT LABELS RECEPTOR. IMPLICATIONS WITH RESPECT TO BIOLOGICAL FUNCTIONS. INTERNAL RECEPTORS OF LIVING, STABLY TRANSFECTED CHO CELLS. Alberto Spisni', Lorella Franzoni', Arianna Benedetti', Primoz Pristovlek2, Clovis R. Nakaie3, Charles E. Spivak', Shaun Hayeslip2, 'Nat'l Institute on Drug Abuse, 5500 Nathan Shock Dr., 'Institute of Biological Chemistry, University of Parma, Via Volturno, 39, Parma, - 43100 Italy, Baltimore, Maryland 21224, 2University ofMaryland 2National Institute of Chemistry, Ljubljana, Slovenia, 3Department of Biophyisics, University of The fluorescent naltrexone analog 6-BNX has been shown to be a potent antagonist of mu opioid Sao Paulo, Brasil receptors (Emmerson et al., Biochem. Pharmacol. 54:1315 (1997)). With its high affinity (70 pM) Protein-protein interactions play a pivotal role in transmembrane signalling by G-protein coupled and the photostability of its BODIPY fluorophore, it seemed an ideal ligand for visualizing the receptors (GPCRs). Although all GPCRs are believed to share a common topographical motif distribution of mu receptors using laser scanning confocal microscopy. consisting of seven transmembrane helices joined by alternating intracellular and extracellular Stably transfected live CHO cells expressing an average of 100,000 mu receptors/cell were loops, we still lack some crucial structural information. The ability of the receptor to select and visualized in a Zeiss 410 laser scanning confocal microscope using a 63x/1.4 objective. Cells couple to a specific G-protein after agonist binding is a necessary step for the activation of the were grown on glass cover slips, which served as the base of a stainless steel bath (0.5 ml intracellular processes. It is accepted that a knowledge of the tertiary structure of the receptor capacity) through which solutions flowed at 1.3 mlmin.. cytosolic domains is required to understand that step. The Ct,B-adrenergic receptor (a,B-AR) Like the probe fluorescein labeled naloxone (FNAL), 6-BNX showed no labeling of mediates the physiological effects of epinephrine and norepinephrine and belongs to the GPCRs nontransfected cells or cells coincubated with 10 M naloxone. However whereas the intensity of family. Some functional domains have been identified in its C-terminal tail as well as in the FNAL association showed an uptake time constant of about I min, the corresponding time second (i2) and third (i3) intracellular loops. These observations prompted us to investigate the constant for the higher affinity probe 6-BNX was about 10 min. Moreover, whereas FNAL structural features of some peptides, spanning regions of i2 and i3, in various solution conditions and specifically and fairly uniformly labeled cell perimeters, 6-BNX was largely internalized often in a using CD, 'H-NMR and restrained molecular dynamics calculations. The results indicate that the punctate pattern that excluded the nucleus. The slow association time constant for 6-BNX, then, is capability to fold into regular structures, mainly helices whose stability and extent can be seen to result from the kinetics ofinternalization instead ofbinding. modulated by the medium, correlates with the biological functions exhibited by the cognate sequences in the full receptor. A model describing the molecular aspects of the signal transduction in the a,B-AR receptor will be proposed.

389- Pos 390 - Pos DEPENDENCE OF G-PROTEIN BINDING BY METARHODOPSIN II ON BILAYER PLASMON RESONANCE STUDIES OF LIGAND BINDING TO THE HUMAN DELTA- LIPID COMPOSITION OPIOID RECEPTOR. Shul-Lin Niul, Drake C. Mitchell2, Burton J Litman', 'National Institutes of Health, 12420 Gordon Tollin', Zdzislaw Salamon', Scott Cowell2, Victor Hruby2, 'Department of Biochemistry, Parklawn Dr., Rockville, MD 20852, 2NIAAA/NIH, 12520 Parklawn Dr., Rockville, MD 20852 University of Arizona, Tucson, AZ 85721, 2Department of Chemistry, University of Arizona, Previous studies show that membrane acyl chain and cholesterol content modulate the extent of Tucson, AZ 85721 metarhodopsin II (MII) formation. How lipid membrane composition effects the extent and kinetics of MII-G formation, which is the initial step in the signal transduction cascade, remains Coupled plasmon-waveguide resonance (CPWR) spectroscopy has been applied to the binding of unknown. In this study, the kinetics and equilibrium of MII formation and MII-G fornation were ligands to the cloned and expressed human delta-opioid receptor. Receptor was incorporated into characterized in reconstituted rhodopsin-containing vesicles with well-defined acyl chain a solid-supported lipid bilayer by detergent dilution. Analysis of the binding isotherm yielded a and cholesterol content flash and UVNis methods. composition using photolysis spectroscopic value of6.8 nm for the receptor thickness and 1200 V 100 A2 for the surface area occupied by one Since the rate of MII-G formation is more than an order of magnitude slower than that of MIT molecule, in good agreement with values for rhodopsin. A typical agonist (DPDPE) binds to the formation, the kinetics of can be resolved into these two The latter G-protein binding components. receptor with an affinity constant of 10.3 V 0.4 nM; a typical antagonist (naltrindole) binds with process occurs via diffusion in the plane of the disk membrane. The presence of 30 mol% an affinity constant of 0.02 V 0.005 nM. These are consistent with literature values obtained using cholesterol in the slows both with a reduction in the rate of lipid bilayer processes, greater MII-G other methods. From the spectral changes, we conclude that agonist binding results in both an formation These indicate that membrane modulates the (by -40%). results lipid composition elongation of the receptor and an increase in the membrane anisotropy. We interpret the controlled MII-G formation. Previous as a diffusionally studies, using diphenylhexatriene (DPH) elongation in terms of a vertical shift of one or more transmembrane helices, with corresponding found that chain were less affected cholesterol in the of probe, acyl dynamics by presence changes in the extramembrane loops and movement of lipids causing an increase in the positive The role of these chains in MII-G formation was also polyunsaturated lipids. acyl modulating curvature of the surface. The anisotropy change can be ascribed to horizontal helix movements examined. Since the visual transduction is a of the system prototype G-protein coupled signal without changes in the lipid phase. In contrast, the binding of antagonist results only in an transduction systems, our findings of lipid modulation of the coupling of receptor and G-protein increase in the refractive index anisotropy, which implies only horizontal helical movements. likely extend toother members of this receptor superfamily. Support provided by NSF grant MCB-9404702.

391 - Pos 392 - Pos CHARACTERIZATION OF ENDOTHELIN RESPONSES IN HL-1 CELLS, A NOVEL PROLONGED EXPOSURE TO ENDOTHELIN ACTIVATES INFLUX OF Ca2+ CARDIAC ATRIAL CELL LINE. THROUGH L-TYPE CALCIUM CHANNELS IN A CARDIAC ATRIAL CELL LINE (HL- Meaghan O'Brien', Michael K. Murawsk/, Gina M. Fadayel2, Anthony Bahinski2, 'Temple 1). University School of Medicine, 2Procter & Gamble Pharmaceuticals Meaghan O'Brien', Michael K. Murawsky2, Gina M. Fadayel2, Anthony Bahinski2, 'Temple HL-1 cells are a novel cardiac atrial cell line (PNAS, 95:2979, 1998) which retain a highly University School of Medicine, 2Procter & Gamble Pharmaceuticals, 8700 Mason-Montgomery differentiated cardiac morphological and biochemical phenotype during serial passage. Our goal Rd., Mason, OH 45040 was to characterize the HL-1 cell response to endothelin (Et), a potent vasoconstrictor implicated HL-I cells are a novel immortalized cardiac muscle cell line (PNAS, 95:2979, 1998), derived from in many pathophysiological conditions including cardiac hypertrophy and failure. Intracellular mouse atrial myocytes, which retain a highly differentiated cardiac morphological and Ca2+ responses to Et isopeptides (Et-l, Et-2, Et-3) were characterized by single and multiple cell biochemical phenotype during serial passage. HL-I cells respond to brief applications of microfluorimetry using either Fura-2 or Fluo4. All three Et isopeptides evoked an endothelin (Et), a potent vasoconstrictor, with a transient release of caffeine-sensitive intracellular immediate/initial transient increase in cytosolic Ca2+ from a caffeine-sensitive intracellular store. Ca2+ stores. We investigated the response of HL-I cells to prolonged exposure of Et. Intracellular Further, prolonged ET exposure resulted in Ca2+oscillations. The EC50 values for transient release Ca2+ was measured using single and multiple cell microfluorimetry. Prolonged exposure to Et of internal Ca2+ stores were 10 nM, 10 nM and 30 nM for Et-l, Et-2, and Et-3, respectively. The isopeptides (Et- I, Et-2, Et-3) caused oscillatory Ca2+ transients. Thapsigargin (1I M) abolished the endothelin-dependent Ca2+ transient was abolished by preincubation with thapsigargin (IAM) but Ca + transient in response to brief application of the Et isopeptides, but did not inhibit the Ca2+ not by removal of extracellular Ca2'. Preincubation with an EtB receptor antagonist, BQ-788 oscillations during prolonged exposure. The Ca2+ oscillations were inhibited by removal of (lsM) did not significantly effect the Ca2+ transient in response to Et-l, Et-2 and Et-3. Yet, extracellular Ca2+and by preincubation with the L-type Ca2+ channel antagonist nifedipine (2pM). preincubation with the EtA receptor antagonist, BQ-123 (IpM), did significantly reduce the Et The Et isopeptide-induced Ca2+ oscillations were also abolished by preincubation with the protein response, indicating a predominance of EtA receptors on HL-I cells. Our studies suggest that HL- kinase C (PKC) inhibitors staurosporine and chelethyrine. These data suggest that the Ca2+ I cells respond to endothelins in a manner similar to native cardiomyocytes and should serve as a oscillations in response to prolonged exposure to Et are 1) due to influx of extracellular Ca2+ reliable model for studying the physiology and pathophysiology of endothelins. through L-type Ca2+ channels and 2) regulated by activation of the PKC pathway. These results sugest that HL- I cells may offer a unique rystem to study the physiological role of myocyte PKC Ca + regulation in response to Et.

67A 393-398 MEMBRANE RECEPTORS & SIGNAL TRANSDUCTION SUNDAY

393 - Pos 394 - Pos DIFFERENCES IN CONFORMATIONAL PROPERTIES OF THE SECOND A SPATIALLY ORDERED SEQUENCE OF INTRAMOLECULAR INTRACELLULAR LOOP (IL2) IN 5HT2C RECEPTORS MODIFIED BY RNA EDITING REARRANGEMENTS OBSERVED FROM SIMULATIONS OF AGONIST-RELATED CAN ACCOUNT FOR THE SILENCING OF CONSTITUTIVE ACTIVITY ACTIVATION OF 5HT2C RECEPTORS Irache Vlsiers', Sergio A. Hassan2, Harel Weinstein', 'Mount Sinai School of Medicine, One Irache Visiers, Juan Ballesteros, Harel Weinstein, Mount Sinai School ofMedicine, One Gustave Gustave Levi Place, New York, New York 10029, 2Dept. Physiology and Biophysics, Mount Sinai Levi Place, New York, New York 10029 School of Medicine, New York, New York 10029 The transition of a G protein-coupled receptor (GPCR) from its inactive to the active state that Adenosine-to-inosine RNA editing events demonstrated for 5HT2C receptors result in an couples to G proteins, involves rearrangements in transmembrane segments 3 and 6 (TMS3 & alteration of the amino-acid sequence at positions 156, 158 and 160 of the receptor's IL2. The TMS6). We carried out molecular dynamics simulations of a validated model of the 5HT2C "edited" receptor isoforms have reduced basal activity, but similar maximal responses to agonist subtype to study conformational changes that may constitute a molecular mechanism by which binding. It has been proposed that the primary effect of editing may be to alter the ability of agonist binding triggers receptor activation. Our results focus on changes related to ligand binding spontaneous isomerization to the active R* conformation rather than affecting the intrinsic ability involving the cluster of aromatic residues in TMS6 (Phe6.44, Trp6.48, Phe6.52) which straddles of receptor isoforms to promote G-protein coupling. We have carried out conformational studies the absolutely conserved proline P6.50. Our findings point to the interaction between the aromatic of the IL2 isoforms using Biased Sampling Monte Carlo simulations with a Screened Coulomb moieties of Phe6.52 and 5HT as a trigger for the conformational change in the highly conserved Potential-based Implicit Solvent Model, to compare the conformational space accessible to these Trp6.48. A change in the orientation of this residue from 6perpendicular6 to OparallelO to the loops in comparison to the unedited version. Our results show that the IL2 of the unedited membrane plane has been shown from spectroscopy to occur in the transition from inactive to (5HT2C-INI) receptor has a slightly larger population of structures oriented towards the 7TM activated rhodopsin. This rearrangement affects the bending angle of TMS6 that is caused by the bundle than the 5HT2C-VGV edited receptor. This difference in preferred orientation can affect proline-kink from P6.50. This coincides with confornational changes in TMS6 described the association of IL2 with other intracellular loop domains involved in G protein coupling, which experimentally to be associated with activation when TMS6 Omoves awayO from TMS3. Thus, a implies a high sensitivity of the system to small changes in the interaction surface presented to spatially ordered sequence of structural rearrangements stabilized by the interaction of the ligand other intracellular loops, and/or the G-protein. with residues in the Obinding pocket6 suggests a molecular activation mechanism.

395 - Pos 396 - Pos DIFFUSION AND ROTATION OF HOMER-MEDIATED mGluR5 CLUSTERS INTERACTION BETWEEN IrADRENERGIC RECEPTOR AND Gr COUPLED Arnauld Serge, Agnes Hemar, Daniel Choquet, CNRS, 146 rue Leo saignat, Bordeaux, 33000 RECEPTOR SIGNALING IN CANINE CARDIOMYOCYTES France, Metropolitan Ylng-Ying Zhou, S-J Zhang, EG Lakatta, R-P Xiao, Lab of Cardiovascular Science, National Homer proteins bind to the intracellular C-terminus of group I metabotropic glutamate receptors Institute on Aging, NIH, Baltimore, USA. (mGluRs) and may play a role in their organization in the membrane. mGluR5 expressed alone by Recently we showed that 02-adrenergic receptors (AR), unlike 3,AR, couple to Gi in addition to transfection in Ptk2 epithelial cells presents a diffusive distribution in the plasma membrane, but is G,. Interestingly, some G,-coupled receptors, such as opioid receptor, interact with only P3AR but aggregated in clusters (0.2micron2) when coexpressed with Homerlb. Cluster formation depends not P2AR in rat myocytes (Pepe et al, Circulation 95: 2122, 1997). This study is to examine both on the C-terminus of mGluR5 and the C-terminal multimerisation domain of Homerlb. whether and how G,-coupled muscarinic (M) or adenosine(A) receptor activation cross talks with We use single particle tracking to study the mobility of myc-tagged mGluR5 (mGluR5-myc) with ISsAR signaling in canine cardiomyocytes, in which 32AR stimulation cannot increase global anti-myc coated 0.5micron beads manipulated with laser tweezers. The trajectory of mGluR5-myc cAMP or PKA (Kuschel et aL Circulation 99: 2458, 1999). While activation of M2 receptor with coupled beads oscillates between pure diffusion and confinement in small areas. In ptK2 cells carbachol (CCh, 5X106M) or A1 receptor with PIA (10-5M) had no direct effect on contractility, it transfected with mGluR5 alone, the mean duration of confined and diffusive events are completely abolished the inotropic and relaxation effects elicited by I5AR stimulation with respectively 10s and 58s. Coexpression of Homerlb increases the duration of confined events to zinterol (104M), as well as those by P,AR stimulation with norepinephrine (5XIOM), in a PTX- 35s and decreases the duration of diffusive event to 35s. In confined domains, the bead seems to sensitive manner. Inhibition of either NO synthase or phosphodiesterase failed to modify these rotate on a slowly diffusing disk (radius=0.23micron, rotational diffusion=12rad2/s and interactions. To determine whether the antiadrenergic effects occur downstream of cAMP translational diffusion=2.0 103micron2/s). This rotative behavior is also directly visualized with signaling, a non-hydrolyzable cAMP analogue, CPT-cAMP, was employed. Both CCh and PIA beads decorated by smaller beads. In hippocampal neurons, mGluR5 expressed alone display reversed the contracile response to CPT-cAMP (104M), and this inhibition was totally prevented confined trajectories, indicative of an endogenous Homer. These data indicate that clusters of by a phosphatase inhibitor, calyculin A (10-4M). We conclude that, unlike opioid receptor, bot M2 receptors are rigid structures, not anchored to the cytoskeleton, with high rotational mobility. and Al receptor activation counteracts P2AR signaling in canine myocytes, at least in part through a PTX sensitive phosphatase pathway.

397- Pos 398- Pos SINGLE MOLECULE IMAGING OF SIGNAL-TRANSDUCTION OF EPIDERMAL SIGNAL PROCESSING AT THE RAS CIRCUIT: WHAT CONTROLS THE TEMPORAL GROWTH FACTOR RECEPTOR (EGFR) STIMULATED BY EGF ON THE SURFACE PATTERN OF RAS ACTIVATION? OF A431 CELLS. Boris N Kholodenko, Oleg Demin, Vladimir Eryomin, Jan B Hoek, Thomas Jefferson University, Yasushi Sako*, Shigeru Minoghchi*, Toshio Yanagida*, t*Departmnent of Physiology and 1020 Locust St, Philadelphia, PA 19107 Graduate School of Osaka Biosignaling, Medicine, University, 2-2 Yamadoaka, Suita 565-0871, In unstimulated cells, switches of Ras oncoproteins between an inactive GDP-binding and active Japan. tSingle Molecule Processes Mino Project, ICORP, JST, 2-4-14, Senba-Higashi, 562-0035, GTP-binding state are controlled by the intrinsic catalytic activities of Ras. Using a kinetic model, Japan we found that the active fraction of Ras is highly sensitive to the intrinsic rate constant of GTP- Single molecule imaging is an ideal technology to study molecular mechanisms of biological hydrolysis, e.g., a 50% decrease in this rate constant led to about 1000/o increase in Ras-GTP. reactions in vitro. Here we extend this technology to real-time observation of binding of Multiple extracelluar stimuli initiate Ras interactions with specific proteins that facilitate either fluorescent dye-labeled epidermal growth factor (EGF) to its receptor (EGFR), dimerization of GTP exchange or GTP-hydrolysis. The kinetic model demonstrated how these multiple signals are EGF/EGFR complexes and autophosphorylation of EGFR in the plasma membrane of A431 integrated and converted into distinct temporal patterns of Ras activation observed experimentally. carcinoma cells. Significant amount of the fraction containing EGFR dimer was formed prior to As a specific example, EGF-induced SOS activation of Ras was examined. We demonstrated that the intracellular calcium signaling induced by fluorescent EGF. Direct arrest of an EGF molecule the switch-offofRas signaling with a characteristic time of 5 to 10 min implies the involvement of in solution onto a pre-formed cell surface complex was the predominant mechanism of the GAP and cannot be accounted for by the complete deactivation of the SOS-signal only, whereas dimerization, suggesting dimers of EGFR were preformed. Fluorescence resonance energy this deactivation can account for a transient Ras activation with a "decay" time of 60 to 90 min. transfer showed that EGF/EGFR complexes indeed formed dimers at molecular level. EGFR was The results suggest the importance of spatial organization of Ras signaling. We provide kinetic found to be phosphorylated after dimerization. Thus, we show mechanism of dimerization of evidence that for Ras activation to occur, SOS should be "piggy-back" bound to the activated EGF/EGFR followed by autophosphorylation of EGFR, which is essential to signal transduction EGFR. Accordingly, to switch-off the Ras signaling, GAP molecules should "ride" the activated of EGFR. EGFR.

68A SUNDAY MEMBRANE RECEPTORS & SIGNAL TRANSDUCTION 399-402

39 - Pos 400- Pos THE SH3 DOMAIN OF BRUTON'S TYROSINE KINASE IS LIKELY TO BE INVOLVED TIME AND REGION DEPENDENT STIMULATION OF STRESS-ACTIVATED IN FORMING A HOMODIMER MITOGEN-ACTIVATED PROTEIN KINASE SUBFAMILIES FOLLOWING Henrik Hansson', C. I. Edvard Smith2, Torleif Hard', 'Royal Institute of Technology, Novum, MYOCARDIAL INFARCTION 141 57 Huddinge, Sweden, 2Karolinska Institute, Dep. of Biosciences at Novum B. Huang, L. Deng, D. Qin, M. Boutjdir, N. El-Sherif. VA Medical Center and SUNY HSC at Brooklyn The hereditary inumunodeficiency X-linked agammaglobulinemia is caused by mutations in the Boyu Huang, Lili Deng, Dayi Qin, Mohamed Boutjdir, Nabil El-Sherif, VA Medical Center and gene coding for Bruton's tyrosine kinase (Btk). Btk appears to play an important role in the SUNY HSC at Brooklyn, 800 Poly Place, Brooklyn, NY 11209 regulation of B cell proliferation and differentiation. Knowledge about the regulation of this We have shown differences between post-infarction and pressure-overload hypertrophy which enzyme on a molecular level is important for understanding how a defective enzyme can cause the may be related to stimulation of different stress-activated mitogen-activated protein kinases disease. The domain composition of Btk resembles that of Src family kinases whose activation (SAPKs/MAPKsXGidh-Jain el al, JMCC, 1998). Regional ischemia induced by ligation of left involves displacement of domains. Btk contains five different regions/domains, namely PH, TH, aterior descending artery (LAD) in rat is an excellent model to compare effects of ischemia (I) SH3, SH2 and CD. versus stretch (S). Within few minutes of ligation, the ischemic zone (IZ) is akinetic and the non- We have previously determined the structure of the SH3 domain from Btk (Hansson et al., isehemic zone (NIZ) identified by Evans blue dye, is subjected to S. We investigated 3 MAPK Biochemistry, 1998). Here we present data that suggest that the SH3 domain is involved in pathways in IZ and NIZ at different time intervals following LAD ligation (n=6) and compared forming a dimer. One of the proline rich sequences within the TH region/domain, previously them to sham. Phosphorylations of MAPKs and JNKs were differentiated by Westem blot using suggested being an intramolecular ligand, bind to the SH3 domain in an intermolecular fashion. phospho-specific kinase antibodies. MAPKs and JNKs were immunoprecipatated and their This has implications for the understanding of the activation ofthe tyrosine kinase. activities measured. Results (Table): 1. p55 but not p46 isoform of JNK activity increased early in NMR spectroscopy in combination with other spectroscopic methods, BiaCore studies and both NIZ and IZ, and continued to increase only in NIZ (*P<.01). 2. p38 MAPK activity was analytical gel filtration have been used in this study. increased early in both IZ and NIZ, peaked at 3 hours and then decreased. 3. p42 but not p44 isoform of MAPK activity was only increased in 1 day-old NIZ. Conclusions: S but not I caused early and continued stimulation of JNK while both S and I caused transient increase of p38. Late stimulation ofp42 MAPK may be a signal for remodeled hypetrophy in NIZ.

401 - Pos 402 - Pos MEASUREMENT OF THE RELATIONSHIP BETWEEN THE FUNCTION OF REAL-TIME KINETICS OF LIGAND CAPTURE BY CELL SURFACE RECEPTORS IN CADHERINS AND THE VISCOELASTIC PROPERTIES OF TISSUES LIVING CELLS: BINDING OF EPIDERMAL GROWTH FACTOR TO THE David H Stocker, Clarkson University, Physics, Clarkson Ave, Potsdam, NY 13699 EPIDERMAL GROWTH FACTOR RECEPTOR. John C. Wilkinson, Cheryl A. Guyer, Joseph M. Beechem, James V. Staros, Vanderbilt Recent work has established a correlation between the strength of cell adhesion and the metastatic University, Box 1820 Station B, Nashville, Tennessee 37235 potential of certain cancers. On the other hand, according to Steinberg's "differential adhesion We describe a system for extending stopped-flow fluorescence anisotropy analysis to the kinetics hypothesis", the strength of particular cell adhesion is related to the surface tension of the of ligand capture by cell surface receptors in living cells. Most mammalian cell lines cannot corresponding tissue. Thus, measuring the surface tension and the viscoelastic properties of survive the shear forces associated with stopped-flow mixing. We determined that a murine cancerous tissues may provide a diagnostic tool for monitoring their metastatic potential. Using a hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing with calculated dead specifically designed apparatus, we have measured the surface tension and the viscoelastic times of -100 msec. Parental 32D cells, which do not express any endogenous epidermal growth properties of embryonic murine L-cell aggregates (i.e. tissues), with controlled expression of N- factor (EGF) receptor or ErbB family members, were used to establish cell lines stably expressing cadherins. We have found a quantitative relationship between the values of the tensions, their the EGF receptor. Flow cytometry and radioligand binding assays showed the receptor to be viscoelastic properties and the number of cadherins expressed by these genetically transfected expressed at the cell surface at approximately physiological levels (5-7 x 104 receptors/cell). cells. This result allows us to establish a functional relationship between the magnitude of Following rapid mixing, binding of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor- effective cadherin-binding, the effective lifetime of a cadherin bond and the variation of functional expressing 32D cells was observed by measuring changes in fluorescence anisotropy over time cell surface cadherin molecules. using T-format steady-state fluorescence anisotropy detection. These measurements were performed at various concentrations of F-EGF in the nM range, and all data were simultaneously analyzed using global analysis techniques. Our preliminary analysis is consistent with the presence of two distinct receptor populations having association rate constants of 1.9 x 107 and 1.4 x 106 M' s-'. Supported by NIH grants RO1 GM55056 and T32 GM08320.

SUNDAY INTRACELLULAR.1 1. . -- v- %J '%,,JJt CAI CIUM SIGNALING I 403-404

403 - Pos 404- Pos HIGH GLUCOSE ELEVATED T-TYPE CALCIUM CHANNEL EXPRESSION AND CA2t-ACTIVATED MEMBRANE DOCKING BY THE UBIQUITOUS C2 DOMAIN: BASAL [CA2+II IN RAT ISLET BETA-CELLS. COMPARISON OF C2 DOMAINS FROM DIFFERENT FUNCTIONAL FAMILIES. Min Zhang, University of south Alabama, Pharmacology, Mobile, AL 36688 Eric A. Nalefski, Mark Wisner, Susy C. Kohout, Nate Malmberg, Joseph J. Falke, University of There are accumulating data from animal models of diabetes and investigations in patients with Colorado, Boulder diabetes revealing that abnormal [Ca2+]i is a common defect in both insulin-dependent (type I) In stimulated eukaryotic cells, many signal-transducing proteins are activated upon binding Ca2+ and non-insulin-dependent (type II) diabetes. In this study, we proposed that T-type Ca2+ channels are responsible for high glucose mediated elevation of basal [Ca2+]i. We used the and relocalization in the cytoplasm. The ubiquitous C2 domain protein module serves as a Ca2+ fluorescent Ca2+ indicator fura-2 to directly measure [Ca2+]i, and chronically treated rat beta- triggered membrane-targeting motif that regulates numerous diverse signalling proteins such as cells with 3.7, 1 1.1 and 33.3 mM glucose for 48 hours, and we found that the basal [Ca2+]i were phospholipases, isoforms of protein kinase C, mediators of secretory vesicle exocytosis including 74.98 14.78, 106.5 23.19 and 138.50 49.42 nM, respectively. The T-type Ca2+ channel blocker synaptotagmin, and regulators that target specific membrane proteins for degredation such as mibefradil (I M) significantly reduced the elevation of basal [Ca2+]i induced by 11.1 and 33.3 Nedd4. Here we demonstrate that in addition to differences in Ca2+ stoichiometry, phospholipid mM glucose. Neither the L-type Ca2+ channel blocker nifedipine (5 M) nor the Na+-Ca2+ headgroup selectivity and mechanism of membrane binding, the Ca2+ and membrane affinities exchanger blocker benzamil (10 M) exerted effect. We also found that the T-type Ca2+ current and kinetics of distinct C2 domains differ widely. Together these parameters adjust the sensitivity density and mRNA levels increased markedly after exposure to 11.1 and 33.3 mM (aIlG) glucose of different C2 domains across a 10-fold range of Ca2+ and modulate activation and deactivation when compared to 3.7 mM glucose treatment. Cells with higher levels of basal [Ca2+]i exhibited timescales over tens and hundreds of milliseconds, respectively. Thus, dispite considerable less calcium transient response to acute glucose stimulation than those with lower basal [Ca2+]i. structural similarity, C2 domains are functionally specialized for specific signalling pathways Our data suggests that the peak Ca2+ transient elicited by 1.1 mM glucose correlated negatively with the basal [Ca2+]I (n = 23, p = 0.0133). These results implicate that T-type Ca2+ channels are initiated by distinct Ca2+signals. involved in the regulation of basal [Ca2+]i in beta-cells. Cells with high levels of basal [Ca2+]i will be less responsive to glucose stimulation.

69A 405 410 INTRACELLULAR CALCIUM SIGNALING I SUNDAY I SUNDAY

405- Pos 406- Pos MODULATION OF CALCIUM HOMEOSTASIS IN LYMPHOBLASTS BY 1I - SKc, CHANNEL BLOCKERS REDUCE THE AMPLITUDE AND ALTER THE HYDROXYSTEROID DEHYDROGENASE (11-HSD). KINETICS OF JURKAT T CELL Ca2+ RESPONSES. Jeffrey P Gardner, UMD-New Jersey Medical School, Hypertension Research Center, 185 S. Christopher M. Fanger', H. Rauer', A. L. Neben', J C Rosa2, C. R. Ganellin2, K. G. Chandy', M. Orange Ave., Newark, NJ 07103 D. Cahalan', 'U. C. Irvine, Dept. Physiology & Biophysics, Irvine, CA 92697, 2University College Glucocorticoids modulate G-protein receptor mediated Ca2+ signaling and store-operated Ca2+ London entry in human B lymphoblast cell lines (LCLs, Gardner J. and Zhang, L. (1999) J. Physiol. We tested the effects of three new Kc, channel blockers on the small conductance Ca2+-activated 514(2):385). Since maximal increases in cortisol-stimulated platelet-activating factor (PAF) K+ (SKc.) channel found in the human T cell line Jurkat. These blockers, bis-quinolinium response occurred at relatively low levels of exogenous cortisol (200 nM), the effects of altered cyclophanes known as UCLI684-F2-2, UCL2079-F2, and UCL-i530-F2-3, exhibit Kd values of cortisol metabolism on Ca2+ signaling in LCLs were studied. Relative to cells grown with 15% -200 pM, -220 pM, and -30 nM, respectively, for block of the SKc, current. We found that like apamin, the best-known previously-described blocker of this channel, is highly the PAF-evoked was UCLt684-F2-2 FBS, peak Ca2+ response reduced following withdrawal of FBS (72 hrs in specific for the Jurkat SKc5 channel, exhibiting a Kd in excess of I MM for IKc,, BKc., and Kv ITS+), but enhanced by cortisol supplementation (200 nM, 48 hrs in ITS+). Addition of channels. Unlike apamin, block by UCL1684-2-2 is reversible. Thapsigargin (Tg) inhibits the carbenoxolone, an inhibitor of II ,-HSD activity, had no effect on the PAF response in cells endoplasmic reticulum Ca2' ATPase, resulting in depletion of intracellualar Ca2+ stores. During grown in ITS+. In contrast, responses were increased 55% in cells grown with 15% FBS single-cell Ca2 imaging experiments, we stimulated cells with Tg in the absence of extracellular containing 0.15 mg/ml carbenoxolone. As dehydrogenase activity is primarily exhibited by the Ca +, then readded Ca2' and observed the effects of UCLI684-F2-2 (10 nM) when applied before type 2 (cortisol-inactivating) form of lIp-HSD, the expression of this enzyme in LCLs was or after Ca2' readdition. When preapplied, UCLI684-F2-2 only slightly reduces the peak examined using a standard PCR cloning strategy with DNase-treated RNA. RT-PCR with primers amplitude of the [Ca2+]i response following Ca2+ readdition, but decreases the plateau [Ca2+]i to designed for exons 2 and 4 indicated two major bands at -600 and -350 bp; analysis of the lower less than half that of untreated cells by causing a rapid decline from peak. Addition of molecular weight band indicated complete sequence homology with a 348 bp portion of the UCLI684-F2-2 after the peak also depresses [Ca2+], levels, but to a lesser degree. Thus, Kc, mRNA transcript for I1 P-HSD type 2. These results suggest I I f-HSD type 2 is present in LCLs channels help to sustain the plateau ofthe Jurkat Ca2+ response. (Supported by NCI training grant and alterations ofCa2+ 5T32CA09054 to C.M.F., a grant from the A.v. Humboldt Foundation to H.R., and NIH grants may regulate glucocorticoid-mediated homeostasis. MH59222 to K.G.C. and M.D.C. and GM41514 to M.D.C.).

407 - Pos 408 - Pos ER/MITOCHONDRIAL INTERACTIONS IN CA2+ SIGNALING IN SYMPATHETIC MODULATION OF MITOCHONDRIAL Ca2+ SIGNALING BY INTRACELLULAR Na+ NEURONS IN VASCULAR ENDOTHELIAL CELLS David D Friel, Stephen L Colegrove, Case Western Reserve University, Neuroscience, 10900 Marina Sedova, Jorg Huser, Lothar A. Blaster, Department of Physiology, Loyola University Euclid Ave, Cleveland, OH 44106 Chicago, Maywood, IL 60153 S.L.COLEGROVE AND D.D. FRIEL We have investigated the effect of intracellular Na+ on the regulation of mitochondrial calcium Dept. ofNeurosciences, Case Western Reserve University, Cleveland, OH 44106 concentration ([Ca"],) in single vascular endothelial cells. [Ca '], was measured by loading the cells with the membrane-permeant Ca2+ indicator fluo-3/AM and washout of cytoplasmic fluo-3 Sympathetic neurons respond to depolarizing stimuli with a rise in free Ca2+ cytosolic after permeabilization with digitonin. Extramitochondrial calcium concentration ([Ca2+,]) was concentration ([Ca2+]i) that is initiated by Ca2+entry through voltage-gated Ca2+channels but is changed by superfusingpermeabilized cells with solutions of intracellular ionic composition strongly influenced by Ca2+uptake and release by intracellular stores. This study examined the containing various [Ca +]. An increase of [Ca2+], from 100 nM so concentrations > 0.8 gM interplay between Ca2+ across the and two in increased the measured fluo-3 signal. This increase in fluo-3 fluorescence was sensitive to transport plasma membrane by organelles defining red or FCCP, the time of and after ruthenium indicatingthat it was due to energy-dependent Ca2+uptake through the course [Ca2+]i during membrane depolarization: the endoplasmic reticulum mitochondrial Ca" uniporter. [Ca ], < 0.8 pM failed to increase [Ca"]0. In the presence of 10 (ER) and mitochondria. In fura-2 loaded cells, depolarization evokes a reversible rise in [Ca2+]i mM extramitochondrial Na+, application ofbriefpulses of Ca2+ ([Ca2" =2-5 AM, duration 5 s, 1 whose onset and recovery kinetics are modified by thapsigargin (Tg) and FCCP. Inhibition of pulse/100-200 s) to simulate cytoplasmic [Ca"2]i oscillations seen during agonist stimulation Ca2+ accumulation either caused transient increases of [Ca"2],. These [Ca2],,-transients were characterized by a fast rising by organelle accelerated [Ca2+]i elevations evoked by weak phase followed by a slow decay. Removal of extramitochondrial Na+slowed mitochondrial Ca2+ depolarization and the recovery following repolarization, implicating reversible Ca2+ efflux dramatically and resulted in a net accumulation of mitochondrial Ca2+ with each [Ca2+], accumulation by both organelles. To investigate potential ER/mitochondrial interactions, the pulse.These results indicate that regulation of [Ca2],,, in vascular endothelial cells critically FCCP-sensitive component of the total cytosolic Ca2+ flux during the recovery was measured +/- depends on cytoplasmic Na' and suggests a crucial role of the mitochondrial Na'/Ca2 exchange Tg. Similarly, the Tg-sensitive flux component was examined +/- FCCP. The results suggest that mechanism for the regulation of [Ca ']m. Furthermore, the data suggest that changes in [Na'], affect [Ca2"], and through this pathway might contribute to the of Ca2+-sensitive the of ER and mitochondrial Ca2+ are not modified regulation [Ca2+]i-dependence uptake by Ca2+ mitochondrial enzymes and mitochondrial energy metabolism. accumulation by the other organelle. However, by modifying [Ca2+]i kinetics, transport by one organelle does influence transport by the other.

409- Pos INACTIVATION OF THE CARDIAC 410- Pos L-TYPE Ca2+ CHANNEL IS REGULATED BY MITOCHONDRIAL UNCOUPLERS SLOW WAVE PROPAGATION IN OP CELLS MITOCHONDRIA. Laurel L. Haaki, SorayaS. Smaili', Heping Cheng2, James T. Russell', 'NIH, LCMNINICHD, Jorge Sanchez, Maria Garcia, Virendra K Sharma, Shey-Shing She., of Rochester Medical Center University 49 Convent Dr, Bethesda, MD 20892-4495, 'NIA, NIH, Baltimore MD 21224 Mitochondria in oligodendrocyte precursor (OP) cells take up and release Ca2+during agonist- evoked Ca2+signals (Simpson et al., 1998; Smaili and Russell, 1999). This may actively promote L-type Ca2+channels play important roles in vital cell functions such as muscle contraction and Ca2+wave propagation by priming nearby IP3receptors. To test this, we evoked waves with the hormone secretion. This channel is inactivated in heart by a Ca2+-dependentprocess. Here we IP3-Coupled agonist methacholine (MeCh, in untreated OP cells or in that inhibition of 3gM) cells treated with report mitochondrial Ca2+uptake mechanism either by ruthenium red (RR, 2-10 either FCCP (5MM) + oligomycin (I,g/ml) or antimycin A (1pg/ml) + oligomycin (I,ug/ml). gM) or by carbonyl cyanide m-chlorophenylhydrazone (CCCP, 1-5 the of Cells were loaded with fluo-4 and levels in OP the gM), delays recovery Ca+ processes were recorded using rapid linescan channel from the inactivation produced by a previous depolarization. The amplitude and time confocal laser scanning microscopy. We found that the mitochondrial uncouplers FCCP and course of Icduring single depolarizations are not affected by RR or CCCP suggesting that they antimycin A both significantly decrease wave speed in OP cells. MeCh evoked waves had a mean have no direct on effects the Ca2+channel itself.In agreement with the electrophysiological data, speed of 0.5+0.4 pm/msec (n=4 cells). After treatment with FCCP, wave speed slowed to we found that perturbing the mitochondrial function decreases Ca2+release only at stimulation 0.0Si0.01tm/msec (n=5cells, p<0.04). Antimycin A showed similar effects: wave speed slowed frequencies at which recovery from inactivation ofthe channel is not We to that L-type complete. suggest 0.04±0.02pm/msec (n=4 cells, p<0.02). Together, these results indicate that IP3-mediated mitochondria play a crucial role in regulating the inactivation of the dihydropyridine- release of Ca" from internal stores in OP cells may be positively modulated by energized sensitive-Ca2+channel (DHPR) by clearing cytosolic Ca2+fromits microdomain. The implications mitochondria. are profound on excitation-contraction coupling, and on the strength and the rate ofheartbeats.

70A SUNDAY INTRACELLULAR CALCIUM SIGNALING I 411416

411 - Pos 412 -Pos THE MITOCHONDRIAL COMPONENT OF IP3 MEDIATED Ca'+ A KINETIC MODEL FOR DIFFERENTIAL TARGET ACTIVATION BY CALMODULIN. Chris Fall, Joel Keizer, University of California Davis, Institute of Theoretical Dynam, One Yan Zhamg, M. Neal Waxham, Ah-Lim Tsai, John A. Putkey, University of Texas-Houston Avenue, Davis, CA 95616 Medical School

little doubt that mitochondria play a role in modulating intracellular Ca2+ signaling. Calmodulin (CaM) is an important calcium-dependent signal transducer that regulates a wide Numerous groups have shown that mitochondrial function affects not only the spatial and variety of essential cellular functions. The classical mechanism of action of CaM involves binding temporal dynamics of Ca2+ signaling but also downstream Ca2+ dependent processes. Here we Ca2+ to free CaM with subsequent binding of Ca2+/CaM to target proteins. However, the effect of present some results of investigations in which we have attempted to incorporate mitochondrial different targets on the off-rate of Ca2+ from N- and C-domains of CaM, coupled with oscillating components into models of IP3 mediated Ca2+ signaling. We show a detailed kinetic model, a Ca2+ levels during stimulation, could lead to differential binding of CaM to targets. To evaluate model, a model which does not rely upon Ca2+ for inactivation of the IP3 receptor, and a this possibility, we have used FRET and stopped flow kinetics to monitor discrete events upon which we begin to address the question of spatial localization of mitochondria and ER removal of Ca2+ from CaM/peptide complexes. Specifically, we wished to determine the rate of separation of the N- and C-domains of CaM, and the rates of dissociation of the N- and C-lobes of CaM from model peptides. To achieve this, we engineered CaM and peptides such that FRET donor/acceptor pairs Wvere attached to the N- and C-domains of CaM, or on the N- or C-domains of CaM and the N- or C- terminus of the model peptide. We found the separation of the CaM domains fit a single exponential rate constant of 3.5-8.5 s -', which was dependent on the CaM binding affinity of the peptide. In contrast, dissociation of CaM from the peptide was a two-phase reaction, indicating initial dissociation of the N-domain and subsequent dissociation of the C- domain.

Pos 414 - Pos ALTERED STRUCTURAL AND FUNCTIONAL STATE OF CALMODULIN CaM KINASE II INHIBITOR, KN-62, DECREASES HALOTHANE- AND CAFFEINE- ASSOCATED WITH CALDESMON IN SMOOTH MUSCLE THIN FILAMENTS INDUCED TENSION TRANSIENTS IN RABBIT SKINNED FEMORAL ARTERIAL Notarianni', Nikolai B Gusev2, Tessa Hill3, Steven B Marston', 'Imperial College Sch. STRIPS. Med., 2Moscow State University, 3University of Warwick Judy Y. Su, AnhKiet Vo, University ofWashington, Seattle, WA 98195 muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon Ca2+ binding protein. Thin filament activation of myosin MgATPase is Ca2+-regulated but CaM kinase II has been shown to modulate the SR Ca2' release channels in striated muscles. The filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or goal of this study was to examine the role of CaM kinase II in halothane-induced Ca2+ release calmodulin (CaM) are not Ca2+ regulated at 25°C/50mM KCI. We isolated the Ca2+ from the SR in vascular smooth muscle. Strips from rabbit femoral arteries were mounted on binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in force transducers and then the sarcolemma were permealized with saponin ("skinned"). The Urea and Phenyl Sepharose chromatography. Thin filaments assembled with this protein were skinned strips were then cycled in four different solutions to load Ca2' into, and then to release Ca2+-regulated at 25°C/50mM KCI. Amino acid sequencing, MALDI-MS and electrospray MS Ca2+ from the SR using 10 mM caffeine, resulting in a tension transient. Each experiment CaBP is identical to CaM. Structural probes indicate it is different: CaM increases consisted offour cycles: (1) a control cycle with 10 mM caffeine, (2) a conditioning cycle with 10 caldesmon tryptophan fluorescence but CaBP does not. The tryptic digestion patterns in Ca2+ and mM caffeine+[KN-62]i (a CaM kinase II inhibitor), (3) a test cycle with [halothane]i or [caffeine]i different. The distribution of in MS are different and charged species electrospray ± [IN-62]i, and finally (4) another control cycle. The area of the test tension transient was skimmer fragmentation patterns are different indication a more stable N terminal lobe for expressed as a percentage of that of the two bracketing controls. We found that halothane dose- CaBP. heating abolishes these special properties of the CaBP. ES MS in aqueous buffer dependently induced tension transients [34.7%, 70.1%, and 108.3%, for 0.3%, 1%, and 1.5% evidence for the presence of any covalent or non-covalently bound adduct. The only halothane, respectively). KN-62 dose-dependently decreased 1% halothane-induced tension remaining conclusion is that CaBP is calmodulin locked in a metastable altered state. transients (90.3%, 67.2%, 44.7%, and 56.1% for 0, 1, 10, and 30 pM KN-62, respectively). KN- 62 (10 pM) decreased 0.3 mM caffeine-induced tension transients. The results suggest that CaM kinase II modulates the ryanodine-receptor SR Ca2' release channel and the halothane-induced Ca2+ release from the SR in vascular smooth muscle. (Supported by NIH-GM48243)

Pos 416- Pos OF PROTEIN KINASE C ON INTRACELLULAR CALCIUM SIGNALING IN rAND Br INHIBIT Ca2'+UPTAKE BY THE CARDIAC SARCOPLASMIC RETICULUM UMBILICAL VEIN ENDOTHELIAL CELLS Margaret E Kargacin, Shi-Jin Zhang, Gary J Kargacin, University of Calgary, 3330 Hospital Li Li, Tania M Szado, Casey Van Breemen, Department of Pharmacology & Therapeutics, Drive N.W., Calgary, Alberta T2N 4NI Canada British 2176 Health University Columbia, Sciences Mall, Vancouver, British Columbia V6T H' efflux from the reticulum of cardiac muscle is to IZ3 Although sarcoplasmic (SR) coupled Ca2+uptake, additional charge compensation is believed to be necessary and may be mediated by Intracellular free calcium concentration ([Ca2+]j) in primary cultures of Human Umbilical Vein influx of Cl into the SR. To test this possibility, we examined the effect of anion substitution on Cells (HUVEC) loaded with fluorescent Ca2'+ indicator fura-2 was measured using SR Ca2+uptake using a fluorometric method to measure Ca2+uptake into ventricular SR vesicles. imaging microscopy. Application of histamine induced an increase in [Ca2l]i, characterized by an We also employed two spectrophotometric methods to measure the ATPase activity of the SR peak followed by a plateau. Application of protein kinase C (IPKC) activator indolactam Ca2e pump. SR Cs2' uptake was inhibited in a concentration-dependent manner when Bre or I- during histamine induced [Cs2+], plateau greatly decreased the [Ca 'i signal, and pretreatment were substituted for Cl in the extravesicular media but not when Cl was replaced by s042-. SR HUVEC with indolactam prevented the histamine induced [Ca2+]i increase. On the other hand, CaW+uptake was inhibited by 60% when I was substituted for Cl- and by 40% when Br- of pretreatment HUVEC with PKC inhibitor staurosporin potentiated the histamine induced replaced Cr-. Neither Br- nor r-directly affected the ATPase activity of the SR Ca2+pump. The while [Ca2+]iplateau, subsequent withdrawal of staurosporin during histamine stimulation lowered inhibitory effect of Ion SR uptake was the same in the presence or absence of ruthenium red, an value. In histamine induced plateau addition, higher [Ca2'], plateau in HUVEC incubated in inhibitor of the ryanodine receptor. Our results are consistent with the hypothesis that an anionic Buffered Saline Solution with a high concentration of glucose (36 mM) for 3 hours in current that accompanies SR Ca2+uptake is reduced by the substitution ofBr or I for Cl-. presence of staurosporin than in HUVEC incubated in the same solution without staurosporin. possible that the mechanism whereby PKC inhibits the histamine-induced [Ca2']isignal is of phospholipase C (PLC). Application of the PLC inhibitor U-73122 was shown to similarly decrease the histamine induced [Ca2+]i plateau. These results suggest that PKC regulates termination of [Ca2+]j response through a negative feedback mechanism, guaranteeing a finely regulation of the signaling cascade. This action is possible through its inhibitory effect on activity. Furthermore the activity of PK C may attribute to altered [Ca2+]i signaling caused by high glucose load in diabetes.

71A 417-422 INTRACELLULAR CALCIUM SIGNALING I SUNDAY

417- Pos 418 - Pos CALCIUM UPTAKE BY THE CARDIAC SARCOPLASMIC RETICULUM IS EFFECTS OF INTRALUMINAL AND CYTOPLASMIC POTASSIUM ON CALCIUM INHIBITED BY TAMOXIFEN UPTAKE BY THE SARCOPLASMIC RETICULUM Gary J Kargacin', Christopher A Ward2, Zenobia Ali', Natashka S Pollock', Margaret E Margaret E Kargacin, Shi-Jin Zhang, Gary J Kargacln, University of Calgary, 3330 Hospital Kargacin', 'University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4NI Canada, Drive N.W., Calgary, Alberta T2N 4N1 Canada 2Queens University It has been hypothesized that K+ crosses the membrane of the sarcoplasmic reticulum We have found that tamoxifen reduces both the magnitude of Ca2' transients in isolated (SR) during Ca2' uptake to partially balance the positive charge moved into the SR by the Ca- myocytes and the amount of Ca2' that can be released from the sarcoplasmic ATPase. We have tested this hypothesis by measuring Ca2' uptake into skeletal muscle SR reticulum (SR) by caffeine. Experiments were, therefore, carried out to determine if these actions vesicles when extravesicular and/or intravesicular K+ was replaced by cesium, choline or a direct effect of tamoxifen on cardiac SR function. The effects of tamoxifen on calcium tetraethylammonium (TEA). Fluorescence measurements of SR Ca2> uptake showed that uptake uptake by SR vesicles and on the ATPase activity of the SR Ca2' pump were determined. SR Ca2 was inhibited in a concentration-dependent manner when extravesicular K+ was replaced by any uptake, measured using fura2, was inhibited by tamoxifen at concentrations > 2.4 PM. The of the three cations. The magnitude of the inhibitory effect was determined by the K+ concentration giving half maximal inhibition was Tamoxifen did not inhibit was ' 5 pM. concentration rather than by the species of the substituting cation. The extent of inhibition uptake by causing a leak of Ca from the SR. Fluorimetric measurements of the ATPase activity independent of the cation present in the intravesicular medium. The rate of SR Ca2+ uptake was of the SR Ca pump showed that tamoxifen did not directly inhibit the pump. Tamoxifen also increased over the control rate (intravesicular and extravesicular cation = K+) when intravesicular reduced the rate that stored Ca2' could be released from the SR by Br-A23187. These results are K+ was replaced by Cs+or choline+ and the primary cation in the extravesicular medium was KW. consistent with the hypothesis that tamoxifen inhibits an anion current that accompanies Ca2' This effect was not seen when intravesicular K+was replaced by TEA. The effect was blocked in movement across the SR membrane and with the known inhibitory effect of tamoxifen on some the presence ofextravesicular TEA and partially blocked by 4-aminopyridine in the extravesicular C1 channels. medium. These results are inconsistent with the proposal that SR Ca + uptake requires the counter movement of K+ out ofthe SR.

Pos 420 - Pos ISOFLURANE ACTIVATES MAP KINASE IN CULTURED VASCULAR SMOOTH Ca2+ CLEARANCE IN ACINAR CELLS AFTER FLASH PHOTOLYSIS OF CAGED MUSCLE CELLS FROM THE RABBIT. COMPOUNDS. Li Zhong, Judy Y. Su, University of Washington, Box 356540, Seattle, WA 98195 David I Yale, Stephen Straub, David R Giovannucci, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642 activation of PKC and CaM kinase II by isoflurane has been implicated in the isoflurane- The mechanismsthat shape transient rises in Ca2+ and restrict local Ca2+ signaling events to the increased submaximum Ca2+-activated force in rabbit skinned femoral arterial strips (Toda & Su, secretory pole ofexocrine cells are not well understood. Because of the high degree of structural 1998). In cultured smooth muscle cells from rat aorta, MAP kinase has been shown to be organization of pancreatic acinar cells, mitochondria, ER, and plasma membrane Ca2+ pumps downstream of ionomycin-activated CaM kinase It (Abraham et al., 1996). The goal of this study may be positioned to sense local domains of Ca2+ release. In the current study, the clearance of was to examine whether activation of CaM kinase II by isoflurane was also via MAP kinase. instantaneous rises in [Ca2+]c evoked by the photolytic release of caged Ca2+ prior to and Using selective cell migration method (Saward and Zahradka, 1997), smooth muscle cells were following selective pharmnacological block of Ca2+ sequestration or extrusion was investigated. from rabbit femoral artery. In passages 5-10 at 80-90% confluence, the cultured cells Repetitive flashesevoked Ca2+-sensitive chloride currents that increased in both peak amplitude growth-arrested for 48 hours before use. The cultured cells were then treated with isoflurane and time to recovery to baseline, the degree ofwhich was dependent on Ca2+ load, flash number, activities were measured by westem blotting using antibody against phosphorylated but not on the time interval following patch rupture. Treatment with cyclopiazonic acid ERKI/2. The effect of isoflurane on ERK1/2 activities was studied at various time periods (time- diminished the rate of Ca2+ recovery, where as treatment with ruthenium red, which blocks course) and at various isoflurane vapor concentrations (0%, 1%, 3%, & 5%) (dose-response mitochondrial Ca2+ import, enhanced the peak current response 2-fold compared to control relationship). Results showed that isoflurane increased ERKI/2 activity in a dose-dependent responses. Cytosolic Ca2+ recovered over a longer time course in the presence of ER and manner and reached plateau at 10min. Itis concluded that isoflurane activates ERKI1/2 which may mitochondrial inhibitors indicating plasma membrane pumps also contribute to Ca2+ clearance. downstream of CaM kinase II. (Supported by NIH-GM48243) These data reveal temporally segregated contributions to Ca2+ clearance by mitochondria, ER, and plasma membrane pumps in pancreatic acinar cells.

Pos 422 - Pos CELL SWELLING ON INTRACELLULAR CALCIUM AND MEMBRANE EFFECT OF INTRACELLULAR AND EXTRACELLULAR CALCIUM AND SODIUM BOVINE ARTICULAR CHONDROCYTES ON THE MTX RESPONSE IN CHINESE HAMSTER OVARY CELLS (CHO). Clare Yellowley', J C Hancox2, H JDonahue', 'Penn State College of Medicine, 500 University Ver6nica Morales, Luis Vaca, Instituto de Fisiologia Celular, Circuito Exterior S/N Ciudad Drive, PO Box 850, Hershey, PA 17033, 2Dept. Physiology, School of Medical Sciences, Universitaria, Mexico, D. F. 04510 Mexico University Bristol, BS8 lTD,UK. Maitotoxin (MTX) is a polyether isolated from the marine dinoflagellate Gambierdiscus toxicus, exist within an environment Chondrocytes osmotic dictated by the nature of the cartilage matrix and is one ofthe entities implicated in ciguaterapoisoning. MTX has been studied extensively as a is influenced by mechanical loading. It is possible that cells may sense mechanical calcium influx activator in a wide variety of cells. The mechanismsby which MTX activates changes environment via changes in osmolarity. In this study, we examined the effect of calcium influx remain obscure. hypo-osmotic stress on early signaling events in chondrocytes, by making measurements of In this study we characterized the effects on the intracellular and extracellular calcium and sodium intracellular calcium [Ca2i]i and of macroscopic ionic currents. Chondrocytes exposed to concentrations on the MTX response. The results presented here indicated that 1) MTX activates hypotonic solution (-450mOsm) swelled in size to a peak at 4 minutes and recovered gradually calcium and sodium influx in a concentration-dependent manner. 2) Extracellular calcium is normal over the following 8 minutes. Cells were loaded with fura-2 AM and exposed required for the sodium influx. 3) Removal of the extracellular sodium did not alter the MTX increasingly hypotonic Tyrode's solutions. When exposed to a decrease in of osmolarity response. 4) The intracellular calcium concentration plays a critical role in the MTX response 75mOsm, 28.7± 3.6% of cells responded with an increase in 43.9±10.7% of 5) [Ca2],, cells MTX, at the concentrations tested, did not affect cell viability in the supernadant of treated cells. responded -IlOmOsm and 73.5± 6.7% to -l5OmOsm. The proportion of cells responding to a hypo-osmotic stress of -150mOsm (73.5± 6.7%) was not affected by nifedipine (20pM), (67.5±11.2%), but was reduced by gadolinium (504M) and thapsigargin (5OnM) to 3.8± 2.6% and 8.1± 3.0% respectively. Inward and outward membrane currents increased over 4-fold when exposed to -lSOmOsm Tyrode's, as measured using the amphotericin perforated patch clamp technique. These effects may be involved in a mechanism by which mechanical loads are chondrcytes.

72A SUNDAY INTRACELLULAR CALCIUM SIGNALING I 423-426

423 - Pos 424- Pos FUNCTIONAL ROLES OF P2X, PURINOCEPTORS IN HUMAN PLATELETS ROLE OF IKCal AND Kv1.3 ON Ca'+ SIGNALING AND PROLIFERATION IN HUMAN Michael G Rolf, Martyn P. Mahaut-Smith, University of Cambridge, Downing Street, Cambridge, T-LYMPHOCYTES. CB2 3EG United Kingdom Heiko Ramer', C. Fanger', A. Neben', M. W. Pennington2, K. G. Chandy', M. D. Cahalan', 'Dept. In human platelets, metabotropic P2Y, and P2YAC receptors stimulate shape change and Physiology and Biophysics, UC Irvine, Irvine, CA 92697, 2Bachem Biosci., King of Prussia, PA aggregation via a combination ofraised [Ca2>]i and lowered cAMP. The functions of ionotropic 19406 P2Xj purinoceptors are unknown. We have investigated the functional roles of the P2X, Voltage-gated (Kv1.3) and Ca2+-activated (IKCal) K+ channels regulate membrane potential in purinoceptor using simultaneous measurements of fluorescence and light transmission in platelet resting and activated human T-lymphocytes. Mitogenic stimulation causes a >20-fold increase of suspensions. [Ca+]i was measured using fura-2 (in saline) or fluo-3 (plasma:saline mixtures). functional IKCal expression in activated T-cells, whereas the number of Kv1.3 channels increases Shape change and aggregation were monitored from the transmission of 578 rm light. Platelets only -2-fold. Kv1.3 blockers inhibit T-cell proliferation, and recently it has been demonstrated were prepared as described previously (MacKenzie et aL 1996 J. Biol. Chem. 271:2879) except that clotrimazole, an IKCal inhibitor, has simnilar effects. Since clotrimazole also targets P-450 that an initial 2 -3 ml blood was discarded; blood was drawn directly into anticoagulant to limit enzymes we exploited two novel toxin analogs ChTX-Glu32 and ShK-Dap22, highly selective purinoceptor desensitisation. The effects of citrate on pH. and [Ca2+]. were compensated for in inhibitors of IKCal and Kv1.3, respectively, to characterize the role of these channels in T-cells in plasma:saline experiments. Stimulation with the P2X-specific agonist ajS-methylene ATP caused more detail. Using 3H-thymidine- and fluorescent CyQUANT-assays we showed that ChTX-Glu32 a transient increase in [Ca2+]i and a delayed, transient decrease in lighttransmission, consistent and ShK-DapP inhibited OKT-3 and PHA-stimulated T-cell proliferation in a dose-dependent with platelet shape change, in both saline and plasma:saline mixtures. The light transmission manner. Thapsigargin (TG) depletes internal Ca2+ stores and elicits Ca2+ influx through CRAC decrease was unaffected by 20 sgml"' Reopro and was abolished by omission of apyrase or channels following re-addition of external [Ca2]. Blockade ofIKCal with ChTX-Glu2 prior to external Ca+; either condition also prevents P2X,-receptor dependent calcium influx. re-addition of external Ca2> reduces the amplitude of the TG-induced Ca+ signal to a greater Experiments conducted at 130C slowed the calcium responses elicited by the metabotropic extent in activated than in resting T-cells. In contrast, block of KvI.3 by ShK-Dap22 causes a receptors compared to the ionotropic P2X, receptors, allowing further elucidation of the relative dose-dependent delay of the Ca2+ influx upon Ca2' re-addition, but does not alter the amplitude of roles of these receptors in the [Ca2+]i signals. the Ca` signal after a certain Ca>ithreshold was reached. These results imply that both K+ channels play an important role in maintaining a high internal [Cas+] during the activation of human T-cells. (Supported by NIH grant MH59222 (KGC MDC), NS14609 (MDC) and the Alexander von Humboldt foundation (HR)).

425 - Pos 426- Pos IONOMYCIN ACTIVATES A BAX-DEPENDENT Ca'+ PERMEABILITY. MOVEMENT OF BAX FROM CYTOSOL TO INTRACELLULAR MEMBRANES IN Agustin Guerrero-Hernandez', Juan Manuel Arias', Lucia Garcia', Andres A Gutierrez2, CELLS UNDERGOING APOPTOSIS. 'CINVESTAV-IPN, Dept. of Biochemistry, Av. IPN 2508, Mexico city, Mexico D. F. 07000 Manjunatha B Bhat', Kaisa M Heiskanen', Anna-Liisa Nieminen2, Jianjie Mal, 'Dept. of Mexico, 2Inst. Nacional de Cancerologia Physiology & Biophysics, Case Westem Reserve Univ., Cleveland, OHIO 44106, 2Dept. of lonomycin (a Ca>+ ionophore) at concentrations that can trigger apoptosis induces the activation of Anatomy, Case Westem Reserve Univ., Cleveland. a Ca+ permeable, voltage-independent, non-selective cation channel of 23 pS conductance in Apoptosis which plays an important role in development and tissue homeostasis is regulated by LNCaP cells (Gutierrez AA etal. 1999. J. Physiol. 517:95). We have studied the role of Bax (an pro- and anti-apoptotic proteins. Bax is a pro-apoptotic protein localized to the cytosol. The ion channel forming and pro-apoptosis protein) in the electrophysiological responses to ionomycin translocation of Bax from cytosol to the mitochondria is thought to be one of the initial events in of LNCaP cells. Bax is a 22 kDa protein expressed in LNCaP cells as shown by Western blot apoptosis, although the mechanism(s) of this movement is not clearly understood. To understand analysis. LNCaP cells responded to ionomycin with an initial hiperpolarization (due to the the molecular basis of Bax-induced apoptosis, we expressed a fusion protein of Bax and green opening of maxiK channels) and a late membrane depolarization (associated with the activation of fluorescent protein (GFP-Bax) in Chinese Hamster Overy (CHO) cells. The subcellular the 23 pS channel). These changes correlated with a biphasic rise in [Ca2+]i; the initial one was due localization of the expressed fusion protein was determined using confocal microscopic imaging. to release from internal stores and the second required Ca?+ entry via the plasma membrane. CHO cells transiently expressing GFP-Bax exhibited diffuse fluorescence suggesting the cytosolic LNCaP cells were more sensitive to ionomycin in the whole cell configuration than in perforated localization of the fusion protein. The fluorescence distribution in these cells became punctate in patch because a higher elevation of the initial [Ca>]i rise and a faster activation of the late -24 hours after transfection along with DNA fragmentation which is one of the characteristic depolarizing-conductance were observed. The presence of anti-bax antibodies (1001sg/ml) in the features of apoptosis. Treatmnent of cells with ATP which induces intracellular calcium release pipette solution did not effect the initial hiperpolarization but, strongly reduced both, the rate of accelerated the translocation of GFP-Bax from cytosol to the mitochondria, suggesting the role of membrane depolarization and the increase in [Ca2+]j. The depolarizing conductance was not calcium in the signal transduction pathway of apoptosis. The movement of GFP-Bax to the activated by adding cytochrome c and dATP to the pipette solution. These results indicate that Bax mitochondria and subsequent cell death were reduced in CHO cells stably expressing the anti- is associated with the activation of a Ca2 permneable conductance in response to ionomycin. apoptotic protein Bcl-xL. The role of calcium and mitochondria in Bax-mediated apoptosis is currently being investigated using agents such as BAPTA (a calcium chelator) and cyclosporin A (an inhibitor of the mitochondrial permeability transition). Supported by NIH and AHA.

SUNDAY ION MOTIVE ATPASES 427-428

427- Pos 428 Pos FUNCTIONAL EXPRESSION OF THE HUMAN NON-GASTRIC H,K-ATPase IN CHANNEL-LIKE BEHAVIOR OF THE TRANSMEMBRANE FRAGMENTS OF A P- INSECT CELLS. TYPE ATPASE. Gall Adams, M. Tillekeratne, C. Yu, N. B. Pestov, N. N. Modyanov, Department of Olliver Radresaa, Erik Goormaghtigh2, Fabrice Hombl', Jean Marie Ruysschaert2, 'ULB, blv du Pharmacology, Medical College of Ohio, Toledo, Ohio 43614-5804 Triomphe, Brussels, 1050, 2Free University of Brussels Previously, expression in Xenopus oocytes was used to demonstrate that the protein product of the Our previous results about the accessibility of the transmembrane segments of the Neurospora human ATPIALI gene can assemble with the n-subunit of the gastric H,K-ATPase (PHKg) as a crassa H+-ATPase are consistent with the presence of a membrane pore delimited by the functionally active ion-pump; a ouabain-sensitive H,K-ATPase. To gain insight into catalytic transmembrane segments. Direct evidence for the presence of such a channel still remained to be functions and reaction mechanism, we have analyzed the ATPlALl/PHKg complex using a established. To study the transport activity of the transmembrane domain of the H+-ATPase, we baculovirus expression system. Recombinant baculoviruses expressing the ATPIALI and the cleaved the cytoplasmic loops of the reconstituted enzyme with an endoprotease Arg-C. After PHKg both together or separately were prepared and tested in several insect cell lines using purification of the hydrophobic segments, proteoliposomes were fused with a planar lipid bilayer. We measured the current through the transmembrane domain in the presence of specific antibodies. The most efficient expression of functional ATPIALI/PHKg ATPase increasing complex was obtained upon infection ofSf21 cells with separate viruses containing ATPlALI and concentrations of KCI, pH 6.8. The identified channel, remains mainly open below 50 mV, and starts cycling between rapid closures and apertures at higher potentials. In PHKg under polyhedrin promoters. Expression of either protein alone did not produce active asymmetrical concentrations of KCI, the ionic selectivity reveals a cation selective behavior. In symmetrical ATPase. The ATPase activity of recombinant ATPlALI/PHKg complex expressed in insect cell conditions ( between 150 mM and 600 mM KCI ), the macroscopic conductance of the channel membranes was found to be sensitive to ouabain (Ki of -55 and to vanadate but resistant IiM) lies in the nanoSiemens range. The conductance/concentration dependency can be fitted by an toward The apparent Km value for ATP was 0.4 mM. The highest level of ouabain- oligomycin. asymptotic curve, so that in these concentrations the channel still remains able to freely transport sensitive ATPase activity was detected at pH 7.4. Assays of ouabain-sensitive ATPase activity of cations. Our results demonstrate that after cleavage, the purified transmembrane fragments of the the did not reveal ATPIALl/PHKg complex within wide ranges of K+ and Na+ concentrations H+-ATPase can reassemble in a functionnal channel-like structure within the hydrophobic core of significant dependence on either cation. The distinctive properties of the ATPlALl/PHKg the bilayer. complex in respect to formation and stability of the phosphorylated intermediate have also been determined. (Supported by NIH grants HL-36573 and GM-54997).

73A 429-434 ION MOTIVE ATPASES SUNDAY

429 - Pos 430 - Pos MULTIPLE PLASMA MEMBRANE Ca2+-ATPASE ISOFORMS EXPRESSED IN RAT PLASMA MEMBRANE CaATPase IN HUMAN RETINAL PIGMENT EPITHELIUM PANCREATIC 0 CELLS. Nancy J Manginfl, Karin U Loeffler2, Brian G Kennedy3, 'University of Illinois at Chicago Adams KAMAGATE, Andre HERCHUELZ, Fran9oise VAN EYLEN, Universit6 Libre de College of Medicine, 1855 W. Taylor St., Chicago, IL 60612, 2Universitslts-Augenklinik, Bonn Bruxelles, 808, route de Lennik, Bruxelles, 1070 Belgium Germany, 3Indiana University School ofMedicine, Gary IN Ca concentration in the subretinal space (SRS) varies with light exposure, and regulation of SRS When stimulated by glucose, the pancreatic 3-cell displays large oscillations of intracellular free [Ca] is critical for retinal homeostasis. The retinal pigment epithelium (RPE), an epithelial Ca2+ ([Ca2+]i). To control [Ca2+]j, the P-cell must be equipped with potent mechanisms for Ca2' monolayer situated between the sensory retina and its choroidal blood supply, regulates ionic extrusion. We studied the expression of the plasma membrane Ca2+-ATPases (PMCA) in 3 insulin composition of the SRS. The plasma membrane CaATPase (PMCA), present in the RPE, is secreting preparations (a pure P-cell preparation, RINmSF cells and pancreatic islet using postulated to function in transepithelial Ca transport, and hence in regulation of SRS Ca content. cells), The work was reverse-transcribed PCR, RNase protection assay and Westem blotting. The 4 main isoforms, present designed to determine expression and subcellular localization of the four PMCAI, PMCA2, PMCA3 and PMCA4 were in the 3 Six known PMCA isoforms (termed PMCA I - 4) in human RPE. Gene expression was assessed by expressed preparations. alternatively RT-PCR and spliced mRNA transcripts, characterized at splice sites A, B and C were detected in the 3 protein expression by western blotting. Confocal immunofluorescent microscopy was used to preparations (rPMCAlxb, 2yb, 2wb, 3za, 3zc, 4xb), plus 2 additional transcripts in pancreatic islet determine subcellular localization. Significantly, transcripts of all four PMCA genes were detected in cells (PMCA4za, lxkb). The latter transcript corresponded to a novel variant of rat PMCA I gene human RPE. Similarly, at the protein level, all four isoforms were also detected. lacking the exon, coding for the 1Od' transmembrane segment, at splice site B. At the mRNA level, By immunofluorescence, PMCA4 reactivity was predominantly localized to the apical cell 5 transcripts predominated (lxb, 2wb, 3za, 3zc, 4xb), although all transcripts could be detected at surface. PMCA1, by contrast, was detected basally. PMCA2 and 3 gave less robust staining, and much ofthe observed was the protein level. Hence, the pancreatic 3-cell is equipped with multiple PMCA isoforms with signal cytoplasmic. (Supported by NIH RO1 EY- I 1308). possible differential regulation, providing a full range ofPMCAs for [Ca +]i regulation.

431 - Pos 432 - Pos THE RATE OF ACTIVATION BY CALMODULIN OF ISOFORM 4 OF PLASMA HETEROLOGOUS EXPRESSION AND CHARACTERIZATION OF THE MEMBRANE CALCIUM PUMP IS SLOW AND IS CHANGED BY ALTERNATE DEAFWADDLER MUTANT OF THE RAT PLASMA MEMBRANE CALCIUM ATPASE SPLICING 2B. Adelalda G. Filoteol, Ariel J Caridel, Nancy L Elwess2, Anil K. Verm'a, Agnes Enyedi3, Bajzer Alan R. Penheiter, Adelaida G. Filoteo, Cynthia L. Croy, John T. Penniston, Mayo Clinic, 200 Zeljko', John T. Penniston', 'Mayo Clinic, 200 1st St SW, Rochester, MN 55905, 2Plattsburg State First St. SW, Rochester, MN 55905 University, 3National Institute ofHaematology and Immunology, Hungary The deafivaddler mutant in mouse was the flirst spontaneous mutant found in the plasma We measured the ATPase activity of specific isofornms of the plasma membrane Ca2+ pump membrane calcium ATPase [Street V.A. et al. (1998) Nature Genet. 19:390-394]. A nucleotide continuously, and the effects ofadding or removing calmodulin in a reconstituted system. The rate substitution in deafwaddler results in an Ala to of Gly transition at amino acid 283 in the small activation by calmodulin of isoform 4b was found to be very slow, with a half-time (at 235 nM cytoplasmic of PMCA 2. Mice that are for are deaf and calmodulin and 0.5 loop homozygous deafivaddler have poor tsM free Ca2+) of about one minute. The rate of inactivation of isoform 4b balance. However, the balance and disorders ofthe mice are less severe when calmodulin was removed hearing deafivaddler than was even slower, with a half-time ofabout 20 minutes. Isoform 4a homozygotes for a functionally null frameshift mutant or homozygous PMCA 2 knockout mice. has a lower apparent affinity for calmodulin than 4b, but its activation rate was surprisingly faster, This difference in indicated that the (half phenotypic severity deafwaddler pump retains partial time about 20 seconds). This was coupled with a much faster inactivation rate, consistent biological PMCA 2b was constructed in the vector with its low affinity. A activity. deafwaddler expression pMM2, truncated mutant of isoform 4b also had a more rapid activation rate, transiently expressed in COS cells, and membrane preparations were assayed for calcium transport indicating that the downstream inhibitory region of full-length 4b contributed to its slow The retained activation. The activity. deafivaddler pump 30% of the specific activity with 21.5 pM Ca2' and was results indicate that the slow activation is due to occlusion of the calmodulin- stimulated 1.5 fold 544nM in the same binding domain of4b, caused by calmodulin, manner as the wildtype. Although calcium by its strong interaction with the catalytic core. Since the activation transport activity was reduced in the deafwaddler total from of 4b occurs on a time scale comparable to Ca2+ spikes, this pump, phosphoenzyme formation phenomenon is functionally ATP (E1P + E2P) was slightly higher for deaJ;vaddler than the wildtype. Fifty pM LaCI3 which important. (NIH Grants GM 28835(JTP) and GM55514, HAS Grant OTKA T023659(AE) and increased the HHMI(AE and steady state level of phosphoenzyme 3.2 fold for the wildtype, had no effect on the JTP)). deafivaddler. Supported by NIH Grants GM28835 and GM55514. Reference Street V.A., McKee-Johnson J.W., Fonseca R.C., Tempel B.L., Noben-Trauth K. (1998) Mutations in a plasma membrane Ca 24-ATPase gene cause deafness in deafwaddler mice. Nature Genet. 19:390-394.

433 - Pos 434 - Pos KINETICS OF Ca2+, H+ AND Mg2+ INTERACTION WITH THE ION-BINDING SITES REPLACEMENT OF SERCA2A WITH THE NON-MUSCLE SERCA2B ISOFORM IN OF THE SR Ca-ATPase. TRANSGENIC MICE. Christine Peinelt, Hans-Juergen ApeU, University ofKonstanz Mark Ver Heyen', Bonaventure Awede2, Jean Lebacq2, Peter Carmeliet3, Mieke Dewerchin3, Electrochromic styryl dyes were successfully applied to investigate charge-translocating reaction Stephane Heymans3, Desire Collen3, Thomas Reed4, Ludo Van Den Bosch', Frank Wuytack', steps in the SR Ca-ATPase, which has a transport stoichiometry of 2/2/1 for Ca/HlATP. The 'Physiology KULeuven, Belgium, Herestraat 49, Leuven, 3000 Belgium, 2Physiology, UCL, mainly contributing reaction steps were ion binding and release of both transported ions. Belgium, 3Center for Transgene Technology and Gene Therapy, KULeuven, Belgium, Therefore, we investigated the mutually antagonistic effects ofCa2' and H+ on binding ofthe other 4Department ofMedicine, Division ofCardiology, University ofCincinnati ion in the El and P-E2 state of the pump. On the cytoplasmic side of the protein we found strictly Transgenic (TR) mice were made carrying mutations in the 3' end of the SERCA2 gene that competitive binding with the sequence, For Ca2+ ions the a H2E,<=>HEI<=>E,<=>CaE,<=>Ca2E,. prevent regulated splicing which controls the formation of the muscle-specific SERCA2a experimentally one-step reaction was detected with a Hill coefficient of 2. The equilibrium isoform. These mice will therefore in cardiac dissociation constants = = express and slow-twitch skeletal muscle the non- were in the order ofK,,2(Ca2) 1 .5pM, K,,2(H) InM (pK 9) and Kl2(H2) muscle SERCA2b isoform instead of SERCA2a. The = 2.5isM (pK 5.6). It was found that Mg2+ ions were also able to enter the binding transgenic mice were viable, appeared sites healthy, and exhibited no outward manifestations of a disease phenotype. We could however (electrogenically) and competed with the transported substrate ions (K10(Mg) = 0.5mM). In the P- observe a reduced spontaneous nocturnal activity of the TR mice. Transgenic mice showed a E2 state, in which the binding sites are facing the luminal side of the sarcoplasmatic reticulum, the reduced SERCA2 mRNA level measured concentration dependence of in the heart and soleus muscle (40-30% of WT), whereas no Ca2+ and H+ binding could be described satisfactorily only difference was detected for the with a branched reaction scheme in which besides the state P-E2Ca also a mixed state, phospholamban (PLB) mRNA levels. The overall SERCA2 protein P-E2CaH2, levels in the heart and soleus are 45% of WT. The PLB protein level is increased in TR hearts exists. Equilibrium dissociation constants could be determined which allowed a consistent (216% of maximum simulation ofall observed antagonistic concentration dependences. WT). The velocity of the oxalate-supported Ca2' uptake on total heart homogenate was reduced by 44% in TR hearts, while the Ko 5 was shifted from 0.28 AM Ca2' for WT to 0.19 PM Ca2+ in TR hearts. Mechanical measurements of skeletal muscle revealed a significant increase in the half relaxation time of transgenic soleus (slow-twitch) muscle whereas no difference was detected for EDL (fast-twitch). The tension developed by soleus or EDL muscle and its maximal rate oftension rise were not modified in transgenic mice.

74A SUNDAY ION MOTIVE ATPASES 435-440

435- Pos 436 - Pos TWO Ca2+ DEPENDENT TRYPTIC CLEAVAGE SITES IN THE CYTOPLASMIC EVIDENCE FOR A DIRECT FUNCTIONAL INTERACTION BETWEEN LEUCINE DOMAIN OF SCALLOP Ca ATPase. ZIPPER RESIDUES OF PHOSPHOLAMBAN AND THE CA-PUMP OF CARDIAC C. Ryan, M. Chen, Peter M.D. Hardwlcke, Dept. of Biochemistry & Molecular Biology, SARCOPLASMIC RETICULUMV Southern Illinois Univ., Carbondale, Illinois 62901-4413 Razvan L Cornea, Joseph M. Autry, Zhenhui Chen, Larry R. Jones, Indiana University School Stabilization of the El form of the Ca-ATPase of the scallop sarcoplasmic reticulum by binding of ofMedicine, 1 11 1 W. 10th St., Indianapolis, IN 46202 Phospholamban (PLB), a small phosphoprotein inhibitor of the cardiac (SERCA2a), Ca2+ to its high affinity sites led not only to exposure of the bond between 15 and Ca-pump ArgI97-Ala198 shows a distinct oligomeric distnbution between monomers and homopentamers that are stabilized 06 in the small cytoplasmic domain (T2 site in L23 loop) to trypsin, as is the case when rabbit through an intramembrane Leu/Ile zipper. Previous studies using Ala-scanning mutagenesis have SERCAla, but in addition caused refolding of the large cytoplasmic domain. Thus, the suggested that residues responsible for pentamer formation are on one face of the PLB conformation of the Elab(iii)c region in the nucleotide binding domain in the large cytoplasmic transmembrane a-helix and cannot participate in SERCA2a inhibition, whereas residues on the domain (L45 ) was altered, as judged by greater accessibility of the Lys582-Phe583 peptide bond opposite face are required for SERCA2a inhibition. We have tested this dual face hypothesis by using mutations to Leu, Ile, Cys, and Phe at the Leu/Ile zipper sites, in addition to Ala. between Na3 and Np4 (T3 site) to trypsin in the El as compared to the E2 state of the enzyme. Several pentamer-enabling isomeric (i.e., L371) or Cys (i.e., L37C) mutations were identified that actually This is associated with formation of an -37 kDa polypeptide. A second site in L45, at Lys727- increased inhibitory potency of PLB relative to the pentamer-disabling L37A. In contrast, the completely pentamer-disabling mutation L37F reduced inhibitory potency relative to L37A. These Ser728 halfway along the second putative a-helical segment of the hinge domain (Ha2) (T4 site), results further support the hypothesis that the transmembrane domain of PLB is essential for was also found to be more exposed in the El state. Proteolysis at that site produced a 25-23 kDa inhibition. However, in contrast to previous suggestions, the leucine/isoleucine zipper region of doublet on SDS gels. Cleavage at the T2 site in the El state took place at a rate comparable to the PLB is not inert, but instead appears to interact with and directly inhibit SERCA2a. Our results in primary cleavage at Lys504-Val505, and an 88 kDa fragment was observed. Differences in combination with previous studies suggest a model in which the PLB transmembrane domain is accessibilty of the tryptic cut sites between the El and E2 states probably reflect alterations in engulfed by SERCA2a, maintaining multiple essential contact sites with SERCA2a around the tertiary as opposed to quaternary structure, since digestion in the E2 state, when the scallop entire circumference ofthe PLB transmembrane helix. enzyme is organized into ribbons of dimers, yields the same products as those produced from the E2-P form of the enzyme, when the Ca-ATPase is not dimeric. Protection of the three Ca2+- sensitive sites in the E2 form of the enzyme may be due to both internal refolding within the two cytoplasmic domains and repositioning of the domains with respect to one another.

437 - Pos 438 - Pos STRUCTURAL DYNAMICS OF THE CYTOPLASMIC DOMAIN OF STRUCTURAL DYNAMICS OF THE TRANSMEMBRANE DOMAIN OF PHOSPHOLAMBAN BY SITE-DIRECTED SPIN LABELING PHOSPHOLAMBAN BY SITE-DIRECTED SPIN LABELING Tara L. Kirby, John D Stamm, David D. Thomas, University of Minnesota, Minneapolis, MN John D Stamm, Tars L. Kirby, David D. Thomas, University of Minnesota, Minneapolis, MN 55455 55455 We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to test and We have used electron parsmagnetic resonance (EPR) spectroscopy to elucidate the interactions refine models for the structure and dynamics of phospholamban (PLB), a 52-amino-acid between the transmembrane (TM) domains of the Ca-ATPase and its regulatory protein, membrane protein that regulates calcium levels in cardiac muscle by inhibiting the sarcoplasmic phospholamban (PLB). PLB consists of 52 amino acids, approximately halfof which are thought reticulum Ca-ATPase (SERCA). This inhibition is relieved upon phosphorylation of PLB. It has to span the membrane in a single a-helix. It has been proposed that functional regulation involves been proposed that the cytoplasmic domain functions as a switch between the inhibitory and physical interactions of both the cytoplasmic and transmembrane domains of PLB with the Ca- noninhibitory forms of PLB, determined by its phosphorylation state. To investigate this ATPase. However, the site and nature of the interactions of PLB's transmembrane domain is hypothesis, we have prepared mutants of PLB purified from E. coli, each containing a single Cys poorly understood. Mutants of PLB, each containing a single cysteine residue in the residue in the cytoplasmic domain of PLB. E. coli-expressed wild-type PLB is structurally and transmembrane domain, were purified from E. coli for spin-labeling and co-reconstitution with the functionally similar to insect cell-expressed wild-type PLB, as shown by SDS-PAGE, EPR Ca-ATPase in lipid bilayers. EPR spectroscopy was then used to determine the mobility and spectroscopy, and inhibition of ATPase activity. We attached a Cys-specific nitroxide spin label accessibility of each spin-labeled site in PLB, as a function of the presence of the Ca-ATPase. to each single-Cys mutant and performed EPR spectroscopy to determine protein structural Data were analyzed to determine the sites on the TM domain of PLB that interact with the Ca- features and changes during functional regulation of the Ca-ATPase by PLB. ATPase.

439 - Pos 440 - Pos OLIGOMERIC STRUCTURE DOES NOT INFLUENCE THE EXTENT OF HOW DOES PHOSPHOLAMBAN DECREASE SR Ca CONTENT? INTERACTIONS BETWEEN PHOSPHOLAMBAN AND THE Ca-ATPase FROM Thomas R. Shannon, E. G. Kranias, D. M. Bers., University of Cincinnati, Cincinnati, OH and SARCOPLASMIC RETICULUM. Loyola University-Chicago, Maywood, IL. W. Christine B. Cecilia Gregory Hunter, Karim, Sheen, George Barany, David D. Thomas, The SR Ca content ([Ca]sRT) depends upon the SR Ca influx and efflux rate through the SR Ca University of Minnesota, MN 55455 Minneapolis, pump (Shannon, et al., BJ, in press. Phospholamban (PLB) phosphorylation in response to agents We have used fluorescence energy transfer (FET) to correlate interactions between such as the 0-adrenergics relieves PLB inhibition ofthe SR Ca pump rate. We have examined the phospholamban (PLB) and sarcoplasmic reticulum (SR) Ca-ATPase, and within PLB oligomers. effect of PLB upon [Ca]sRT. Membrane vesicles from PLB knockout (KO) and wild-type (WT) Our principal goal is to test the hypothesis that the extent of association of PLB with the Ca- mice were made. The time course of thapsigargin-sensitive ATP-dependent 45Ca influx into and ATPase is influenced PLB's structure and by oligomeric phosphorylation state. Donor fluorescent efflux out of the membrane vesicles was measured at 100 nM [Ca];. No oxalate was present. were attached the domain probes to cytoplasmic of PLB and non-fluorescent acceptor moieties Ruthenium red and oligomycin were added to inhibit mitochondrial Ca uptake, and digitonin was were attached to the cytoplasmic domain of the Ca-ATPase in co-reconstituted vesicles. FET present to prevent SL Ca uptake. The SR Ca leak rate is relatively small (Bassani and Bers, BJ measurements were performed using pentameric wild-type PLB and synthetic analogs of PLB that 68:2015, 1995; Shannon, et al. BJ, in press) and is unaffected by PLB (Shannon and Bers, BJ had transmembrane cysteine substitutions which resulted in destabilization of pentameric 76:A297, 1999). Therefore the efflux from the SR is primarily due to backflux through the SR Ca structure. ATPase measurements in activity PLB/Ca-ATPase co-reconstituted vesicles were pump. When compared to KO mice, we found that PLB in WT mice decreased the initial rate of performed to correlate function of each PLB with inhibitory analog its association with the Ca- SR Ca uptake (0.11 vs.0.39 nmol/mg/s) and decreased steady-state SR 45Ca content (0.9 vs. 5.8 ATPase. results indicate that PLB is Preliminary closely associated with the ATPase regardless of nmol/mg). Furthermore, when measured at the same [Ca]sRT in both mice, the rate of Ca efflux in phosphorylation state, oligomerization, or binding of antibodies specific for the cytoplasmic WT was increased (0.088 vs. 0.040 nmol/mg/s at 0.9 mnmo/mg). We conclude that the decreased domain of PLB. These results that PLB suggest oligomerization states do not reflect levels of [Ca]sRT in WT mice is caused by decreased SR Ca influx rate, unaltered leak rate and an increased association with the Ca-ATPase. The of these observations on implications the oligomerization rate of backflux. PLB therefore decreases the Ca gradient that the SR Ca pump can generate and model of PLB mechanism will be discussed. hence its energetic efficiency.

75A 441-443 ION MOTIVE ATPASES SUNDAY

441 - Pos 442- Pos THE SMF FAMILY OF METAL-ION TRANSPORTERS-GLIMPSE INTO THEIR MOLECULAR MODELLING AND SIMULATION STUDIES ON SUBUNIT C FROM MECHANISM OF ACTION F*F, ATPASE Nathan Nelson, A. Sacher, Department of Biochemistry, Tel Aviv University, Ramat Aviv, 69978 Lucy R Forrest', Georg Groth2, Mark S P Sansom', 'University of Oxford, South Parks Road, Tel Aviv Oxford, OXI 3QU United Kingdom, 2Heinrich-Heine Oniveraitlt, Dusseldorf Transition metals are essential for many metabolic processes and their homeostasis is crucial for life F-ATPase from E. coli catalyses the formation ofATP at the expense of an electrochemical proton processes. Aberrations in the cellular metal-ion concentrations may lead to cell death and severe gradient. The catalytic Ft domain is extramembranous and its structure is known to atomic diseases. Metal-ion transporters play a major role in maintaining the correct concentrations of the resolution. The proton-translocating membrane integrated domain, Fo, has subunit stoichiometry various metal-ions in the different cellular compartments. Recent studies of yeast mutants revealed ab2cg 12. The structure of subunit c has been determined in chloroform:methanol:water by NMR key elements in metal-ion homeostasis including novel transport systems. The studies of yeast (Girvin M.E. et al. 1998, Biochemistry 37: 8817-8824), and has been shown to consist of two metal-ion transporters helped to unravel the molecular mechanism of macrophage defense against helical transmembrane domains connected by a short loop. Molecular dynamics simulations of the bacterial infection and hereditary diseases. It was demonstrated that DCTI cotransports Fe2+ monomeric subunit in different solvents have been used to assess the suitability of the NMR together with H+ with a stoichiometry of one. Replacing Na+ by choline and Clr by NO3. or SCN- structure for simulation and modelling studies. Simulations in methanol and in water give some had no effect on the Fe2+ transport. DCTI expressed in Xenopus oocytes induces a proton leak at insight into potentially functionally important structural changes. Significantly, the structure low pH and under certain conditions may operate as a H' uniporter. The yeast Smflp, expressed in shows minimal conformational changes in a phospholipid bilayer (POPE) environment. Several Xenopus oocytes, exhibited quite similar properties to DCTI. The most striking difference was models of the oligomeric state of subunit c have been proposed previously. These place the observed for the uncoupled leak that was specific for Na' and not H+. Oocyte expressed DCTI subunits so that the helices form two concentric rings around a central pore. However, uncertainty exhibits large presteady-state currents but only at positive potentials. Addition ofmetal-ion results in remains as to which helix forms the outer ring of the functional protein. Furthermore, the number the disappearance of the presteady-state current either by preventing the inward proton movements of subunits remains an issue. In vacuo simulations have been used to explore these factors and or under competent transport conditions by releasing the proton and the metal-ion from the models have been assessed according to the experimental evidence. This model has been fitrther membrane to the cytoplasm. Recently we observed that substituting Cl in the transport solution with refined by MD simulations in a phospholipid bilayer. gluconate or isethionate results in the appearance of large presteady-state currents not only at positive but also negative applied potential. Addition of metal-ions prevented the presteady-state currents but no steady-state current could be observed. Therefore, the metal-ion transport is dependent on the presence of Cl- or other small unions such as NO3. or SCN but not SO4. We propose that the metal ion is cotransported with Cl- and the steady-state current results from the transport of positive charges of H+. The kinetic experiments with Xenopus oocytes expressed DCTI and Smflp together with the available yeast mutants provide a solid foundation for a detailed study ofthe mechanismof metal-ion transport across membranes.

443 - Pos PHOTOPHOSPHORYLATION IN ALKALOPHILIC HALOBACTERIAL CELLS CONTAINING HALORHODOPSIN Armine Vregi Avetisyan, Andrey D. Kaulen, Vladimir P. Skulachev, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State Univetsity, Moscow, 119899 Russian Federation Light-driven ATP synthesis was observed in halorhodopsin-containing cells of alkalophilic bacterium Natronobacterium pharaonis, providing that respiration is suppressed. No decrease of photophosphorylation was detected as long as the light was on (at least for 90 min). The ATP synthesis was stopped by switching off the light and inhibited by FCCP, a protonophore uncoupler, or by TPT, a CrlOH antiporter. It was shown that the rate of ATP hydrolysis depends on chloride concentration. The ATP hydrolysis was not detected in sulfate-containing medium, but was restored by addition of 100 mM NaCl. DCCD inhibits both photophosphorylation and ATP hydrolysis. Photophosporylation was also observed in bacteriorhodopsin - depleted mutant strain of Halobacterium salinarium containing halorhodopsin from Natronobacterium pharaonis. Comparison of the data obtained with N.pharaonis and H.salinarium cells containing H+-ATPase revealed that ATP-synthase from N.pharaonis is proton-independent. We suggest that the electrochemical potential of the chloride ions formed in Natronobacterium pharaonis by the halorhodopsin is utilized by Cl-ATP-synthase.

444-445 NA, K-ATPASES SUNDAY

444 - Pos 445- Pos AN IN VITRO APPROACH TO THE ASSEMBLY OF THE NA,K-ATPASE SUBUNITS. TRAFFICKING OF Na,K-ATPase IN INSECT CELLS: FUNCTIONAL ACTIVITY IN Long Ha Huynh, Douglas M. Fambrough, Johns Hopkins University, Biology, 3400 N. Charles ISOLATED MEMBRANE FRACTIONS. Street, Baltimore, MD 21218 Craig Gattol, Scott M. McLoud2, Jack H. Kaplan2, 'Illinois State Univ., Dept. of Biol Sci., The Na,K-ATPase is a heterodimer consisting of an a- and a n-subunit whose assembly is critical Normal, IL 61790, 2Dept. ofBiochem. & Mol. Biol., Oregon Health Sci. Univ. for the targeting of the functional enzyme to the plasma membrane. Immunoprecipitation and The Na,K-ATPase, is an alpha-beta heterodimer, with ten putative transmembrane segments in the yeast two-hybrid studies have shown that the extracellular loop between transmembrane helices 7 a-subunit and one such segment in the P-subunit. It is the plasma membrane enzyme responsible and 8 of the a-subunit (aEC59) and the ectodomain of the P-subunit (ecto-,B) are involved in the for the active transport ofNa and K and exists in nearly all eukaryotic cells. However, it has been assembly of the two subunits. We designed yeast two-hybrid screens to examine this subunit shown that some insect cells (e.g. Sf9 and Hi5 cells) have no (or extremely low) Na,K-ATPase. interaction more critically. From the libraries, we isolated mutants of aEC59 that show no We expressed sheep kidney Na,K-ATPase in Hi5 cells via the Bac-to-Bac bacuolovirus system interaction with the ecto-P and a minimal region in ecto-I that is still capable of interacting with (GibcoBRL). For expression, log phase Hi5 cells are infected with viral stocks (MOI = 10-15) wild-type a. Both a- and f-subunits exist as multiple isoforms that can assemble in various and harvested 3-4 days post-infection. Cells are collected, disrupted (via dounce homogenization), combinations. We hypothesize that the level of expression of these isozymes on the cell surface is and cell debris removed by centrifugation. The endoplasmic reticulum, golgi apparatus, and dependent, in part, upon the strength of interactions of the subunit isoforms. To approach this plasma membrane fractions are then separated on a 5-step sucrose gradient (0.8-2.OM sucrose). issue, we have developed an in vitro binding assay. For this assay, aEC59 is purified as a Characteristics of Na pump function (i.e. ATPase activity, [3H]ouabain binding, turnover number, glutathione S-transferase fusion protein on glutathione beads, and ecto-J is secreted by mouse L and K+ dependence) were measured for each fraction and found not to differ between the various cells into the culture medium. In 10/o dimethyl sulfoxide and PBS (pH 7.4), specific a-S membrane pools. Furthermore, quantitative electroblot analysis suggests that all of the Na,K- ATPase which is in the plasma membrane is fully functional and thus measurements from this interaction occurs. We are currently characterizing the assembly of the aEC59 mutants as well as fraction are not complicated by the presence of non-functional heterologously expressed enzyme. the minimal 3-subunit region with their respective wild-type partners. We are also determining the We thank Dr. Elmer Price (Univ. of Missouri) for sheep Na,K-ATPase cDNA. Supported by NIH binding affinity of the two subunits under these conditions. This in vitro binding assay will allow grant HL303 15 to J.H.K. and NIH grant HL09972 to C.G. us to measure the binding constants for all combinations of a-S isoforms and address the question of isozyme expression in the cells.

76A SUNDAY NA, K-ATPASES 446-451

446 - Pos 447 - Pos INTRAMEMBRANE MOIETY OF NA, K-ATPASE: SPATIAL STRUCTURE PROBING NEW ASPECTS OF NA/K-ATPASE; ACID LABILE ATP AND/OR ADP/PI BINDING TO BY ZERO-LENGTH CROSS-LINKING. THE TETRAPROTOMER. Alexander V. Ivanov, H.Zhao, N. N. Modyanov, Department of Pharmacology, Medical College K. Taniguchi', S. Kayal, T. Yokoyama', K. Abe', T. Katoh', M. Yazawa', Y. Hayashi2, S. Mardh3, of Ohio, Toledo, Ohio 43614-5804 'Hokkaido University, N-10, W-8, Sapporo, Hokkaido 060-0810 Japan, 2Kyourin University, Oxidative zero4ength cross-linking with Cu-o-phenantroline was used to obtain information on 3Linkoping University spatial arrangement of membrane helixes of sodium pump. Previous studies on native and The mechanism of Nae and K' dependent ATP hydrolysis has been extensively studied. The digitonin solubilized 19 kDa membrane preparation of sodium pump revealed two major cross- hydrolysis appears to occur, via the Na+-Enzyme-ATP complex (NaElATP), ADP sensitive linking products: 33 kDa (between N and C-termini ofa-subunit) and -80 kDa product (consists phosphoenzyme (NaEIP), the K' sensitive phosphoenzyme (E2P) and the K' occluded enzyme of 13-subunit TM and of in a or which TM7-1O a-subunit) (Sarvazyan et aL. JBC, 270, 26528-32, 1995 and 272, (KE2) protomer oligomers consists ofax- and 1- chains. We found that the enzyme 7855-8, 1997). Solubilized preparation yields more of high MW product and native 19 kDa bound I and 0.5 molof 32P/mol a -chain in the presence [a-32P]ATP and [y-32P]ATP, respectively, membranes yield more of 33 kDa product. Above products and 19 kDa fragment were isolated, accompanied by a maximum accumulation of 0.5 mol of NaEIP followed by a slow steady treated with CNBr and resulting peptides were analyzed. Results revealed two disulfide bridges liberation of Pi and E2P accompanied with a rapid 0.25 mol of acid labile Pi burst. The enzyme (TM8 ofa-subunit - TM of 1-subunit and TM9-TMIO) in high MW product Studies of 33 kDa occluded a maximum of 2 mol of Rb (K congener)/mol ofa-chain. The addition of Nae + Mg2 cross-link resulted in discovery of multiple disulfides (one between N- and C-termini ofa-subunit completely induced deocclusion. Further addition of [c-32P]ATP and [y-32P]ATP, respectively, and one (or two) within TM7-10. Experiments on 19 kDa fragment (TM7-10) showed the induced 0.5 mol of 32P binding to occlude mol of "Rb+/mol a-chain accompanied by a rapid 0.5 presence few disulfide bonds as well. We suggest that TM8 segment plays an essential role as a mol of Pi burst. Electron microscopy of Na/K-ATPase preparations indicated the presence of bridge between 5-subunit and the N-terminal domain of a-subunit. A new model of tetraprotomeric structures (ap)4. These data and others suggest that Na/K-ATP hydrolysis occurs transmembrane arrangement of sodium pump is proposed. (Supported by NIH grants HL-36573 via four parallel paths accompanying conformational changes, the sequential appearance of and GM-54997). (NaEIP:E'ATP NaElP:E.ATP), (E2P:E.ATP : E2P:EADP/Pi) and (KE2:E.ADP/Pi KE2:E.ADP/Pi), each of which has been previously referred to as NaEIP, E2P and KE2.

448 - Pos 449- Pos ERYTHROSIN ISOTHIOCYANATE, AS WELL AS FITC, CAN MODIFY ALL cX1 FRET EVIDENCE FOR TWO ATP SITES ON THE SODIUM PUMP cc-SUBUNIT PROTOMERS OF MEMBRANE-BOUND Na,K-ATPase Larry D. Faller', V. N. Kasho', I. N. Smirnova', S-H. Lin2, R. A. Farley3, 'UCLANAMC WLA, D. Owen, M. Taylor, T.J.H. Walton, Josh D. Cavieres, University of Leicester 11301 Wilshire Blvd., Los Angeles, CA 90073, 2University of Texas, Galveston, 3USC School of Fluorescein and its derivatives have been used as nucleotide site ligands. At pH 9.2, FITC Medicine inactivates high-affinity but not low-affinity ATP reactions of Na,K-ATPase, despite a random Evidence for two nucleotide sites on a single a-subunit of Na,K-ATPase has been obtained by modification of ct subunits (Ward & Cavieres (1998) J.B.C. 273, 14277). Conversely, erythrosin combining fluorescence resonance energy transfer (FRET) with stopped-flow (SF) measurements isothiocyanate (ErITC) prevents the activation of SR Ca,Mg-ATPase by millimolar ATP but does to estimate the maximum distance between fluorescein covalently attached to lysine-501 with not affect phosphorylation by, or turnover at, micromolar ATP (Huang, Negash & Squier (1998) fluorescein 5-isothiocyanate (FITC) and cobalt in simultaneously bound tetrammine complexes Biochemistry 37, 6949). These findings suggest the presence of two ATP sites per a subunit of P- with ADP or ATP. The kinetics of the reaction and differences between the distance estimates in type ATPases. Contradictory evidence maintains that, at pH 7.25, only 0.5 mol each of FITC and apoenzyne and holoenzyme provide evidence for "induced fit" following fornation of the ErITC can be specifically incorporated per at subunit of Na,K-ATPase, with a long distance collision complex. The estimated distances are all shorter than the minor axis of the asyssuetric between probes (Linnertz et al. (1998), J.B.C. 273, 28813). We have inactivated Na,K-ATPase structure identified as the a-subunit in image re-constructions of two-dimensional crystals of with either FITC or ErITC, at both pH 9.2 and 7.25, and solubilised to apc protomers with C,2Es. Na,K-ATPase. Fluorescein and Co(NH3)4ATP binding to the same site is unlikely because In all instances, we find that the fractional specific Na,K-ATPase activity ofthe soluble protomers predicted energy transfer between cobalt in the nucleotide analogue and fluorescein attached to (ratio to side-by-side controls) is no different from that with the parent membrane enzyme, i.e. cysteine-457 with 5Y-iodoacetamide (IAF) is not observed. Binding to different cc-subunits related there is no partial reactivation. This excludes the possibility that FITC or ErITC access only one by approximately 180' rotation is unlikely because energy transfer is also observed with half of a membrane and furthers the two-site a hypothetical (ap>2 dimer, hypothesis. Supported by solubilized enzyme. Therefore, the data are consistent with two nucleotide sites on a single cc- research grantfrom The Wellcome Trust. subunit of Na,K-ATPase. There is precedent for this interpretation in solved structures of other enzymes that catalyze phosphoryl group transfer and bind two nucleotids like adenylate kinase (AK) and adenylosuccinate synthetase (AMPSase). Supported by VA and NIH DK52802.

450- Pos 451 - Pos BETA SUBUNITS OF THE Na ,K+-ATPase IN XENOPUS LAEVIS. A NOVEL MUSCLE-SPECIFIC ISOFORM OF THE MAMMALIAN X,K-ATPASE Sandeep Sagar, Brett Pazdur, Robert F. Rakowski, Finch University ofHealth Sciences SUBUNIT The ,Bsubunit of the Na+,K+-ATPase was cloned from both adult Xenopus laevis brain and oocytes N. B. Pestov', G. Adams', M B. Kostina2, M. I. Shakhparonov2, Nikolai N. Modyanov', using 3' and 5' RACE reactions. The cDNA sequence from oocytes is nearly identical to the 'Department of Pharmacology, Medical College ofOhio, Toledo, Ohio, 2Shemyakin-Ovchinnikov published Xenopus A3 subunit from embryonic nervous tissue. Three sequence differences were Institute ofBioorganic Chemistry, Moscow, Russia found in the ectodomain of the oocyte protein, T99P, N206S and S241L. The 13 subunit from We have identified the fifthmember of the mammalian X,K-ATPase P-subunit gene family. The adult brain differs markedly from the embryonic P3 subunit. We have amplified and subcloned the new human gene contains 8 exons and is located on X chromosome. The human, rat, and pig coding regions of both 03 subunits into the PGEMHE vector which contains the 5' and 3' cDNAs have been cloned and sequenced. Two alternative splice forms of each cDNA, one of untranslated regions of the Xenopus f1-globin gene. We are currently using a two microelectrode them lacking 12 bp, were identified. All these genes are largely expressed in skeletal muscle and voltage clamp to study the effect of the sequence differences on the kinetics of pump current ata lower level in heart. The deduced long variants ofhuman, rat, and pig proteins designated as activation by external cations. The oocyte 13 subunit has also been subcloned into the pET-2 1(a) O3m (Pmuscle), consist of357, 356, and 355 amino acid residues, respectively, and exhibit 89-92 % vector for overexpression and purification of the protein. The oocyte 3' RACE reaction product identity. The tetrapeptides which are absent in the short protein variants, are located in the produced three bands on an agrose gel which when sequenced suggests three different AATAAA junction between cytoplasmic and membrane domains. The sequence homology of pm proteins polyadenylation sites. The 5' untranslated region (UTR) of the oocyte 13 gene also differs from the with known Na,K- and H,K-ATPase 1-subunits are 30%-39%. Unlike other f1-subunits, putative embyronic gene. The 5' and 3' UTR of the P3 subunit in oocytes may play a role in differential 13m proteins have large N-terminal cytoplasmic domains containing long Glu-rich sequences development. Supported by NIH grant NS-22979. which, according to a secondary structure prediction, formtwo long a-helices. The data obtained indicate the existence of hitherto unknown X,K-ATPase (most probably Na,K-ATPase) isozymes in muscle cells. (Supported by NIH grants HL-36573 and GM-54997).

77A 452-457 SUNDAY NA, K-ATPASES SUNDAY

Pos 453 - Pos y OF NA,K-ATPase THE NATURE OF THE CONDUCTANCE INDUCED BY THE HUMAN NA,K-ATPase Bernhard', Alla Shainskaya2, Mann', Karlish2, 'Odense Univ., Odense, GAMMA SUBUNIT IN XENOPUS OOCYTES Denmark, 2Weizmann Inst. of Science, Rehovot, Israel Qun Sha, Robert W Mercer, Colin G Nichols, Washington University School of Medicine, 660 S. Euclid Ave, Box 8228, St.Louis, MO 63110

y protein single trans-membrane segment , expressed primarily in The y subunit is a specific component of the plasmalemmal Na.K-ATPase. Like structurally kidney, specific regulator Na,K-ATPase. SDS-PAGE, the y subunit related single membrane-spanning proteins such as cardiac phospholemman, Mat-8, and renal (Mr 8, 9kDa). spectrometric analysis has been performed on the two y CHIF, large ion conductances are activated when human y-subunits are expressed in Xenopus Na,K-ATPase whether there are post-translational oocytes. We here report critical properties of the y-activated conductance, including non-selective sequences. purification and development of cationic and anionic permeation, extremely slow kinetics, and inhibition by millimolar in-gel tryptic digestion, determination on the extracellular divalent ions like Ba2+ and Ca2+. Using 3H-labeled 2-Deoxyglucose (MW-180) or y chains, peptide mapping peptide sequencing of tryptic fragments reveals: (I) yl inulin (MW-5000) as tracers, it is clear that quite large molecules, as well as [3H]spermidine, can (carbamidomethyl cysteine) corresponds exactly published efflux through the y-activated conductance pathway. Moreover, similar conductances, permeable sequence Thus, yl modified; (2) y2 has mass 7353.8 Da, to large molecules are activated by extreme hyperoplarization (>-150 mV) of uninjected oocytes. sequence N-terminally acetylated. Seven N-terminal We conclude that the human Na,K-ATPase y subunits activate Ca2+- and voltage-gated, non- yl, TELSNAH, replaced by Ac-MDRWYI/L y2, but otherwise the chains are selective, large diameter pores that are intrinsically present within the oocyte membrane. identical. appear splice variants search

(Sweadner, (1999). Na,K-ATPase, August 17-23, Sapporo,

Japan).

Pos 455 - Pos EVIDENCE ISOFORM-SPECIFIC REGULATION OF THE P-ADRENERGIC STIMULATION OF Na+/K+PUMP ACTIVITY IN RAT AND GUINEA Na+-K* PIG CARDIAC MYOCYTES. Kerrie Buhagiar, Hansen, Bewick, Helge H Rasmussen, Royal North Shore Joseph R. Stimers, Stephanie L. Hastings, Maxim Dobretsov, Univ Arkansas for Medical Hospital, Highway, Leonards, 2065 Sciences, 4301 W. Markham St., #611, Little Rock, AR 72205 previously rabbits with the angiotensin converting enzyme The.p1-adrenoreceptor agonist, isoproterenol (ISO), was reported to strongly stimulate Na+/K+ inhibitor, captopril (cap), Na+-K+ pump current (I,). This can be pump current (IQ)in rat cardiac myocytes and has weak negative or positive effects (depending on by reexposure angiotensin (All) in vitro. Since protein kinase C (PKC) regulates Ca++) on Ipin guinea pig myocytes. We previously demonstrated in rat myocytes that intracellular Na+-K+ pump, AII reported activate PKCe, we examined if PKCe was involved Cas' did not modulate the effect of ISO on Ii. This discrepancy may indicate a real species- signal transduction pathway linking All receptor to the Na+-K+ pump. We isolated dependent difference in organization of the adrenergic control of the heart or it may result from myocytes (con) rabbits treated with cap for 8 days. Myocytes were differences in protocols and experimental conditions in these studies. To resolve this question, voltage clamped MCI which contained 10 mM using patch pipettes Na+. Ip was effects of ISO on Ipin both rat and guinea pig ventricular myocytes was measured under identical holding by 100 AM 0.6 conditions with intracellular Ca:' buffered to <10 nM. ISO stimulated Ipby 52±9% in rat and by ouabain. cap statistically 18&+9%in guinea pig (range 0-50%). In guinea pig myocytes ISO also stimulated an inward (likely significant (p 0.05) Ip compared (see 0 chloride) current that was positively correlated and overlapping in time with Ipstimulation. The figure). Superfusion myocytes from cap-treated T correlation between these currents could be due to damage to intracellular signal transduction AII whole-cell configuration mechanisms during cell isolation, affecting both currents, or to a modulation of Ipby the ISO- significant I,. This v0.2- (6 () (8) (9) activated inward current. In conclusion, our results suggest that in both rat and guinea pig cardiac pipette myocytes 3-adrenergic stimulation activates the Na+/K' pump. Supported in part by NIH grant isoform-specific blocking peptide (pep) 00 HL44660. for binding of PKCe to This anchoring proteins (see figure). ca csp + cp + implicates regulation Na+-K+ pump by All AUI+pep All.

457- Pos CATALYTIC PROPERTIES OF THE al, a2 AND at3 ISOFORMS OF THE Na+/K+-ATPase AS A SIGNAL TRANSDUCER: PROTEIN TYROSINE NA,K-ATPASE. PHOSPHORYLATION IS AN EARLY EVENT IN THE LINKAGE OF Laura GROWTH- Segall', Daly', N. Boxenbaum', L. K. Lane2, R. Blostein', 'McGill University, 1650 RELATED GENES TO CARDIAC SODIUM PUMP. Avenue, Montreal, Quebec H3G IA4 Canada, 2Univ. Cincinnati Col. Med. Michael S. Haas, A. Askari, Z. Xie, Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614-5804 describes comparative study of the ca1, a2 and a3 catalytic isoforms of the Partial inhibition of Na/K-ATPase by ouabain stimulates hypertrophic growth of the rat neonatal Na,K-ATPase. Two systems were used: (1) HeLa cells expressing the three isoforns cardiac myocytes through the activation of the Ras/RaflMEK/MAPK cascade (Kometiani et al. individually described by Jewell & Lingrel (J. Biol. Chem. 266, 16925-30, 1991), with ax2 and JBC, 273, 15249, 1998). Since protein tyrosine phosphorylation is involved in Ras activation by a c3 relatively resistant to ouabain by mutagenesis and (2) native tissues, namely kidney number of other receptors, we wished to determine if increases in tyrosine phosphorylation also (cr11O1), axolemma ( 70%cra311), pineal gland (a312). Analysis of the kinetic properties precede ouabain-induced activation of the above cascade. Pre-incubation of myocytes with suggests that, compared to the ubiquitous alIisoform, a2 and a3 have reduced (2-3-fold) catalytic genistein, a tyrosine kinase inhibitor, repressed ouabain-induced activation ofp42/44 MPAK in a turnovers, V,,=/maximal phosphoenzyme (EP,.,). In the case of a2 but not a3, this is dose-dependent manner, suggesting the involvement of protein tyrosine phosphorylation in shift in E,/E2 conformational equilibrium in favor of Elas evidenced in studies ouabain-activated signaling. When protein tyrosine phosphorylation was determined by Westem of(i) change effected by oligomycin which traps the enzyme as E,P(Na), (ii) sensitivity blot analysis of myocyte lysates using an anti-phosphotyrosine antibody, it was found that non- inhibition by vanadate, a probe of the E2 conformation and (iii) the K'-sensitivity profile of Na- toxic concentrations of ouabain increased tyrosine phosphorylations of several proteins in the where -> -> range of 30-200 micromolar ATP, E2(K) El is rate-limiting. N-terminal deletion of a2 and kDa, including p42/44 MAPK. These effects were time-dependent;with some peak effects occurring after 2 (ca2M30; a3M26), described previously for al(alM32), reduces turnover further ( 2-fold) min. Interestingly, increases in tyrosine phosphorylation of p42/44 case, is associated with a MAPK appeared to lag behind those of several other shift in equilibrium towards El. Preliminary experiments proteins in the range of 60-120 kDa. Like suggest that the decrease in of ouabain, inhibition of the pump by lowering extracellular K' also stimulated turnover a3 is not a consequence of either the mutation to ouabain protein tyrosine resistance transfection since phosphorylation. These data suggest that Na+/K+-ATPase catalytic turnover of both axolemma and pineal are lower than that ofcardiac myocyte membrane acts as a signal transducer through interaction(s) of its subunits with some receptor kidney. (Supported by grants from the MRC, Quebec Heart & Stroke Foundation and the tyrosine kinases and/or NIH). nonreceptor tyrosine kinases. (Supported by AHA and NIH HL-36573).

78A SUNDAY NA, K-ATPASES 458463

458 - Pos 459 - Pos Na+/K'-ATPase AS A SIGNAL TRANSDUCER: RELATION OF Ca2'+ AND REACTIVE Na'/K'-ATPase AS A SIGNAL TRANSDUCER: COMPARISON OF OUABAIN- OXYGEN SPECIES (ROS) AS SECOND MESSENGERS WITHIN THE GENE ACTIVATED SIGNAL PATHWAYS IN CARDIAC MYOCYTES AND A7r5 SMOOTH REGULATORY PATHWAYS THAT ARE LINKED TO CARDIAC SODIUM PUMP. MUSCLE CELLS. J. Liu', J. I. Shapiro2, Anmir Askaril, Z. Xiel, 'Departisent of Pharmacology, Medical College of Peter Kometiani, M. S. Haas, J. Liu, Z. Xie, Department of Pharmacology, Medical College of Ohio, 2Department of Medicine, Medical College of Ohio Ohio, Toledo, Ohio 43614-5804 We have shown (JBC, 271, 10372, 1996; 273, 15249, 1998; 274, 19323, 1999) that the gene Partial inhibition of Na'/K+-ATPase by ouabain in cultured neonatal rat cardiac myocytes regulatory effects of the nontoxic concentrations of ouabain in neonatal rat cardiac myocytes are regulates the transcriptions of several growth-related genes through the activation of dependent on (a) extracellular Ca2' and ouabain-induced rise in [Ca2'],; and (b) ouabain-induced Ras/Raf0MEK/MAPK caacade, preceded by stimulation of protein tyrosine phosphorylation generation of intracellular ROS. The aim of this work was to assess the relation of [Ca2+], and (Kometiani et al., JBC, 273:15249, 1998; and Haas et al., these proceedings). To explore the ROS within the pathways that are linked to Na+/K+-ATPase. Measuring [Ca2+], and ROS with the cellular specificities ofthese ouabain effects we have compared such regulatory effects of ouabain use of appropriate fluorescent probes, we found that while ouabain-induced rise in [Ca2+], was in cardiac myocytes and A7r5 cells. We confirmed that ouabain inhibition of Na+/K+-ATPase in dependent on the presence of extracellular Ca2+ as expected, the ouabain-induced rise in ROS was A7r5 cells caused a rise in [Ca]i as it did in cardiac myocytes. Also, ouabain rapidly increased independent of the presence of extracellular Ca2'. When the pump was partially inhibited by tyrosine phosphorylations of severa proteins in both cell types. However, in contrast to the case lowering of extracellular K+, changes in [Ca2']j and ROS were the same as those induced by of cardiac myocytes, ouabain failed to activate p42/44 MAPK in A7r5 cells. In A7r5 cells, c-fos ouabain. Lowering of extracellular Nae without change of extracellular K+ or Ca2e concentrations, was rapidly induced by ouabain. In contraat to induction of cardiac c-fos, induction of c-fos in increased [Ca2']i as expected, but caused no significant increase in ROS generation. These A7r5 cells by ouabain (a) did not require the presence ofextracellular Ca2+; (b) was not prevented findings suggest that partial inhibition of cardiac Na+/K+-ATPase (or increase in the fraction of the by the Ca2+/calmodulin antagonist W-7; and (c) was not attenuated by the MEK inhibitor enzyme that is not pumping) leads to activation of two parallel pathways: One that is dependent PD98059. These findings, in conjunction with previous data on other cell types (Peng et aL., JBC, on the well-established rise in [Ca2']i resulting from the effect of pump inhibition on Na+/Ca2+ 271:10372, 1996), demonstrate the ubiquitous role of sodium pump as a signal transducer. They exchanger; and the other a ROS-generating branch that is directly linked to Na+/K+-ATPase also support our previous suggestion that the signal pathways that link cardiac Na+/K+-ATPase to without the involvement of [Ca2+]i or the Nae/Ca2' exchanger. (Supported by AHA and NIH grant the growth-related genes of myocytes may differ significantly from the gene regulatory pathways HL-36573). that re linked to Na+/K+-ATPase in cells other than cardiac myocytes. (Supported by AHA and NIH grant HL36573).

460- Pos 461 - Pos Nae/K+-ATPase AS A SIGNAL TRANSDUCER: COMPARISON OF THE EFFECTS OF Na+/K+-ATPase AS A SIGNAL TRANSDUCER: COMPARISON OF OUABAIN- LOW EXTRACELLULAR K' AND OUABAIN ON THE REGULATIONS OF GROWTH- ACTIVATED SIGNAL PATHWAYS IN NEONATAL AND ADULT CARDIAC RELATED GENES IN NEONATAL CARDIAC MYOCYTES. MYOCYTES. Jiang Liu', J. I. Shapiro2, Z. Xie', 'Department of Pharmacology, Medical College of Ohio, Zijian Xie, J. Tian, Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614- Toledo, Ohio 43614-5804, 2Department of Medicine, Medical College of Ohio 5804 Partial inhibition of Na+/K+-ATPase by ouabain in cultured neonatal rat cardiac myocytes We have shown that partial inhibition of Na/K-ATPase by ouabain activates multiple signal activates Ras and mitogen-activated protein kinases (MAPK), increases production of reactive pathways and regulates growth and growth-related marker genes in neonatal rat cardiac myocytes oxygen species (ROS), and regulates several early- and late-response genes that are markers of (JBC, 273:15249, 1998). The aim of this work was to determine similarities and differences in cardiac hypertrophy (Kometiani et al., JBC, 273:15249, 1998; Xie et aL., JBC, 274:19323, 1999). ouabain-activated signal pathways in adult and neonatal rat cardiac myocytes. As in neonatal To explore if inhibition of the sodium pump by means other than ouabain also causes such myocytes, ouabain increased intracellular Ca2' in a dose-dependent manner in cultures of Ca2'- changes, we have determined the effects of low K+ on MAPK, ROS production, and on early- and tolerant adult rat cardiac myocytes. Exposure of these adult cells to non-toxic concentrations of late-response marker genes in neonatal rat cardiac myocytes. As expected, inhibition of Na+/K+- ouabain activated p42/44 mitogen-activated protein kinases (MAPK) in a dose and time-dependent ATPase by gradual lowering of extracellular K+ from 5 to 0.3 mM increased intracellular Ca2+ manner. Expression of a dominant negative Ras or preincubation with PD98059, a mitogen- concentrations. Like addition of ouabain, lowering of K+ also stimulated ROS production in a activated protein kinase kinase (MEK) inhibitor, blocked ouabain-induced activation of p42/44 dose-dependent manner. Exposure of myocytes to low extracellular K' (change from 5 to 1 mM MAPK, establishing that ouabain activates the Ras/Raf/MEK/MAPK pathway in adult cells as it K') caused a rapid activation of p42/44 MAPK (peak at 5 min) lasting for at least 30 min. Also does in neonatal cells. However ouabain effects on p42/44 MAPK were more transient in adult similar to ouabain exposure, lowering of K+ caused inductions of c-fos and skeletal a-actin gene, than in neonatal cells; lasting less than 15 min in the former and more than 45 min in the latter. and repression of the gene of the a3 subunit of Na+/K+-ATPase. These findings clearly show that To see ifactivation ofthese pathways affects marker gene expression, effects ofouabain on the oa2 the gene regulatory effects of ouabain in cardiac myocytes are indeed due to its well-established subunit of Na/K-ATPase in adult cells were determined. Like a3 in neonatal cells, a2 protein interaction with Na+/K+-ATPase, and not caused by previously unknown actions of ouabain. levels were decreased by ouabain in adult cells. PMA, another growth stimulus for the neonatal (Supported by AHA and NIH grant HL-36573). cells, also reduced a2 expression in adult cells, as it down-regulated a3 expression in neonatal cells. These data shown that Na+/K+-ATPase of the adult myocyte also acts as a signal transducer, and suggest that the gene regulatory roles of the sodium pump in the intact heart should be explored. (Supported by AMA and NIH grant HL-36573).

462 - Pos 463 - Pos Na+/K+-ATPase AS A SIGNAL TRANSDUCER: OUABAIN-INDUCED ACTIVATION OF EFFECT OF ENDOTHELIN-1 ON Na+-K+PUMP CURRENT IN CARDIAC MYOCYTES. PROTEIN KINASE C (PKC) IN RAT NEONATAL CARDIAC MYOCYTES. Clyne Fernandes, Nerida L Bewick, Helge H Rasmussen, Royal North Shore Hospital, Pacific Kamiar Mohammnadi, Z. Xie, A. Askari, Department of Pharmacology, Medical College of Ohio, Highway, Sydney, NSW 2065 Australia Toledo, Ohio 43614-5804 Endothelin-l (ET-1), an important cardiovascular hormone, activates tyrosine kinase (TK). Since Our previous studies on ouabain-induced regulations of growth-related genes in neonatal cardiac TK activation increases the apparent affinity of the Na+-K+ pump for intracellular sodium (Na'i), the involvement of PKC in the myocytes suggested signal pathways that are activated by the we examined if ET- I regulates the pump. Rabbit ventricular myocytes were voltage-clamped at - inhibition of partial Na+/K+-ATPase (JBC, 273, 15249, 1998). The aim of this work was to seek 4OmV using wide-tipped patch pipettes containing 10 or 80 mM Na+(Na+'ip). Na+-K+ pump current direct evidence for such a role of PKC. We studied the effects of the exposure of myocytes for 10 (Ip) was identified as the shift in holding current induced by 104M ouabain. When Naepp was min to ouabain, or to a phorbol ester (PMA) as a positive control, on PKC activities in particulate 10mM mean Ip(±SE) of 13 myocytes exposed to ET-1 (0.49±0.02pA/pF) was significantly larger and cytosolic fractions. PKC activities were conventional after assayed by procedures, partial than in 8 controls (0.36±O.O2pA/pF). When Na+p'p was 80mM mean I, of 10 myocytes exposed to purification on DEAE cellulose, from ATP into histone using 32Pi-incorporation III-S in the ET-1 (199+0OO4pA/pF) and of 11 control myocytes (1.94±0.09pA/pF) were similar. ET-IA presence of Ca2+, or into a peptide substrate of in the absence of Ca2+. PMA had the PKC,o,,, receptor is the predominant ET-I receptor in cardiac myocytes. We exposed 10 myocytes to both expected effects. Ouabain (0.1 mM, the pump about 500/a in these caused inhibiting by cells) ET-I and 104M of the specific antagonist BQ-123 10mM). Mean (0.32±O.O2pA/pF) was significant increases in the and decreases in the (Na+p;p Ip particulate cytosolic activities, using either significantly smaller than in myocytes exposed to ET-1 only. To examine if TK is induces the substrate. When in similar Westem blot were done experiments analyses using isoform-specific effect of ET-1 we included 104M of the TK inhibitor Tyrphostin-A25 (Tyr-A25) in pipette antibodies, significant ouabain-induced translocation of PKC,pi,,,,, but not PKCd,,l, into the solutions. Mean of 16 myocytes exposed to ET-1 and Tyr-A25 (0.31i±O.O2pA/pF) was particulate fraction was noted. To date, the data on have been inconsistent. We conclude Ip PKC,,0. less than exposure that activation is indeed involved in significantly myocytes exposed to ET-1 alone. We conclude that brief of PKC,,,,,,,i ouabain-induced gene regulatory pathways in these cardiac to NIH myocytes ET-1 increases the apparent affinity of the pump for Na+i. This involves the myocytes. (Supported by grant HL-36573). ET-1A receptor and TK.

79A 464-468464-468 NA,NA,K-ATPASESK-ATPASES SUNDAYSUNDAY

464 - Pos 465- Pos A MECHANISM EXPLAINING THE VOLTAGE AND ION DEPENDENCE OF EFFECTS OF FORSKOLIN ON THE Na+-K+ PUMP CURRENT-VOLTAGE TRANSPORT BY E779A-SUBSTITUTED Na,K-ATPase. RELATIONSHIP IN GUINEA-PIG VENTRICULAR MYOCYTES. R. Daniel Peluffo, Joshua R. Berlin, UMDNJ-New Jersey Medical School, 185 S. Orange Ave., C. 0. Lee, M. Sakaguchi, D. C. Gadsby, Pohang Univ. of Science and Technology, Pohang, Newark, NJ 07103 Korea, and Rockefeller Univ., New York, N.Y. 10021 The substitution E779A in the Na,K-ATPase al subunit dramatically increases ATP hydrolysis in Na'-K' pump current was measured at -36°C as strophanthidin-sensitive whole cell current in the absence of K' (Vilsen, Biochemistry 34: 1455, 1995; Koster et al., J. Biol. Chem. 271: 2413, ventricular myocytes internally perfused with the solution containing 50 mM Na' and 100 nM 1996) due to acceleration of electrogenic Na-Na exchange (ArgOiello et al., J. Biol. Chem. 271: Ca2+. 40-ms voltage pulses from the 0 mV holding potential were used to determine steady-state 24610, 1996). When this exchange process was examined in voltage-clamped HeLa cells with levels of membrane current between-100 and +30 mV. The steady-state Na+-K+ pump current- saturating intracellular Na', activation of electrogenic Na-Na exchange had a biphasic dependence voltage relationship (I-V curve) was determined as the difference of the membrane I-V curves in on extracellular Na' (Na'o) with a high affinity (K°5.5, Na'o concentration for half-maximal the absence and the presence of strophanthidin. Effects of forskolin (2-3 pM) on the I-V curve and activation at 0 mV, of 13.4 ± 0.6 mM) and a low affinity component (Koo.5 of 121 ± 19 mM, in 37 their time courses were investigated. The c-AMP dependent protein kinase activation was seen by cells). Both exchange components were 'M-dependent, dissipating 30 ± 3% and 82 ± 6% of the increased membrane current at the 0 mV holding potential. 4 types of result were observed. (1) In membrane dielectric, respectively. The low affinity component was also highly cooperative (nH = 7 cells from 22 cells tested, the control I-V curve was crossed over with that in the presence of 2.2 ± 0.2). Inclusion of 2 mM intracellular ADP had little effect on the high affinity exchange forskolin. It appeared that the time course in the change of the control I-V relation was different component but inhibited the low affinity component. These data suggest that electrogenic Na-Na from that in the presence of forskolin. (2) In 5 cells from 22 cells, the Na'-K' pump current was exchange is activated by Na', acting as a K' congener and by Na"o acting with low affinity via a increased by forskolin at the whole voltage range tested. With time after breaking into the cell, the process that is distinct from electroneutral Na-Na exchange. To explain these data and our increased effect of forskolin was reduced. (3) In 5 cells from 22 cells, the steady-state Na+-K+ previous results showing that electrogenic Na-K exchange is also altered in this mutant enzyme pump current was little affected by forskolin. With time after breaking into the cell, the Na+-K' (Arguello et al., ibid.), we developed a branched pseudo 6-state model that reproduces our data by pump I-V relationship in the absence and the presence of forskolin was little changed. (4) In 5 changing the kinetics ofNa', and K', binding reactions without altering the VM dependence of ion cells from 22 cells, the Na+-K+ pump currents in the absence and the presence of forskolin were transport. This model suggests that the E779A substitution alters the relative affinities for Na' and similarly rundown. K' at K+, binding sites and accelerates enzyme turnover after Na' binds to a Na',-specific site even when the enzyme is not fully Na'-loaded.

466 - Pos 468 - Pos LOW CONCENTRATIONS OF DIHYDROOUABAIN STIMULATE a2- AND a3- ISOLATION OF AN ENDOGENOUS DIGITALIS-LIKE FACTOR (EDLF) FROM ISOFORMS OF THE Na/K ATPase BUT NOT THE al-ISOFORM. KIDNEYS. J. Gao, J. Wang, R. Wymore, R.T. Mathias, I.S. Cohen, G.J. Baldo, SUNY at Stony Brook, NY Frederic Mandel, Baylor College ofMedicine, One Baylor Plaza, Houston, Texas 77030 11794 We have previously reported that low concentrations of dihydroouabain (DHO) stimulate Na/K Growing evidence indicates that EDLFs play a role in renal, cardiovascular, visual, central pump current (Ip) in cardiac myocytes from human (ac-, a2- and a3-isoforms), canine (ac- and a3- nervous system and other pathologies and have been implicated in the pathogenesis of various isoforms) and guinea pig (a,- and a2-isoforms). In the present study, we investigated which forms of hypertension in humans and animal models. In a previous study, a ouabain-like isoform mediates this stimulation. In guinea pig, stimulation of Ip was enhanced by a-adrenergic factor(OLF) was extracted from human plasma. Due to the low concentration of OLF present in activation and insensitive to f-adrenergic activation, suggesting that only the a2-isoform was plasma, only - 50 micrograms (net) were obtained from approximately 1,000 liters of human involved, since a-activation increases transport by the a2- but not al-isoform, whereas J- plasma. At best, one microgram per ten liters of plasma was isolated. The objective of this project activation affects only the cc,-isoform. Some batches of guinea pigs have ventricular myocytes was to explore the possibility that kidneys are a more abundant source of EDLFs Our that express the a,- but not the a2-isoform. These myocytes also lack the stimulation of Ip by low hypothesis was that as kidneys are a very rich source of the Na,K-ATPase enzyme, it would act as concentrations of DHO, consistent with stimulation of the but almost no cc,-isoform. a sink for EDLF(s). Based on the concentration of NKA molecules known to be present in a2-isoform our calculations indicated that it should be to isolate EDLF Canine ventricular myocytes also show the stimulation, however they express the a3- instead of kidneys, possible -I pg per kg of kidneys. Thus, a protocol was developed that led to the isolation of an EDLF from lamb kidneys. a2-isoform. Thus the a3-isoform appears to also posses the stimulatory site for DHO. Based on The factor isolated was chromatographically similar to human plasma OLF. Furthermore, the yield the data from guinea pig, maximal stimulation nearly doubles the activity of the a2-isoform. A of- 1.2 pg per kilo of kidneys was >10-fold the yield per liter of plasma parallel binding model was employed to interpret our data. In this model, we assume the a2- isoform has two DHO-binding sites, a stimulatory (R') and inhibitory (R-) site. R' and R are competitive for DHO, but R' has the higher affinity. However, once R- is occupied, R' is not available. With this assumption, the dissociation constants of R' and R- are 2 nM and 148 nM DHO respectively. Supported by the American Heart Association, HL28958, HL54301 and HL20558.

469-470 ELECTRON TRANSFER SYSTEMS SUNDAY

469 - Pos 470- Pos PHOTOINDUCED ELECTRON-TRANSFER BETWEEN THE RIESKE IRON-SULFUR STEADY-STATE KINETIC PROPERTIES OF CYTOCHROME CAA, SUPPORT A PROTEIN AND CYTOCHROME cl IN THE BOVINE CYTOCHROME bc, COMPLEX. ROLE FOR CONFORMATIONAL CHANGES DURING CATALYSIS. Robert C. Sadoski', G. Engstrom2, H. Tian', L. Zhang', L. Yu', C.-A. Yu3, B. Durham2, F. Bruce C. Hill, Jianfeng Kong, Queen's University, Kingston, ON K7L 3N6 Canada Millett2, 'Arkansas Tech University, Russellville, Arkansas 71801, 2University of Arkansas, One of the cytochrome oxidases expressed by the aerobic bacterium Bacillus subtilis is of the caa3 3Oklahoma State University type. Cytochrome caa3 has the two core subunits common to all members of the cytochrome Electron transfer between the Rieske iron-sulfur protein (2Fe-2S) and cytochrome cl was studied oxidase superfamily. Subunit I contains three of the five redox active metal centres, cytochrome using the ruthenium dimer complex, Ru2D, to either photoreduce or photooxidize cyt cl within 1 a, cytochrome a3 and CUB. Subunit II contains the traditional dinuclear copper centre, CuA, and a ps. Ru2D has a charge of +4 which allows it to bind with high affinity to the bc, complex. Flash 100 amino acid extension that forms a cytochrome c domain. Electrons can be delivered with high photolysis of a solution containing oxidized beef cyt bci, Ru2D, and a sacrificial donor resulted in efficiency using ascorbate plus N,N,N'N'-tetramethyl-p-phenylenediamine (TMPD) to the reduction of cyt c] within 1 pLs, followed by electron transfer from cyt cl to 2Fe-2S with a rate cytochrome c domain, and then to the other centres of the oxidase, as follows; Asc - TMPD - constant of 9 x 104 S-a. Flash photolysis of reduced beef bcl, Ru2D, and a sacrificial acceptor C - CUA - a - a3-CuB. The enzyme exhibits biphasic steady-state properties as a function of resulted in oxidation of cyt c,, followed by electron transfer from 2Fe-2S to cyt ci with a rate electron input rate, and the kinetic parameters are modulated by solution pH. As TMPD constant of 1.5 x 104 S-' at 25 'C. The rate constant decreased to 5 x 103 s at 6 °C, suggesting that concentration is increased the enzyme changes from a low-activity to a high activity state. Since electron transfer is limited by the rate of a change in the conformation of the Rieske protein. the association with the cytochrome c domain is unchanged, we cannot invoke binding Oxidant-induced reduction of cyt bH was observed with a rate constant of 300 sa' in the presence of considerations as an explanation, and support models that advocate conformational changes as part antimycin. These studies provide kinetic evidence for a change in conformation of the Rieske of the oxidase's catalytic cycle. The two kinetic forms of the oxidase have different pH optima, protein as it transfers electrons from quinol to cyt cl. Supported by NIH grants GM20488 and which further support the idea of different conformers occurring during catalysis. (Supported by GM30721. an operating grant from MRC Canada to BCH).

80A SUNDAY ELECTRON TRANSFER SYSTEMS 471-479

471 - Pos 473 - Pos RAPID CALCIUM ACTIVATION OF OXIDATIVE PHOSPHORYLATION: THE EVIDENCE FOR A FUNCTIONAL ROLE FOR CONFORMATIONAL CHANGES IN KINETICS OF mV02, NADH AND LIGHT SCATTERING IN CARDIAC SUBUNIT m (SII5) OF BEEF HEART CYTOCHROME c OXIDASE (COX). MITOCHONDRIA. Lawrence J. Prochaska, E. Ogunjimi, A.J. Lincoln, C.N. Pokalsky, L.A. Shroyer, N. Donat, Paul R Terrlto, Stephanie A French, Robert S Balaban, National Institutes of Health, 10 Center Wright State University, Dayton, OH 45435 Drive, I0/BlD-400, Bethesda, Maryland 20892 The role of SIl in COX functioning and its ability to undergo functionally important conformational changes are both unknowns. We have monitored changes in SIB conformation by Parallel activation of mitochondrial biochemical work by Ca2' has been shown to involve the Ca2' using N, N'-dicyclohexylcarbodiimide (DCCD) chemical modification of SIII and site-directed sensitive dehydrogenases (CaDH) and the FJ/F,ATPase. This activation results in elevated antibodies to hydrophilic domains that connect the a-helices in Sm. Control and DCCD modified [NADH], and the driving force for ATP synthesis, along with a net increase in ATP synthesis. In COX were treated with a-chymotrypsin at pH 8.5 in Triton X-100 and we found that SIII of the the current study we hypothesize that the response time of Ca2' stimulation of oxidative DCCD modified COX was more labile to digestion than SIII of control COX. Also, SIII of phosphorylation (ox-phos) is rapid enough to support step changes in workload (-lOOmSec). To test DCCD modified COX was more reactive with iodoacetyl-biotin than SIII ofcontrol enzyme. The these hypothesis, rapid kinetics with [substrates] +/- [Ca2+] for mVO2 , light scattering (e), and intersubunit cross-linking of SRI of COX induced by the reagent, N-succinimidyl-(4-azidophenyl NADH were acquired with a rapid scanning spectrophotometer at lOOmSec time resolution in 1,3'-dithio)-propionate, was inhibited by DCCD modification of SIll. Site-specific antibodies absorbance and fluorescence modes respectively. Experiments were conducted in isolated porcine against each of four hydrophilic regions in SIu (AA 57-66, AA 119-128, AA 148-159, AA 180- heart mitochondria oxidizing glutimate+malate (G/M) at 370C. Response times of mVO2 to Ca2+ at 189) stimulated COX electron transfer (10-50%) without affecting the proton pumping activity of state 3, with Hb as an optical probe of [02], was dose dependent with an optima at 535nM the enzyme. These results suggest that retaining the native conformation of Silt is important for (0.2±0.lsec), and was significantly shorter than 800pM ADP (2.5±0.2sec; p<0.05, n=7) and maximum functional activities of COX and that both DCCD modification of SIII and antibody 21U/ml ATPase (3.7±0.5sec; p<0.05, n=12). The Ca2+ effect was reversible with ruthenium red binding to SIll cause conformational changes that disrupt SIII-subunit I interactions to modify the (2.5±0.3sec; pS0.05, n=12) or EGTA (1.5±0.2; p.0.05, n=6) illustrating matrix Ca2+ dependence. functional activities ofthe enzyme. (Supported by American Heart Association-Ohio Affiliate). Response times for NADH and £ were similar and approached the detection limits (-IOOmSec) of the system at 535nM [Ca2+]. These data demonstrate matrix accumulation, swelling, and activation of the metabolic driving force by Ca2+ is rapid. Rapid swelling may play an important mechanism in this process. Overall, these data illustrate Ca2+ is capable of modifying ox-phos on a time scale adequate to tightly regulate metabolism during work transitions.

474 - Pos 476 - Pos EFFECT OF THE Qo SITE INHIBITOR ON THE INTRAMOLECULAR ELECTRON NON-PRODUCTIVE COMPLEXES WITH ANABAENA FERREDOXIN ARE INDUCED TRANSFER BETWEEN THE IRON-SULFUR CLUSTER AND HEME C, IN THE BY MUTATIONS AT GLU 139 IN ANABAENA FERREDOXIN- NADP REDUCTASE AT BOVINE CYTOCHROME BC, COMPLEX. LOW IONIC STRENGTH Li Zhang', C-H Tai2, D B Jordan3, Linda Yu', Chang-An Yu', 'Oklahoma State University, John K. Hurley', Tammy B. Brodie', Merche Faro-Tomls2, Milagros Medina2, Carlos G6mez- Stillwater, OK 74078, 2University of Oklahoma, 3Dupont Stine-Haskell Research Center Moreno', Gordon Tollin', 'University ofArizona, Tucson, AZ 85721, 2Departmento Bioquimica y The effect of famoxadone and azoxystrobin, the Qo site inhibitors, on the pH induced Biologia Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain intramolecular electron transfer between 2Fe2S and heme c, in partially reduced cytochrome bc, The E139K, D and Q mutants ofAnabaena 7119 ferredoxin-NADP' reductase (FNR) have been complex is investigated. These inhibitors arrest the movement of the head domain of the iron- constructed and their electron transfer (ET) properties with reduced Anabaena 7120 ferredoxin sulfur protein (ISP) at the fixed position in the oxidized complex. The E. of ISP increases by 7 (Fd) studied by transient kinetic measurements. For E139K and E139Q at low ionic strength, k", mV upon famoxadone treatment while decreases by 21 mV when the complex is reacted with for the reduction of these mutants by wt Fd increases non-linearly as the FNR concentration azoxystrobin. The Kis for famoxadone and azoxystrobin are 0.3 and 5 )sM, respectively. increases. A mechanism consistent with these results involves the formation of a complex in Competitive binding study of the Qo site inhibitors shows the following order of binding affinity: which the mutual orientation of the proteins is so far from optimal that intracomplex ET does not stigmatellin > famoxadone > azoxystrobin > UHDBT. The effectiveness of these inhibitors is occur. As the concentration ofFNR is increased, free FNR is reduced by the reduced Fd present in only slightly pH-dependent. Famoxadone and azoxystrobin cause no apparent effect on the rate of the unreactive complex (a temary reaction), thereby resulting in non-linearly increasing kw, pH (drop) induced electron transfer from heme c, to 2Fe2S. The rates of electron transfer from values. As a consequence, E139K is highly hindered at low ionic strengths relative to wt FNR, 2Fe2S to heme cl, induced by pH jump, however, is greatly decreased in these two inhibitors whereas E139Q is significantly less impaired. E139D is much like wt FNR at all ionic strengths treated complexes. The t1,, for pH induced cl reduction reaction increases from 1-2 ms in examiined. At high ionic strengths, saturation kinetics are observed for all the mutants, as expected untreated complex to 35 ms and 110 ms in famoxadone and azoxystrobin treated complexes, for a two-step mechanism involving complex formation followed by ET. We conclude that E139 respectively. This work was supported in part by a grant from NIH (GM30721). is a critical residue for orienting the two proteins for ET. Supported by grants from the NIH (DK15057 to G.T.) and from CICYT (project B1097-0912C02-01 to C.G.-M.

477 - Pos 479- Pos INVESTIGATING THE PUZZLE OF OXYGEN EVOLUTION IN CYANOBACTERIA LOCALIZATION OF REGIONS OF SUBUNIT IV ESSENTIAL FOR INTERACTION LACKING PHOTOSYTEM I WITH THE THREE-SUBUNIT BC, CORE COMPLEX FROM RHJODOBACTER Qingun Wang', Ladislav Nedbal2, John Whitmarah3, 'University of Illinois at Urbana- SPHAEROIDES. Champaign, 2Institute of Microbiology, Czech Republic, 3University of Illinois at Urbana- Shil-Chis Tso, Byron N Quinn, Linda Yu, Oklahoma State Univerisity Champaign & USDA/ARS, 197 ERML, 1201 W. Gregory Dr., Urbana, IL 61801 Recombinant subunit IV mutants which identify the regions essential for restoration of bc, activity In cyanobacteria, photosystem I (PSI) accepts electrons generated by Photosystem II (PSII) from to the three-subunit core complex ofR. sphaeroides were generated and characterized. IV(1-109) water oxidation. Surprisingly, the mutants of Synechocystis 6803 lacking PSI can sustain high had reconstitutive activity similar to that of the wild-type IV, indicating that residues 110-124 are rates of oxygen evolution for over 30 minutes. On a chlorophyll basis the rate is about 25% of the not essential. IV(1-85) had no reconstitutive activity, indicating that residues 86-109 are essential. WT rate. The fact that we observe a net oxygen evolution indicates that the final electron acceptor As expected, mutants IV(1-76) and IV(1-40) showed no reconstitutive activity because both lack for electrons cannot be oxygen. Efforts to detect light-driven hydrogen production yielded null residues 86-109. IV(1-109) is associated with the core complex in the same manner as the wild- results, indicating that protons are not the terminal electron acceptor. Carbon isotope experiments type IV while mutants IV(1-85), IV(1-76) and IV(140) do not associate with the core complex, show a light-stimulated CO2 uptake accounts for about 15-20% of the electrons produced by indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly oxygen evolution in the mutants. In PSI-deletion mutants oxygen evolution is inhibited by DCMU, into the bc, complex. Since IV(86-109) has little reconstitutive activity, some region(s) in but not by DBMIB, suggesting the plastoquinone pool is the branching point to the unkmown residues 1-85 are required for bc, activity restoration after subunit IV is incorporated into the electron transfer pathway. To further investigate the role of the plastoquinone pool we monitored complex The interacting regions are identified as residues 80-85 and 41-53, since mutants IV chlorophyll fluorescence using multiple saturating light flashes. In PSI-deletion strains, QA_ (80-124), IV(77-109), IV(54-109), IV(41-109), and IV(21-109) had 53, 53, 53, 98, and 95% ofthe reoxidation was significantly slowed by KCN the half-time increased from 30 ms to I s. Our reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic observations indicate that in the absence of PSI electrons from water oxidation can reduce the side of the chromatophore membrane. This work was supported in part by a grant from NSF plastoquinone pool, which in tum can be reoxidized in the light or dark via a pathway that is (MCB9630413). independent of oxygen and is inhibited by KCN.

81A 480-485 ELECTRON TRANSFER SYSTEMS SUNDAY

480 - Pos 481 - Pos PROBING THE ROLE OF THE RIESKE NECK IN Qo SITE CATALYSIS. ELECTRON ENTRY INTO CYTOCHROME c OXIDASE: EFFECTS OF MUTATION Elisabeth Darrouzet, Maria Valkova-Valchanova, Fevzi Daldal, Plant Science Institute OF TRP143 AND TYR144. The bel complex is a key component of both respiratory and photosynthetic electron transfer Loia Geren', Y. Zhen2, L. Ma', S. Grinnell', J. Yang2, H.-A. Chu2, S. Ferguson-Miller2, B. Durham', F. Millett', ofArkansas, AR 72701, State University chains. Surprisingly, recent crystallographic structures have revealed different positions for the 'University Fayetteville, 2Michigan Rieske subunit head group within the complex. However, none of these conformations display Trp-143 is a highly conserved residue ofcytochrome c oxidase located on the surface ofsubunit II distances between the cofactors compatible with the observed electron transfer rates. Thus, the just above, and in van der Waals contact with, the CuA site via its ligand, Met-263. The potential idea that the Rieske subunit may move during catalysis has emerged. A comparison of various role of Trp-143 in mediating electron transfer between cytochrome c and CuA was investigated structures suggests that this movement would require conformational changes of the flexible using mutants of both Trp-143 and the adjacent Tyr-144. The purified mutants had the same domain (the neck) connecting the tail and the head of the Rieske subunit. However, whether or not visible, CuA EPR, and Mn EPR spectral properties as wild-type oxidase. The binding constants this movement, and in particular the flexibility of the Rieske neck, is essential for the activity of for cytochrome c were unchanged. Electron transfer between laser-reduced Ru-55-Cc and the the bel complex remained unkmown. Mutant bcl complexes were engineered genetically in mutants W143F, W143A, W143Y, W143H, and W143Q at low ionic strength had intracomplex Rhodobacter capsulatss in order to investigate the properties of this region. These studies revealed rate constants of 85 s', 32 s-', 15 s-1, 28 s-', and 5 s in comparison to 38,000 s-' for native more that a shorter neck yields functional but poorly assembled bel complexes. An exces of rigidity or oxidase. Tyr-144 mutations resulted in native-like rate constants, around 15,000 s'. It a in this therefore appears that Trp-143, but not Tyr-144, plays critical role in electron transfer from the flexibility region has major effects on the activity, but can be overcome by a shorter linker heme group of cytochrome c to CUA. These results are consistent with steady-state kinetic results domain. Most interestingly, a longer neck is also deleterious for the function and we were able for and computational docking analysis. Supported by NIH grants GM20488' and the first time to kinetically resolve and demonstrate the movement which is slowed down in these GM269162. mutant enzymes. (Supported by NIH grant GM 38237).

482 - Pos 483- Pos THE LOSS OF CYTOCHROME BC, COMPLEX ACTIVITY UPON FORMATION OF SITE-DIRECTED MUTAGENESIS OF ARG 91 IN CYTOCHROME bs REDUCTASE. AN INTERSUBUNIT DISULFIDE BOND. Christopher C. Marohnic, Jason D. Palcic, Li-June Ming, Michael J, Barber, University ofSouth Kunbong Xiso', Linda Yu2, Chang-An Yu', 'Oklahoma State University, 2Oklahoma State Florida, Biochemistry and Molecular Bio, 12901 Bruce B. Downs Blvd. MDC 7, Tampa, FL Univerisity, Stillwater, OK 74078 33612 To confirm the essentiality of the movement of the head domain of the iron-sulfur protein (ISP) in Cytochrome b5 reductase (cb5r) catalyzes the reduction ofcytochrome b5 using NADH as the bc, catalysis, R. sphaeroides mutants expressing cytochrome bcl complexes with pairs of electron donor. CB5R belongs to the "ferredoxin:NADP+ reductase" family containing a cysteines engineered (one cysteine each) on cytochrome b and ISP in close proximity (interface) conserved sequence motif, RXYT/sXXS/N involved in FAD binding. Site-directed mutagenesis has were generated and characterized. These are: A185C(b)K70C(ISP); 1326C(b)G165C(ISP), and been used to probe the role of R91 in a recombinant form of rat cb5r. UV/visible spectra of wild- T386C(b)K164C(ISP). The I326C(b)G165C(ISP) mutant is unable to support the photosynthetic type, R91K and R91A mutants showed the latter exhibited a red shift in the flavin maxima from growth because the G165 in ISP is a critical residue. The 1326C(b)G165C(ISP) mutant has the 461 to 458 mn while fluorescence spectra indicated decreased flavin quenching in the order photosynthetic growth behavior, the cytochrome bc, complex activities in chromatophores and in WT>R91A>R91K. Initial-rate kinetic analyses (pH 7, 23 'C) of NADH:ferricyanide reductase purified enzyme similar to those of wild-type cells. The A185C(b)K70C(ISP) mutant has retarded activities yielded V, values of 48, 35 and 23 smoles NADH consumed/min/nmole FAD for WT, photosynthetic growth rate and reduced be, complex activity in chromatophore and purified R91K and R91A, respectively, with K, values of6, 10 and 183 tsM for NADH. 3'P NMR spectra enzyme. This activity decrease results from the formation of disulfide bond between cytochrome indicated downfield shifts in the FAD phosphate resonances for both R9IA (-8.14 and -9.48 ppm) b and ISP, as evident from (I) the appearance of a protein band with an apparent molecular weight and R91K (-8.85 and -10.32 ppm) when compared to wild-type (-7.07 and -9.06 ppm). 5'-ADP of 66 kDa accompanied by the disappearance of the cytochrome b and ISP bands in SDS-PAGE of addition to WT and mutants did not perturb the FAD resonances, however, the ADP resonances mutant bc, complex in the absence of il-mercaptoethanol (B-ME); (2) addition of B-ME to this were shifted upfield for R91K (-8.07 and -10.37 ppm) and R91A (-6.84 and -10.32 ppm) mutant complex restores the bc, activity and resolves the 66 kDa protein band into cytochrome b compared to WT (-6.92 and -8.98 ppm). Therefore R91 is not essential for FAD binding in cb5r and ISP. This work was supported in part from NIH (GM30721). (supported by NIH and AHA).

484 - Pos 485 - Pos THE LOCATION OF THE EXTRAMEMBRANE DOMAIN OF THE IRON-SULFUR ON THE QUESTION OF INTERMONOMER ELECTRON SHARING BETWEEN b PROTEIN IN THE CYTOCHROME BC, COMPLEX IS REDOX STATE DEPENDENT. HEMES OF THE CYTOCHROME bc, COMPLEX Shib-chia Tsol, D. Xia2, L. Zhang', B. N. Quinn', J. Deisenhofer3, L. Yu', C-A. Yu', 'Oklahoma Glenda M Soriano', W. A. Cramer2, 'Purdue University, Biological Sciences, Lilly Hall, West State University, 2NIH-NCI, Bethesda, MD 20892, 3Howard Hughes Medical Institute and Lafayette, IN 47907, 2Dept. of Biol. Sciences, Purdue University, W. Lafayette, IN 47907 University of Texas, Southwestern Medical Center The observation that the distance in the be, complex between the Fe atoms of the two hemes bp, The structural and mutational of mitochondrial that analyses cytochrome bel complex suggest ca. 21 A, is approximately the same as that between heme bp and bm (Science., 277, 60, 1997), and movement of the extramembrane domain of the Rieske iron-sulfur protein (ISP) occurs during the presence of aromatic residues in the dimer interface that might facilitate inter-monomer heme electron transfer. Anamolous scattering analysis of the indicates that in the tetragonal crystals bprbp electron transfer (ET), raise the question of electron sharing between the two hemes bp, and oxidized complex less than 50 % of the 2Fe-2S of ISP is located at the fixed 31A position, away ofthe function ofthe dimer. To consider the relative probabilities ofelectron transfer, one needs to from heme c,. The rest is at the released position, somewhere between the fixed position and the consider the dependence of ET rates on specific pathways. The term (TDA)2 includes so-called "cl-position". In the fully oxidized complex, the relative peak height for 2Fe2S in the contributions from covalent bonds, H-bonds, and through-space jumps to the decay of the ET rate anomalous difference map is 0.47, normalized to the peak of heme bH (1.0). In the partially constant reduced complex (20% of the c, and ISP reduced), the 2Fe-2S is mostly located at the released (Science, 252, 1285, 1991). (TDA)2 for the inter-monomer heme bp-'bp ET ranges from 2.4 x 10-' position with a relative anomalous peak height of 0.29. This result accommodates well the pH to 2.8 x 10-12 while that for intra-monomer bp-b. ranges from 3.7 x 10 ''to 2.4 x 10~'5. The induced fast electron transfer between heme c, and 2Fe-2S in a partially reduced complex. In the smallest value of(TDA)2weighted by the Franck-Condon factor for heme bp-bp electron transfer is c, and ISP both reduced complex, the 2Fe-2Scluster is mostly at the fixed position with a relative 2.2 x 10"16, an order of magnitude larger than that of heme bp-bnET. Inter-monomer bp-bp ET anomalous peak height of 1.15. These results that the of the extramembrane suggest position seems feasible on structural grounds implying that electron sharing between hemes bp must domain of ISP is on the redox states of the be dependent complex. This work was supported in part considered in mechanistic descriptions of function of cytochrome bc, complex. [Supported by by NIH (GM3072 1. NIH GM-38323].

82A SUNDAY ELECTRON TRANSFER SYSTEMS 486 SUNDAY VOLTAGE-GATED NA CHANNELS 487-491

486- Pos 487 - Pos PHOTOINDUCED ELECTRON TRANSFER IN CYTOCHROME OXIDASE- SEVOFLURANE DELAYS ONSET OF INACTIVATION AND RECOVERY FROM CYTOCHROME c COMPLEX USING THIOUREDOPYRENETRI-SULFONATE- INACTIVATION IN THE CARDIAC SODIUM CHANNEL LABELED CYTOCHROME c. Kurt M Klippel, Wai-Meng Kwok, Zeljko J Bosnjak, Medical College of Wisconsin, 8701 Jenny Cappucciol, I. Szundi', Alexander Kotlyar?, 0. Einarsd6ttir', 'Department of Chemistry Watertown Plank Road, Milwaukee, Wisconsin 53226 and Biochemistry, UC Santa Cruz, CA 95064, 2Department of Biochemistry, Tel Aviv University, Israel. Local anesthetic effects on the sodium channel are well documented. In contrast the mechanism of Intramolecular electron transfer in the cytochrome c oxidase/cytochrome c complex was volatile anesthetic effects on the cardiac sodium channel is not known. We have previously shown investigated using a photoactivatable dye. Laser photolysis of thiouredopyrene-trisulfonate that volatile anesthetics have a holding potential-dependent effect on the cardiac sodium channel. (TUPS), covalently linked to cysteine 102 on yeast iso-1-cytochrome c, generates a triplet state of In the current study we investigated the effects of sevoflurane, a newer volatile anesthetic, on the the dye, which donates an electron to cytochrome c, followed by electron transfer to cytochrome inactivation characteristics of the sodium channel. We used the whole-cell patch-clamp oxidase. Time-resolved optical absorption difference spectra were collected at delay times from methodology on enzymatically isolated guinea pig ventricular cardiomyocytes. Onset of 100 ns to 200 ms between 325-650 nm. On the basis of singular value decomposition (SVD) and inactivation and recovery from inactivation were determined with standard two-pulse protocols multiexponential fitting, four processes with apparent lifetimes of24 ± 5 ts, 100 + 15 ,us, 2.0 ± 0.5 under control conditions and after addition of0.6 mM sevoflurane. ms and 180 ± 40 ms were resolved. A sequential kinetic mechanism is proposed from which the Sevoflurane increased the time constant for the onset of inactivation from 134.0 ± 9.7 ms to 175.6 microscopic rate constants and spectra of the intermediates were determined. The triplet state of ± 10.2 ms (n=6). The effect on the recovery from inactivation was holding potential dependent. the TUPS donates an electron to cytochrome c with a forward rate constant of 1.8 as 104 s-1. This From a holding potential of-120 mV sevoflurane did not significantly increase the recovery rate is followed by faster electron transfer from cytochrome c to CuA. Subsequently CuA equilibrates constant . From holding potentials of-70 mV and -50 mV, T increased from 97.6 ± 8.1 ms to with heme a. On a millisecond time scale the oxidized TUPS retums to the ground state and heme 130.9 ± 13.9 ms (n= 1) and 92.0 ± 7.3 ms to 149 ± 9.9 ms (n=5), respectively. a becomes reoxidized. The extracted internediate spectra are in excellent agreement with model The results suggest that sevoflurane interacts with the inactivation gate ofthe sodium channel. spectra ofthe postulated intermediates, supporting the proposed mechanism.

488 - Pos 489 - Pos MECHANISMS OF SLOW INACTIVATION IN HUMAN SKELETAL MUSCLE AND STRUCTURAL STUDY OF THE SODIUM CHANNEL INACTIVATION GATE HUMAN CARDIAC SODIUM CHANNELS. PEPTIDE INCLUDING AN ISOLEUCINE-PHENYLALANINE-METHIONINE MOTIF Yuriy Y Vilin', E Fujimoto', F J Repscher', A L George2, Naomasa Makita3, Peter C Ruben', AND ITS ANALOGOUS PEPTIDE (PHENYLALANINE/GLUTAMINE) IN 'Utah State University, 2Vanderbilt University, 3Hokkaido University TRIFLUOROETHANOL SOLUTIONS AND SDS MICELLES Slow inactivation is less complete and slower in hHl sodium channels than in hSkMl. We used Yoshihiro Kuroda', Kazuhide Miyamoto', Mazaru Matsumoto', Yoshitaka Maeda', Kenji the macropatch technique on Xenopus oocytes to study slow inactivation in chimeras in which Kanaori2, Akira Otakal, Nobutaka Fujii', Terumichi Nakagawa', 'Kyoto University, Sakyo-ku, single S5-S6 linkers (P-regions) were interchanged between hSkMI and hHl. The DIIS5-S6 Kyoto, Japan 606-8501 Japan, 2Kyoto Institute ofTechnology linkers impart properties of slow inactivation that correspond with the isoforms from which the The structures of the Na+ channel inactivation gate related peptide which includes IFM motif (Ac- linkers originated. DI, DIII and DIV S5-S6 linkers do not confer parental slow inactivation KKKFGGQDIFMTEEQKK-NH2; K1480-K1496 in rat brain type-IIA Na+ channels, MP-3A) and properties. P-region identity also strongly alters slow inactivation time constants of all chimeras. its F/Q(Gln) substituted one (MP-4A) in trifluoroethanol (TFE) and sodium dodecyl sulfate (SDS) The hSkMl/hHlDIIS5-S6 chimera expresses slow inactivation time constants similar to wild type micelles have been studied by circular dichroism (CD) and 'H-NMR spectroscopies. Based on hHl. This suggests that the DIIS5-S6 linker is the structural entity underlying differences in slow observed NOE constraints, three-dimensional structures of MP-3A and MP-4A were determined inactivation between hSkMl and hHl sodium channels. Removal of fast inactivation using by simulated annealing molecular dynamics/energy minimization calculations. In TFE, no IFM-+QQQ in the hSkMI/hHlDIIS5-S6 chimera enhances slow inactivation, but does not affect appreciable differences in the structure were observed by either CD or NMR spectra. In SDS activation. This suggests an interaction between the DIII-IV cytoplasmic linker and DIIS5-S6. micelles, the two peptides exhibited definitely different structures from each other. In MP-3A, the Charge neutralizations (R219C and K228C) in the voltage sensor of domain I alter the apparent residues 11488 and T1491 were spatially proximate with each other owing to the hydrogen voltage dependence of slow inactivation. Homologous mutations in domains II, III and IV have bonding between the amide proton of 11488 and the hydroxyl oxygen atom of T1491, whereas in no effect on the voltage dependence of slow inactivation. These data suggest that DIS4 is coupled MP-4A, F/Q substitution separated them owing to conformational changes. The solvent- to a pore-blocking movement of the DII P-region to produce slow inactivation, the probability of accessible surfaces calculated for the structures of MP-3A and MP-4A showed that the former has which is limited by fast inactivation. a smoother interaction surface to the hydrophobic docking site than the latter. In conclusion, the conformational changes, as well as decreased hydrophobicity around the IFM motif owing to the F/Q mutation, can be a reason why F1489Q mutated channels cannot inactivate almost completely.

490 - Pos 491 - Pos U-SHAPED INACTIVATION IN A MUTATED Na+ CHANNEL. MUTATION OF THE SELECTIVITY FILTER DOMAIN ALTERS RECOVERY AND Hannes Todt', K. Hilber', S. C. Dudley2, 0. Kudlacek', R. J. French3, J. W. Kyle4, H. A. IMMOBILIZATION OF GATING CHARGE IN RAT BRAIN IIA SODIUM CHANNELS Fozzard4, 'Institute ofPharmacology, University ofVienna, Austria., Vienna, Austria, 2Division of Frank J.P. KUhn, Nikolaus G. Greeff, Universitat Zurich-Irchel, Winterthurerstrasse 190, Zurich, Cardiology, Emory University/VAMC,Decatur, GA, 3Department of Physiology and Biophysics, Zutrich CH-8057 Switzerland Univ. of Calgary, Calgary, Canada, 4Cardiac Electrophysiology Laboratories, The Univ. of Voltage-gated ion channels display two main functions, the gating mechanism i.e. the opening and Chicago, Chicago, IL. closing of the conducting pathway and the permeation mechanism i.e. the selection and The residues K1237 and A1529 are located in the outer vestibule of the il Na+ channel. The translocation of the ions. Initially, these two processes were regarded to be structurally and mutations K1237E and A1529D enhance entry into an ultra-slow inactivated state (USI). USI is functionally independent. In fact, several mutations have been described that affect either characterized by time constants of very slow recovery from prolonged depolarizations of 100-200 activation or inactivation gating without changing the permeation properties of the channels (e.g. s and entry into this state can be reduced by binding to the pore of a mutant p-conotoxin GIIIA Chahine et al., Neuron, 12:281). However, recently a series ofexperimental data has demonstrated (Biophys. J. 76, 1335-1345). Thus, USI may reflect a structural rearrangement of the outer a linkage between gating and permeation (e.g. Balser et al., J. Phys., 494:431; Townsend and vestibule. Here we report that K1237E and A1529D exhibit striking differences in the voltage Horn, J. Gen. Physiol., 113:321). Hitherto, such studies were mainly performed by ionic current dependence of USI. The channels were inactivated by pulses of 300 s duration to various voltages. analysis. In contrast, we have investigated both ionic and gating currents ofrBIIa sodium channels Thereafter, the potential was returned to the holding potential of -120 mV and recovery from that contain a single point mutation (D384N) in the selectivity filter domain (Pusch et al., Eur. inactivation was monitored by repetitive 30 ms test pulses. In K1237E, the fraction of channels Biophys., J. 20:127) using high expression in Xenopus oocytes and a modified TEVC. Compared recovering from USI increased monotonically with depolarization. In A1529D, however, USI to wild-type, the mutant channel clearly displays a faster recovery of gating charge and a reduced reached a maximum at about -50 mV. At more depolarized potentials the fraction of channels degree of immobilization, thus demonstrating some similarity to the paramyotonia congenita- recovering from USI became smaller, resulting in a U-shaped voltage dependence of the entry into linked mutation RIC/H in voltage sensor S4D4. We propose that both mutations influence the the USI state.The results suggest that in A1529D USI occurs preferentially from "partially movement ofS4D4 by changing electrostatic interactions. activated" closed states, and that the outer vestibule may change its conformation during the process ofactivation.

83A 492-497 VOLTAGE-GATED NA CHANNELS SUNDAY

492 - Pos 493 - Pos SODIUM LOAD OF XENOPUS OOCYTES DURING HIGH EXPRESSION OF RBIIA IMMOBILIZING THE MOVING PARTS OF VOLTAGE-GATED SODIUM CHANNELS. SODIUM CHANNELS REDUCES THE RATIO OF ION PERMEATION TO GATING Richard Spike Horn', Shinghua Ding', Hermann Gruber2, 'Jefferson Medical College, CHARGE Philadelphia, PA 19107, 2Inst. ofBiophys., Johannes Kepler Univ., Linz, Austria Nikolaus G. Greeff, Frank J.P. Kuihn, Universitat Ztrich-Irchel Voltage-dependent gating of ion channels typically involves two types of physically distinct Gating currents (Ig) recorded with a fast TEVC from highly expressed rat brain IIA voltage-gated participants, voltage sensors that move in response to changes of membrane potential, and gates sodium channels display an unexpected large size compared to the corresponding ionic currents that control access to the permeation pathway. Although there are excellent candidates for each in (IN.) (Greeff et al., Biophys. J., 74:A402). I. showed normal properties and was already used to specific regions of the channel protein, little is known how the movement of one affects the characterize the inactivation process from mutant channels (Ktlhn and Greeff, J. Gen. Physiol. conformation of the other. To begin to tackle this problem we have developed a method of 114:167). We undertook further studies to understand the small ratio of IN./Q, (Q, = integrated IB). systematically immobilizing either voltage sensors or gates of a sodium channel using a We hereby noted a continuous decrease of the sodium equilibrium potential EN. during the photoactivated crosslinker. The bifunctional reagent we designed, benzophenone-4- experiment suggesting an influx of sodium from the bath solution into the cell. The intracellular carboxamidocysteine methanethiosulfonate, covalently attaches the benzophenone (BP) moiety sodium concentration [Na]i was calculated from the Nermst equation. A systematic study in several via a negatively charged linker to cysteines that we introduce into specific locations by oocyte batches demonstrated that the ratio of PN,/Q5 strongly correlates with [Na]i (permeability mutagenesis. Subsequent UV irradiation results in the insertion of the BP ketone into a PN. was used instead of IN. to account for different sodium concentrations). This was further neighboring C-H bond with an efficiency up to -70%. We show that the BP-labeled inactivation validated in both directions by partially changing choline vs. sodium in the bath solution. gate can be trapped open or shut selectively by UV irradiation, and that specific voltage sensors Probably, the influx of sodium at high channel expression permanently forces the oocyte to pump (S4 segments) play unique roles in gating. Immobilization of the S4 segment ofeither domain 1 or sodium ions out which represents an energetic stress for the cell. Consequently, the probability for 2 at a hyperpolarized voltage prevents channel opening. Depolarization of the S4 segment of open channel pores decreases by an as yet unknown mechanism, whereas the gating machinery domain 4 causes a movement of at least two-steps, the first coupled to the activation gate and a remains undisturbed. subsequent step coupled to fast inactivation. Blocking the initial step prevents opening; blocking the second step slows the rate ofinactivation.

494 - Pos 495- Pos MUTANTS OF DOMAIN III S45 IN THE HUMAN CARDIAC Na CHANNEL INCREASE HALOTHANE RESTORES FAST INACTIVATION TO THE INACTIVATION INTRINSIC LIDOCAINE AFFINITY. DEFICIENT MUTANT OF HUMAN HEART hHla SODIUM CHANNEL. Bin Ye, Carnen R Valdivia, Jonathan C Makielski, University of Wisconsin-Madison, Rm 24, Anna Stadnieka', H A Harlmann2, Z J Bosnjak', Wai-Meng Kwok', 'Medical College of 1300 University Avenue, Madison, WI 53706 Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, Baylor College ofMedicine The S45 segments of Domain III and IV play a critical role in inactivation of the Na channel, and inactivation is known to be an important determinant of local anesthetic binding. We used The effects of halothane (HAL) on native and cloned Na channels suggest complex interactions alanine scanning mutagenesis for 12 residues (M1319 through 11333) to study the structure- with the inactivated state. The underlying mechanism was investigated in the hHla III-IV linker function of inactivation kinetics and lidocaine block (300 pM). Wild type (WT) or mutant mutants with impaired (F1485Q) or deficient (IFM-QQQ) fast inactivation. The channels were channels were stably transfected into HEK293 cells and Na currents (IN.) were recorded using the expressed transiently in HEK293 cells, and currents were recorded using the whole cell patch whole-cell patch-clamp technique. Compared to WT the mutations shifted the midpoint of the hI,, clamp technique. In FQ mutants, HAL (1.0 mM) decreased ratio of non-inactivating to peak relationship to depolarized potentials by -7 mV for all channels except for M1319, V1322, current (0.38±0.05 to 0.25±0.04) and increased current decay (T3.4±0.4 to 1.9±0.2 ms, at 0 mV). V1323, and L1326 which were unchanged. Activation midpoints were not affected for any HAL rendered the steady-state fast inactivation more complete, and slowed the recovery from fast mutant. Tonic block was 19% for WT and tended to be greater for the mutants (M1319A 43%, R1320 V1322 V1323 L1326 and 11333 inactivation (Tf 0.18+0.01 to 0.46±0.05 ms, T, 15±1.8 to 26±0.8 ms). In QQQ mutants, HAL 45%, 33%, 37%, 58%, 38%). Use-dependent block for a further decreased ratio of non-inactivating to peak current (0.97±0.02 to and induced 10 Hz train was 70% for WT and for V1322 V1323 and 0.56±0.05), pulse significantly greater 78%, 89%, current decay (r 28±3.3 ms, at 0 mV). While absent in control and after HAL washout, the L1326 95%. The shift in the h. in lidocaine index of inactivated state steady- relationship (an affinity) state fast inactivation and the recovery from fast inactivation 38±5.2 were measured in the also tended to be for and 11333. was the (T ms) greater especially R1320, V1322, L1326, S1332 only presence ofHAL. These results suggest that HAL may restore inactivation to 3Q mutant channels. mutation that showed decreased block by all measures. Inactivation kinetics were altered, if at all, in the direction which would tend to decrease lidocaine The increased block Because substitutions in the fast inactivation regulating IFM cluster modify HAL effects, we depolarizing affinity. a interaction of HAL with the putative inactivation in for these mutations, an of lidocaine for the hypothesize hydrophobic receptor particle, therefore, represents intrinsically higher affinity the absence ofcompeting hydrophobic residues ofthe III-IV linker. channel. The DIII S45 segment is likely to form part of or be near to the local anesthetic binding site.

496- Pos 497- Pos DEACTIVATION FROM THE OPEN AND INACTIVATED STATES OF SKELETAL CHARGED RESIDUES IN THE DIII-DIV LINKER CONTROL THE LEVEL OF MUSCLE SODIUM CHANNELS. STEADY-STATE SLOW INACTIVATION IN SODIUM CHANNELS. James R. Groome', E Fujimoto2, P C Ruben2, 'Harvey Mudd College, 1250 N Dartmouth Ave, Elizabeth Spackman, Esther Fujimoto, Yuriy Vilin, Peter C Ruben, Utah State University CA 2Utah State Claremont, 91711, University Fast inactivation appears to limit the level of slow inactivation (Featherstone et al., 1996). Since We investigated the relationship between deactivation from the open state (O to C) and the DIII-DIV cytoplasmic linker comprises the structural basis of fast inactivation, we explored deactivation from the inactivated state (I to C) in skeletal muscle sodium channels. Open-state the role of charged amino acids in the DIII-DIV linker on slow inactivation in human (hSkMI) deactivation was measured from tail current decay, while inactivated-state deactivation was and rat skeletal muscle (rSkMl) sodium channels. Macroscopic current recordings were made determined from the delay in onset to recovery from fast inactivation. We compared R1441C and from on-cell patches on Xenopus oocytes expressing wild type and mutant sodium channels. The R1441P, for which biophysical profiles differ in open-state deactivation, but not for activation or effects of three mutations on slow inactivation were investigated: EE1314QQ in hSkMl, inactivation. We reasoned that differences in the deactivation kinetics of R1441C and R1441P 1295KKKl297->QQQ in rSkMI, and 1295KKK12974QQQ + K1301Q in rSkMI. Using compared to wild type should be similar for both the 0 to C and I to C transitions if these two steady-state slow inactivation protocols (60 s prepulses), we found that the level of slow processes are similar. At 12 'C, both mutations exhibited a greater open-state deactivation tau and inactivation was reduced in EE1314QQ relative to wild type hSkM1, whereas positive charge an abbreviated delay to onset of recovery, with respect to wild type channels. Deactivation tau neutralizations had no effect on the level of steady-state slow inactivation compared to rSkMI. from the open state was greater in R1441P than in R1441C, and time of delay to the onset of These data suggest that electrostatic interaction between the DIII-DIV linker and other sodium recovery from fast inactivation was greater in R1441P than in R1441C. These data suggest that channel structures may be important in determining the level of slow inactivation. We speculate deactivation from the open state or from the inactivated state represent similar translocations for that the negative charges on the DIII-DIV linker may interact with positive charges within one or DIVS4 segments. Additionally, they suggest that effects of these mutations on charge more S4 segments, inducing a secondary conformational change in the S4s that leads to slow immobilization contribute to observed biophyscial defects. inactivation, as explored in an accompanying abstract (Vilin et al.). These negatively charged residues might be at least part ofthe previously described interaction between fast inactivation and slow inactivation.

84A SUNDAY VOLTAGE-GATED NA CHANNELS 498-503

498 - Pos 499 - Pos THE ROLE OF THE PUTATIVE INACTIVATION LID IN SODIUM CHANNEL IMPORTANT ROLE OF THE IV/S6 SEGMENT OF THE SODIUM CHANNEL IN FAST GATING CURRENT IMMOBILIZATION INACTIVATION Michael F Sheets', John W Kyle2, Dorothy A Hanck2, 'University of Utah, 2University of Aelai Kirilov Alekov, Ernst Derra, Nenad Mitrovic, Frank Lehmann-Hom, Holger Lerche, Chicago University ofUhm, Helmholtzstr. 8/1, Ulm, D- 89075 Germany Cha et al. (Neuron 22:73-87, 1999) showed that voltage sensors from DIII&IV accounted for the Fast inactivation of voltage-gated Nae channels is proposed to function in a hinged-lid fashion. slow recovery of gating charge (charge immobilization) during repolarization (OFF-Q) in human Possible candidates for a receptor site of the IFM-motif are cytoplasmic regions of the channel skeletal muscle Na channels. We investigated the contribution of the putative "inactivation lid" to protein important for inactivation. When expressed in tsA201 cells and studied using the whole charge immobilization in a mutant of the human heart Na channel (hHla) that had the IFM region cell patch clamp technique, a double cysteine mutant (Fl586C/Il596C=FI/CC) of the human in the DIII-IV linker mutated to ICM. After transient expression in fused tsA201 cells (AJP muscle Nae channel destabilized the inactivated state, inducing a persistent Na+ current of 12±2% 269:C1001, 1996), the cysteine was modified by 2.5 mM MTSETi. Before modification the ofpeak current and an acceleration ofrecovery from inactivation. Application of the thiol-reagent mutant retained some fast inactivation, but after modification almost all fast inactivation was MTSES induced a strongly voltage-dependent increase in persistent Na+current to 60±3% ofpeak abolished. OFF-Q was recorded at -180 mV following conditioning steps to 0 mV. In WT-hHla current. The voltage-dependence of the reaction rate was close to the steady-state inactivation the fast component of OFF-Q disappeared with a tau of 2.8 ± 2.3 ms (n=5), similar to the tau of curve of the FI/CC mutation. MTSET had no obvious effect on FI/CC. In inside-out patches, inactivation ± development of IN, (2.1 0.3 ms, n=7). After conditioning durations longer than MTSET prevented the reaction with MTSES suggesting an important role ofthe negative charges, 10 ms, only 47% of the total OFF-Q was in the fast component. For ICM-hHIa after MTSET, which needs further investigation. For the triple mutation F1586CN1589C/M1592C M, OFF-Q still contained two components with the tau of the fast component (2.5 A 1.4 ms, n=4) inactivation was also markedly disrupted by MTSES (two-fold slowing of fast inactivation time similar to WT-hHla, but accounted for 69% of OFF-Q. After inhibition of movement of the DIV constant, 1.8±0.1% persistent current). Altogether this region plays an important role in fast Nae voltage sensor by the site-3 toxin Anthopleurin-A in ICM-hHla after MTSET, OFF-Q was nearly channel inactivation and may participate to form a receptor site for the IFM-motif identical in both time course and magnitude to the fast component in ICM-hHla after MTSET. We conclude that immobilization of the voltage sensor in DIII is dependent upon binding of the inactivation lid, while immobilization of the voltage sensor in DIV is independent of the lid.

Pos 501 - Pos AMINO ACIDS IN TRANSMEMBRANE SEGMENT IIIS6 OF THE VOLTAGE- AMINO ACIDS IN TRANSMEMBRANE SEGMENT IIIS6 OF THE SODIUM CHANNEL DEPENDENT SODIUM CHANNEL a SUBUNIT ARE IMPORTANT FOR NORMAL a SUBUNIT FORM PART OF THE RECEPTOR FOR LAMOTRIGINE AND COUPLING OF ACTIVATION TO INACTIVATION ETIDOCAINE. Vladimir Yarov-Yarovoy, Jacob Brown, Elizabeth Sharp, William A. Catterall, Todd Scheuer, Vladimir Yarov-Yarovoy, Jacob Brown, Elizabeth Sharp, Todd Scheuer, William A. Catterall, Dept. of Pharmacology, University of Washington Dept. ofPharmacology, University ofWashington Voltage-gated sodium channels are responsible for the initiation and propagation of action Voltage-gated sodium channels are the molecular targets for block by local anesthetic and certain potentials in neurons. We used alanine-scanning mutagenesis to examine the functional role of anticonvulsant drugs. Specific amino acids in transmembrane segments IS6 and IVS6 affect block amino acid residues in transmembrane segment IIIS6 of the rat brain type IIA Na+ channel a by local anesthetics and anticonvulsants. To determine whether amino acids in segment IIIS6 of subunit in activation and inactivation gating of the channel. a and 11 Na+channel subunits were the rat brain type IIA Nae channel a subunit also are involved in block by local anesthetics and coexpressed in Xenopus oocytes and Na+current was monitored using two microelectrode voltage anticonvulsants, alanine was substituted individually for each of the native residues in IIIS6 clamp. Mutations throughout the IIIS6 segment caused significant depolarizing and stretching from V1454 to 11473. Wild-type and mutant Na2 channel a subunits and wild-type ,1 hyperpolarizing shifts in the voltage-dependence of activation gating. Mutations in the subunits were coexpressed in Xenopus oocytes and Nae current was monitored by two extracellular half of IIIS6 had relatively small effects on the voltage-dependence of inactivation. microelectrode voltage clamp. Individual mutants were identified that either increased or Mutation of residues in the cytoplasmic half of IIIS6 caused strong hyperpolarizing and decreased the affinity of inactivated sodium channels up to 10-fold for the anticonvulsant depolarizing shifts in the voltage-dependence of inactivation. In contrast to similar mutations in lamotrigine and its congeners GW227c89, GW4030w92 and GW619c89 as well as for the local IVS6, no alanine substitution in IIIS6 disrupted inactivation from open states. For several IIIS6 anesthetic etidocaine. The same mutations also affected use-dependent block by these mutants, opposite shifts in activation and inactivation voltage-dependence were observed compounds. Interestingly, the efficacy of individual mutants depended on the compound being indicating that these mutations altered the coupling of activation and inactivation. Evidently, tested. Thus, residues in transmembrane segments IS6, IIIS6, and IVS6 of the Nae channel several of the native amino acids in IIIS6 are critical for normal coupling of activation to contribute to distinct but overlapping receptor sites for local anesthetic and anticonvulsantdrugs. inactivation, especially in the negative voltage range where inactivation is primarily from closed Supported by NIH NS15751 and Glaxo-Wellcome. states. Supported by NIH NS15751 and Glaxo-Wellcome.

502 Pos 503 - Pos INACTIVATION KINETICS OF Na+, Ca2+, and Ba2+CURRENT THROUGH CARDIAC A PENTAPEPTIDE FROM THE HUMAN BRAIN ACTING AS A NA CHANNEL Na+CHANNELS BLOCKER. Eric A Sobie, Silvia Guatimosim, Jader Cruz, W.J. Lederer, University of Maryland School of HeinrichBrinksmeier', P Aulkemeyer', K H Wollinsky2, R Rildel', 'University of Uln, Dep. of Medicine and Medical Biotechnology Center, 725 W. Lombard St., Baltimore, MD 21201 General Physiology, Albert-Einstein-Allee 11, Ulm, D-89069 Germany, 2Rehabilitation Hospital TlTX-blockable Ca2` current (IN&(O)) can be observed in heart cells in the absence of extracellular Ulm and intracellular Na+(Lemaire et al., 1995, Receptors & Channels 3:71; Cole et al., 1997, AJP Block of axonal voltage-gated sodium channels was suggested to contribute to the symptomsin 273:H128; Aggarwal et al., 1997, J. Physiol. 505:353). We used the whole-cell ?atch clamp some inflammatory demyelinating neurological diseases, such as multiple sclerosis. We have technique to examine the inactivation kinetics of TTX-blockable Na, Ca2, and Ba + current in earlier provided evidence (Brinkmeier et al, Muscle Nerve 19:54-62, 1996), that in the isolated rat ventricular myocytes. We found (see Figure): 1) Under all ionic conditions, cerebrospinal fluid of these patients an endogenous Na2 channel blocking factor exists that has inactivation time constants decreased with increasing potential between -60 and -40 mV; 2) When properties of a local anaesthetic. Using mass spectrometry we have now determined the factor's Ca2' or inactivation time IN,Zo) by Ba2%, constants were similar, varying from around 10 exact molecular weight to 609.2 Da. Peptide sequencing from C-terminus and N-terminus and msec at -60 mV to approximately 3 msec at -40 mV; 3) fragmentation in a mass spectrometer revealed the same sequence of a pentapeptide. Isolated and Inactivation was approximately twice as fast when the current was synthesised peptides both blocked the Na+currents of NH15-CA2 neuroblastoma x gliomacells Na+than when 10 , .ca' carried by it was carried by Ca2+or Ba2+. Our data by shifting the steady-state inactivation curve to more negative potentials. Current/voltage curves

No' permeant cation E hypothesis can were not affected. At 10 MM, the synthesized peptide shifted the inactivation curve by -10 mV influence the gating behavior of cardiac Na+ channels. An without showing use-dependence (at IHz stimulation). The tonic block of30-400/ seen when the alternative (but still is that a untested) hypothesis novel channel cells were stimulated from a holding potential of -85 mV was completely abolished after with a similar pharmacological profile to the Na+channel but with hyperpolarization to -135 mV. These blocking characteristics are very similar to those caused by -60 ^40ev permeability properties is expressed in heart. lidocaine. A permuted control peptide was completely ineffective. The pentapeptide, for which we so far could find no precursor, may well contribute to the symptoms seen in certain immune- mediated inflammatory neurological diseases.

85A 504-509 VOLTAGE-GATED NA CHANNELS SUNDAYSUNDAY

504 - Pos 505- Pos ENHANCED EFFECTS OF BETA-SCORPION TOXINS ON SODIUM CHANNELS DO VOLTAGE-DEPENDENT ION CHANNELS CONSTITUTE A FERROELECTRIC WITH MUTATIONS IN THE IIS4 VOLTAGE SENSOR LIQUID CRYSTAL SYSTEM? Sandrine C. CESTELE', Massimo MANTEGAZZA', Marie-France MARTIN-EAUCLAIRE2, H. Richard Leuchtag, Olivier Helluin, Herve Duclohier, Dept. of Biology, Texas Souther Univ., Todd SCHEUER', William C. CATTERALL', 'University of Washington, Box 357280, Seattle, Houston TX, Univ. of Pennsylvania Sch. of Med., Philadelphia,, PA, and UPRES-A 6026 CNRS, WA 98195, 2Universite de la Mediterran6e, CNRS UMR 6560, 13916 Marseille, France Universit6 de Rennes I, France Beta-scorpion toxins bind to the at subunit of voltage-dependent sodium channels at receptor site Peptide-strategy experiments, in which isolated parts of ion channels were inserted into lipid 4, which includes the S3-S4 extracellular loop in domain II, and negatively shift the voltage bilayers, yielded highly voltage-dependent I-V characteristics. Analogues of S4L45 (III) voltage dependence of activation, favoring channel opening. This effect requires prior depolarization and sensor segments formed tetrameric aggregates that conducted Na+ ions. Replacement of the Ile is proposed to occur because the toxin traps voltage sensor IIS4 in the outward position. To and Leu next to the 3rd and 5th Args with unbranched residues -MeAla or Ala resulted in a severe elucidate the molecular mechanism of voltage sensor trapping, we investigated effects of the beta- loss of voltage sensitivity. The conditions for a ferroelectric liquid crystal are: chirality, e.g., an toxin CssIV on IIS4 using rat brain type IIA sodium channels in which glutamine was substituted asymmetric carbon atom; a dipole-moment component perpendicular to the long axis of the for basic residues in the IIS4 segsnent. Only neutralization of the two outermost arginines (R850 molecule; and a smectic (layered) phase with nonzero tilt angle. Ferroelectric liquid crystals and R853) strongly modified the effects of CssIV. In the WT channel CssIV induces a weak and containing amino acids with branched alkyl groups, Val, Leu and Ile, possess large spontaneous partial negative shift of activation, and this requires a depolarizing prepulse. For mutants R850Q polarizations (Yoshino & Sakurmi, Ferroelectric Liquid Crystals, Goodby et al., eds., Gordon & and R853Q, the negative shift ofactivation was greater and was complete. Moreover, this effect of Breach 1991). The side chains of the amino acids provide dipole moments perpendicular to the CssIV was observed without depolarizing prepulses when R853 was neutralized. Both mutations axis of an helix. Ion-channel segments with nonzero tilt have been widely reported. The strongly increased CssIV-induced tail currents following repolarization, indicating slowed return evidence appears to support the hypothesis (Leuchtag & Bystrov, Ferroelectrics 220, 157-204, of segment IIS4 to its resting position. Our results are consistent with the conclusion that 1999) that a voltage-dependent ion channel is a ferroelectric liquid crystal system. neutralization of R850 and R853 stabilizes IIS4 in an outward configuration, slows deactivation of the channel, and favors voltage sensor trapping by CssIV.

506 - Pos 507- Pos A CRITICAL RESIDUE FOR ISOFORM DIFFERENCE IN TETRODOTOXIN MOLECULAR MODEL OF THE Na AND Ca CHANNEL PORES BASED ON AFFINITY IS A MOLECULAR DETERMINANT OF THE EXTERNAL ACCESS PATH MUTATIONAL STUDIES, KcsA CRYSTAL STRUCTURE, HOMOLOGY, AND FOR LOCAL ANESTHETICS THROUGH THE CARDIAC SODIUM CHANNEL PORE. ENERGY MINIMIZATION Akihiko Sunamil, I. W. Glaaserl, S. C. Dudley2, H. A. Fozzard', 'University of Chicago, 5841 S. Gregory M Lipkind, H. A. Fozzard, University of Chicago, 5841 S. Maryland, Chicago, IL Maryland Ave. (MC6094), Chicago, IL 60637, 2Emory University, 1670 Clairmont Rd (It IB), 60637 Decatur, GA 30033 We examine the implications for the Na and Ca channels of using two features of KcsA channel Membrane-impermeant quaternary derivatives of lidocaine (QX222, QX314) block cardiac Na+ - the alpha-helix - strand for the P-loop and the teepee structure for S5 and S6 helices. The P channels when applied from either side of the membrane, but block neuronal and skeletal muscle loops were aligned and organized into alpha helix-turn-beta-strand structures, with the beta-strands Na+channels poorly from the outside. To find the molecular determinants of the cardiac external facing the pore. The four domains assembled radially to form the same relationship of the C-ends QX access path, mutations of the adult rat skeletal muscle (p 1) and rat heart (rHl) Nae channels as required for formation of the TTX/STX binding sites (Lipkind and Fozzard, 1994). The S5 and were studied by two-electrode voltage clamp in Xenopss oocytes. Mutating the I domain I P- S6 helices were aligned and the backbones assigned the corresponding coordinates of KcsA loop Y401, which is the critical residue for isoform differences in tetrodotoxin (TTX) block, to the (Doyle et al., 1998). The P loop vestibules were then docked into the teepee structures to permit residue found in heart (Y401C) allowed QX222 block from the outside, but its mutation to brain optimal dense packing of the amino acid residues. The result was a closed model of the outer type (Y401F) showed little block. Y401C accelerated recovery from block by internal QX222. vestibules of the Na and Ca channels with the P loops of domains I-IV interdigitated between the Block by external QX222 in Y401C was diminished by binding of methanethiosulfonate respective S5 and S6 helices. The Na channel selectivity filter DEKA ring was located just above ethylammonium (MTSEA) to the outer vestibule or by a double mutant (Y40IC/F1579A), which the putative local anesthetic drug binding site. The selectivity filter structures for Na and Ca altered the putative local anesthetic binding site. The reverse mutation in heart, rHl-C374Y channels implied possible mechanismsofinteraction ofthe permeating and blocking ions. reduced outside QX314 block and slowed dissociation of internal QX222. We conclude that the isoform-specific residue (Tyr/Phe/Cys) in IP-loop plays an important role in drug access as well as inTTX binding.

508 - Pos 509- Pos ELECTROSTATIC AND STERIC CONTRIBUTIONS TO Is-CONOTOXIN GIIIA BLOCK OF SINGLE SODIUM MODULATION OF SODIUM CHANNEL GATING BY A PARTIALLY BLOCKING CHANNELS DERIVATIVE OF t-CONOTOXIN GIIIA Kwokyin Hui, Robert J. French, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N Canada Ivan Slerralta', Yue-Kun Ju2, Robert J. French', 'University of Calgary, 3330 Hospital Dr NW, 4NI Calgary, ABT2N 4N1Canada, 2The University ofSydney ji-Conotoxin GIIIA is a (pCTX) 22-residue, basic peptide toxin (net-charge, +6) which blocks Sodium channels from skeletal muscle are blocked in an all-or-none fashion by the 22 amino-acid skeletal muscle sodium channels at nM concentrations. Chang et al. (1998, Biochem. showed 37:4407) peptide, p-conotoxin GIIIA. Arginine-13 is critical for block, and the derivative R13Q allows recently Argl3 to bind near the channel's selectivity filter, intimately interacting with -30% of the normal to flow Glu758 in the channel's outer single-channel current through peptide-bound channels. This enables vestibule, and earlier data suggested Argl3 to be essential for direct study ofionic currents passing through peptide-bound channels. Here, we report the actions pCTX's all-or-none block of the unitary current (Becker et al., 1992, Biochem. 31:8229). Here, of R13E (net charge, +4), in which charge at the critical residue 13 has been reversed. Whole-cell we examine electrostatic and steric factors contributing to pCTX's block of the unitary current in patch-clamp was used to record currents through rSkMIsodium channels expressed in HEK-293 batrachotoxin-activated channels in lipid bilayers. Block of single-channel current becomes cells. Whole-cell current was blocked by R13E with an IC50 of 13 ± 3.5 PM and the residual progressively lesscomplete as the sidechain length, or charge (q13), of residue 13 is diminished by conductance at maximal block was 36 ± 4%of the maximal conductance (mean ± SEM, lumped substitution of ArgI3 with positive, neutral or negative amino acids. This suggests that IsCTX data from 16 cells). The residual is slightly smaller than the value observed for single, block depends critically on q13 and its position relative to selectivity filter. We have also batrachotoxin-modified examined the influence channels in planar lipid bilayers (- 48%). For [Rl3E]>l0PM, a positive of three other toxin residues on block. For these positions, charge- shift in voltage-dependent activation occurred, consistent with an electrostatic interaction between substitutions had a weaker on changing influence f,,, than did residue-13. This suggests that the toxin and the channel's voltage sensor, and comparable with observations for RI3Q (French et placement of charge al., 1996, Neuron In a shift in residue slo linear 16:407). contrast, negative steady-state inactivation was observed, Property, # -f re_ession near residue 13 is a even at [RI3E]ovlpM. These data suggest that a higher affinity, non-blocking interaction may be Charge, 13 0.19±0.002/elementary charge critical determinant of responsible for the effects ofthe toxin on inactivation. Charge, 2, 12, or 14 <0.09/elementary charge completeness of block Side-chainlength,13 atom of the single-channel 0.059±0.002/heavy current.

86A SUNDAY VOLTAGE-GATED NA CHANNELS 510-515

510- Pos 511 - Pos CONTRIBUTIONS TO PERMEATION OF CHARGED RESIDUES BETWEEN THE CHARGED RESIDUES IN P-s6 LINKERS CRITICAL FOR p-CONOTOXIN BLOCK OF SELECTIVITY FILTER AND S6 SEGMENTS IN THE RAT SKELETAL MUSCLE NA RAT SKELETAL MUSCLE NA CHANNEL PORE. CHANNEL. Ronald A. Li, P. V6lez, G. F. Tomaselli, E. Marban, The Johns Hopkins University School of Ronald A. Li, P. Velez, N. Chiamvimonvat, G. F. Tomaselli, E. Marban., The Johns Hopkins Medicine, Baltimore, MD 21205. University School of Medicine, Baltimore, MD 21205. s-Conotoxins (p-CTX) are site I blockers that specifically block Nae ion flux by occlusion of the The deep regions of the Nae channel pore around the selectivity filter have been studied Na channel pore. In the present study, we examined the contribution of outer charged residues extensively, however, little is known about the adjacent linkers between the P-segments and S6. located in the P-S6 linkers from all four domains to p-CTX block. Neutralization ofthe negatively The presence of numerous conserved charged residues hints that these linkers may play a role in charged DII residues D762 and E765 of the pI-2 Nae channel increased the IC5" for p-CTX block permeation. To characterize their structural topology and function, we neutralized all the charged by -100-fold. Restoration and reversal of charge by extemal modification of the cysteine mutants residues in these linkers of the 1tl-2 Na+ channels individually by cysteine substitution. Several with methanethiosulfonate reagents did not affect p-CTX block compared to unmodified. In cysteine mutants displayed enhanced sensitivities to Cd2' block and/or were modified by external contrast, the charge-conserved mutations D762E and E765D conferred wild-type blocking sulfhydryl-specific methanethiosulfonate reagents when expressed in HEK 293 cells, indicating behavior while the charge-reversed D762K and E765K mutants worsened -CTX block even that these residues reside in the permeation pathway. While neutralization of positive charges did further, suggesting that the intersctions between these DII residues and t-CTX are largely not alter single-channel conductance, negative charge neutralizations generally reduced electrostatic. Further kinetic analysis reveals that the changes in toxin sensitivity are largely due to conductance suggesting that such charges facilitate ion permeation. The electrical distances for changes in the toxin dissociation (koft) but not the association (k,) rates. We conclude that both Cd2' binding to these residues indicate that the P loop-S6 (P-S6) linker dips back into the the negative charges at positions 762 and 765 and their localization within the pore are critical for membrane and forms part of the pore lining, at least for DII and IV. Our findings reveal significant optimal toxin-channel interactions, and that these residues exert their effects by stabilizing the structural and functional roles of these charged residues. final toxin-bound channel complex.

512 - Pos 513- Pos USE-DEPENDENCE OF TOXIN BLOCK IN MUSCLE Na-CHANNEL: EFFECT OF ON THE MECHANISM OF ACTION OF THE SCORPION TOXIN Cn2 ON rSkml Na+ POINT MUTATIONS AT THE DEKA LOCUS. CHANNELS. Chien-Jung Huang, Edward Moczydlowski, Dept. Pharmacology, Yale Univ. Sch. of Med., New F. G6mez-Lagunas', M. E. Ramirez-Dominguez', G. Gurrola', L. D. Possani', R. G. Kallen2, Haven, CT 06520 'Facultad de Medicina y Instituto de Biotecnologia. UNAM. M6xico, 2University of Pennsylvania Conti and coworkers (1996, 1999) have proposed that use-dependent block of Na-channels by School ofMedicine. Philadelphia, PA. USA. tetrodotoxin (TTX) and saxitoxin (STX) is due to an interaction between guanidinium toxin and a Scorpion toxins that act on Nae channels are thought to belong to one of two groups: alpha, that cation trapped in the closed pore such as Ca2+. The K(III) residue of the DEKA selectivity locus affect the inactivation of the channels; and beta, that modify the voltage dependence of activation, functions in preventing inward permeation of Ca2+. Thus, it might be expected that mutations of making the channels to open at more negative potentials. Cn2 had been clasified within the beta K(III) that enhance Ca2+-permeability would abolish TlX/STX-use-dependence. Whole-cell group. We took a closer look to its mode of action on rSkmil Na channels expressed in HEK currents were recorded from HEK293 cells expressing the native rat muscle p1l Na-channel cells. To our surprise we found that: (1) Cn2 does not change the voltage dependence of (DEKA), a Ca-permeable DEAA mutant and a Ca-impermeable DERA mutant. Cells were activation, thus Cn2 departs from the expected behavior of a beta toxin.(2) Cn2 strongly reduces depolarized to -lOmV for 10 ms from a holding potential of -120 mV at an initial rate of (I/30) Hz the amplitude of the Na+ current at all voltages, without changing the rate of fast inactivation nor prior to and after reaching steady-state of tonic block, and thereafter, at rates of 4, 2, 1, 0.5, 0.2, the steady state inactivation ofthe channels. The effect ofCn2 is not use-dependent. and 0.1 Hz, with each test was separated by 3-min of (1/30) Hz recovery stimulation. The tonic The results suggest that Cn2 might be occluding the pore ofthe channels. half-blocking concentrations (IC50) of DEKA, DERA, and DEAA channels were 31, 820, and 7000 nM for ITTX, and 5.7, 400, and 5600 nM for STX, respectively. Under rapid simulation (>1 Hz), DEKA and DERA channels exhibited an exponential decay to a state of increased binding affinity (-4-fold limiting value) for both TIX and STX. In contrast, use-dependent block of the DEAA channel by 1TX and STX was insignificant, showing less than 10% extra block at highest stimulation rate of 4 Hz. Apparent loss of toxin use-dependence by the DEAA channel may be due to an alteration of toxin binding kinetics or an alteration of the Ca2+-toxin interaction in the Ca2+-trapped closed state. Supported by NIH GM-51172.

514- Pos 515- Pos ,81-SUBUNIT CAUSES A LOCALIZED CONFORMATIONAL CHANGE IN THE D4S6 SINGLE-CHANNEL RECORDING FROM NATIVE SODIUM CHANNELS OF SEGMENT OF hHl SODIUM CHANNELS, NORMAL AND DISEASED HUMAN ATRIUM AND VENTRICLE. Dominic Deschenes', Michael E OILeary2, Mohamed Chahine', 'Dept. of Medicine, Laval Thomas Boehle, Mathias C. Brandt, Dirk J. Beuckelmann, University of Cologne, Cologne, D- University, Ste-Foy, Quebec, Canada GIK 7P4, 2Dept. of Anatomy, Pathology and Cell Biol. 50924 Germany Jefferson Med. Coll. Philadelphia, PA 19107 Single native human cardiac sodium channels were recorded (pulse frequency 10 Hz) at low noise (bandwidth 10 kHz; sampling frequency 100 kHz; thick-walled patch pipettes: outer diameter 2 Introduction: PI3-subunits are well known to modulate the inactivation gating of many voltage- mm, inner diameter 0.25 mm ofthe raw material) with ultra-clean patch-pipette tips, obtained only gated Na channels. The mechanisms underlying these changes in gating are not known. Recent seconds before the actual gigaseal formation. Cells were isolated from normal and diseased data indicate that residues of the D4S6 segment are exposed within the internal vestibule of the (Ischemic/dilated cardiomyopathy) atrium and ventricle of human myocardium. No differences in channel and may contribute to inactivation gating. single-channel current amplitude of atrial and ventricular myocytes were observed. For different and the mean Mehtods: In this study the effects of the P1-subunit on the orientation of residues of the D4S6 patches cells, single-channel current amplitude at -40 mV was constant at about 2 segment was investigated. Human heart (hHI) Na channels were expressed in tsA201 cells and pA. In patches containing a single channel, open-time and first-latency ranged from about 100 ps whole-cell Na currents measured. Cysteines were substituted for 11756, F1760 and Y1767 of the to about 2 ms and from about 70 ps to about 3 ms, respectively, indicating that different gating D4S6 segment and the sensitivity to internally applied methanethiosulfonate reagent (MTSET) modes of sodium-channel action (BDhle et al., Biophys. J. 75, 1998) were present. The constant was examined. single-channel current amplitude during the time course of the experiment proves, that the driving force was unaltered, hence excluding an artifact to be the cause for the alteration in open-channel Results: Applying MTSET ( 1mM) to the cytoplasmic side of the mutant channels results in a life time and first-latency. We conclude, that native human cardiac sodium channels may switch slow time-dependent reduction in current amplitude (T = 153 the F1767C s). Co-expressing between different gating modes, which may be interesting for understanding ofarrhythmias. mutant with the P1-subunit completely abolishes these effects of MTSET indicating that this residue is either not accessible to the reagent or no longer extends into the vestibule. The effect of MTSET on the Y1767C mutant was not altered by PI-subunit. I1756C, F1760C and Y1767C of hHI are exposed within the internal vestibule and modification with MTSET blocks the channel. Co-expression with the 13-subunit selectively reduces the MTSET inhibition of the F1760C channel.

data suggest that a localized conformational change at position 1760 of D4S6 may well known effects of 31-subunit on the inactivation of human heart Na channels.

87A 516-521 VOLTAGE-GATED NA CHANNELS SUNDAY

516- Pos 517-Pos SUBCELLULAR LOCALIZATION OF THE HUMAN HEART SODIUM CHANNEL MODULATION OF BRAIN SODIUM CHANNELS BY ASSOCIATED RECEPTOR AND ITS GFP-LINKED SUBUNITS EXPRESSED IN HEK293 CELLS PROTEIN TYROSINE PHOSPHATASE B. Thomas Zimmer', C. Biskup', S. Dugarmaa', F. Vogel2, K. Benndor4, 'Friedrich Schiller Charlotte F. Ratcliffe', Yusheng Qul, Kimberly A. McCormickl, Victoria C. Tibbs', Jack E. University, Teichgraben 8, Jena, 07740 Germany, 2MDC for Molecular Medicine, Robert-Rossle- Dixon2, Todd Scheuerl, William A. Catterall', 'University of Washington, Mailstop 357280, Str. 10, Berlin-Buch, Germany Seattle, Washington 98195,2University of Michigan Medical School, Ann Arbor, Michigan The a subunit of the human heart sodium channel (hHI) and itsPI subunit were heterologously 48109-0606 expressed in HEK293 cells and the subcellular localization of both subunits was investigated by We have found that sodium channels are tyrosine phosphorylated and are modulated by a novel immunoelectron and laser-scanning confocalmicroscopy. Overexpression of hHl led to large interaction with the catalytic domains of receptor protein tyrosine phosphatase (RPTP,B), whole-cell Na' currents, suggesting correctly targeted channel molecules. However, the resulting in a positive shift in the voltage-dependence of inactivation and an increase in current. ultrastructure of such transfected cells showed a massive proliferation of tubular and stacked Co-immunoprecipitation experiments show that sodium channels interact with RPTP,B in neonatal endoplasmic reticulum (ER) membranes acconmnodating most of the overproduced protein. To rat brain but not at later developmental stages. In postnatal day 16rat brain, sodium channels study hHl localizationthree-dimensionally in living cells, hHl was labeled at its C-terminus with associate with phosphacan, a secreted isoform of RPTP,B lacking the catalytic phosphatase the green fluorescent protein (GFP). Whole-cell and single-channel currents of this hHI-GFP domains, which localizes to nodes of Ranvier. In vitro matrix binding techniques demonstrate that fusion construct were the same as those of the wild-type protein. HEK293 cells essentially purifiedrat brain sodium channels, consisting of the pore forming a subunit and auxiliary 1 and overexpressing hHI-GFP showed a proliferation of tubular ER and stacked perinuclear P2 subunits, interact with the extracellular carbonic anhydrase domain present in both and membranes similar to hHl cells. a RPTPI transfected Also, only very small portion of the hHI-GFP phosphacan. Immunoprecipitation of sodium channel subunits expressed in tsA-201 cells shows channels was targeted to the plasma membrane. In contrast to this, expression of the GFP-linked that a andPI subunits interact with an endogenous RPTPP isoform and with co-expressed RPTPP subunit led to a of the membrane, as revealed by laser P, predominant staining plasma scanning intracellular phosphatase domains while subunits do not associate. Modulation of sodium confocal microscopy and simultaneous staining of the plasma membrane with the dye FMI-43. J32 channels by RPTPJ3 may occur early in development, while extracellular association with When coexpressing hHI-GFP with the 1, subunit labeled with a blue-shifted GFP variant, phosphacan may RPTPP and sodium channel interactions with the identical fluorescence were observed for and blue emission. The most intensive displace thereby regulate patterns green catalytic domains of the phosphatase. These interactions sodium channel function fluorescent patterns were localized intracellularly and resembled those structures obtained after may regulate and localization in of neurons like nodes of Ranvier. expression of hHI-GFP only. This result indicates that an efficient Na' channel subunit assembly specialized regions may occur already within the ER.

518-Pos 519 - Pos VOLTAGE-GATED SODIUM CHANNELS BIND CALMODULIN. NITRIC OXIDE MODULATES CARDIAC SODIUM CHANNELS John H. Caldwell, Kristin L. Schaller, Katharine A. Reese, Univ. Colorado HSC, 4200 E. 9th Gias U. Ahmmed, Yan Fang Xu, Pei Hong Dong, Nipavan Chiamvimonvat, University of Ave., Denver, CO80206 Cincinnati Proteins that bind to the cytoplasmic domains of sodium channels were identified by the yeast two-hybrid The domains of Nav 1.4 muscle channel) and Nav assay. large cytoplasmic (skeletal We have previously shown a direct inhibition of expressed cardiac L-type Ca2' channels by nitric 1.6 (CNS and PNS were used to screen muscle and brain cDNA libraries. Two full- channel) oxide (NO) donors. In contrast, NO donors have no observed effects on expressed Na' channels. length clones of calmodulin were isolated when the carboxy-terminal domains were used as bait. We directly tested the effects of NO on Na' channels in isolated guinea pig (GP) and mouse (M) A for CAM motifs revealed an CAM binding sequence that is present in seareh binding "IQ" the ventricular myocytes. Saturated NO solution was prepared fresh daily and pH adjusted. Na' carboxy of all NaCh beads were in a "pull-down" terminus isoforms. CAM-agarose used assay current was recorded using whole-cell patch-clamp techniques. In contrast to the expressed Na' and demonstrated that NaChs in rat membrane fractions bound CAM. This binding was specific channels, NO (-200 pM) significantly reduces peak whole-cell Na+ current (current density in since the addition of CAM eliminated this binding. Western blot analysis with CAM exogenous pA/pF using a test potential of-35 mV); GP: before, -24.6±4.6 and after NO, -17.2±2.9, n=5; M: antisera showed that CAM was co-immunoprecipitated with NaChs using NaCh-specific before, -37.2±5.2 and after NO, -26.9±4.3, with no in antibodies. C-terminal domains of NaChs Nav 1.1, 1.4, 1.5, and 1.6 were expressed as S protein n=3; significant changes voltage-dependent of activation or inactivation. of 8-bromo-cGMP mimics the fusion proteins and tested for to CAM. All of the fusion proteins bound to CAM, Application (200 pM) inhibitory binding effects of NO. of NO cGMP leads indicating a direct binding, consistent with the yeast two-hybrid results. These fusion proteins are Moreover, application after to a but smaller reduction in the current being used to determine the Ca dependence of CAM binding. In addition, mutations of the NaCh 2.0 fsrther (Fig 1). does not carboxyl domains are being generated to determine amino acids required for CAM binding. 2.26 5mM DTT Sulfhydryl reducing agent (dithiotreitol, DTT) -3.0 mtF1 mM OTT reverse the effect of either NO or in Electrophysiological studies are being done to identify possible functional consequences of CAM cGMP. Thus, contrast to 4.5 N0 the effects of NO on can occur binding. Supported by NIH & MDA &, Ca2' channel, which via direct .4.0 or indirect pathways, our data show that NO modulates Na' -4.5 channel by an indirect pathway through activation of cGMP CouP 0 10 20 30 40 50 and possibly through another second messenger pathway. Time(min) Supported by American Heart Association.

520 - Pos 521 - Pos ROLE OF THE SODIUM CHANNEL C-TERMINUS IN PRODUCING SUSTAINED AN OXYGEN-SENSING SODIUM CHANNEL IN RAT HIPPOCAMPAL NEURONS SODIUM CURRENT DUE TO G-PROTEIN 1y SUBUNITS. Anna K.M. Hammarstrom, Peter W. Gage, PO Box 334, JCSMR, ANU, Mills Rd, Canberra, ACT Massnimo A. Mantegazza, Frank Yu, William A. Catterall, Todd Scheuer, U. of Washington, 2601 Australia Dept. of Pharmacology, Box 357280, Seattle, WA 98195-7280 Hypoxia causes Ca2` accumulation and abnormal electrical activity in neurons. Na+ channel Sustained (non-inactivating) sodium currents can have dramatic effects on neuronal activity. G blockers inhibit increases in [Ca2+]i and improve recovery of population spikes (1). Hence, Na' protein Iy subunits (GI3) increase sustained sodium current when cotransfected with brain type channel activity is an early step in oxygen sensing and cell damage. A persistent Na+ current (IN.P) IIA Na' channel into mammalian tail of the channel contains a tsA201 cells. The C-terminal increases during hypoxia and cyanide exposure in intact hippocampal neurons (2). Here we sequence ('1'6QXXER) that has been implicated in modulation by GPy. To identify regions of the examined whether this increase in IN,e occurs via intracellular second messengers or is membrane- C terminal tail in producing GIy modulation, we produced deletion mutants in the C-terminal tail delimited. of the rat brain IIA sodium channel. The sodium current in the absence type amount of sustained Single channel recordings were made from inside-out patches from cultured hippocampal neurons and presence of cotransfected was cell voltage clamp. Cotransfection of GJy measured by whole (Vp=+30 mV, +23'C). Mean currents were calculated from 0.5-4min recordings sampled at GP2y3 produced a 4-fold increase in sustained current due to coexpressed WT type IIA sodium OkHz. channels. I skeletal muscle sodium channels were not modulated by Gf3y. However, modulation p Hypoxia (95%N2:5%02) caused -20-fold increase in average mean current (-0.015 to -0.343 pA, of type IIA sodium channels was retained when the proposed GJ3y regulation site was converted to n=21). The activity increased with duration ofhypoxia. I0-IOOpM lidocaine, 5mM dithiothreitol that of the pI sodium channel. When the C-terminus was truncated, Gj3y modulation was (DlT) or 5-20mM reduced glutathione reversed the effect of hypoxia. 5mM NaCN significantly maintained only for truncations C-terminal to S1977. Conversely, internal deletion of the segment increased average mean current (-0.038 to -0.173 pA, n=7) and this effect was abolished by between S1891 and S1977 retained full modulation. Thus, sequences within the last 28 amino lidocaine and DIT. acids of the C-terminal tail of type IIA sodium channel are required for modulation. GMy In conclusion, enhanced activity is triggered by hypoxia and CN via a Supported by NIH NS34801 and the HFSP. IN.p membrane-delimited pathway. Curiously, the effects of hypoxia and CN were both blocked by reducing agents suggesting that the redox-reaction involves at an associated membrane-protein which in turnregulates sodium channel activity. 1. Fried, E., et al.,. (I1995). J.Physiol.489, 557-565. 2. Hammarstrfm, A.K.M. & Gage, P.W. (1998). J.Physiol. 510, 735-741.

88A SUNDAY VOLTAGE-GATED NA CHANNELS 522-527

522 - Pos 523 - Pos DOES DOMAIN IH OF THE a-SUBUNIT OF SODIUM CHANNEL PLAYS A ROLE IN INCREASED LATE Ns+ CURRENT IN MYOCYTES FROM A CANINE HEART THEaelSUBUNIT INTERACTION? FAILURE MODEL AND FROM HUMAN HEART FAILURE IsabeUe Deschenes', Roland G Kallen2, Eric Carbonneau', Hassan Manouzi3, Mohamed Carmen R Valdivia, Robert A Haworth, Mathew R Wolff, Jonathan C Malielski, University of Chasine', 'Laval university, Dept. Medicine, Laval Hospital, Research Center 2725 chemin Ste- Wisconsin, B-24 1300 University ave, Madison, WI 53706 Foy, Ste-Foy, Quebec GIV 4G5 Canada, 2Dept. Biochem. and Biophys. U. Pennsylvania, 3Laval Alterations in various ionic currents have been proposed as mechanisms underlying arrhythmias in University, Dept. Mathematics heart failure. Previous reports have shown no change in the peak transient Nae current (INT), but others have suggested increased late Nae current (INJL) in heart failure. We studied ventricular at 250 and Voltage-gated sodium channels are generally composed of a- and -subunits. The a-subunit is myocytes from failing hearts (HF, n=5 dogs), after 4-6 weeks of rapid-pacing bpm, formed by 4 transmembrane domains, and contains the activation and inactivation gates as well as sham operated controls (NH, n=5 dogs), and also from 7 explanted failing human hearts and two heart. The whole-cell was used to measure Na+ currents for the the pore of the channel. The P-subunit contains only one transmembrane segment and contributes non-failing patch clamp technique transient and was identified as the mean current measured between to the channel activity by enhancing the surface expression and modulating the a-subunit. peak cunrent (IN.T) IN.L IN.L 650 and 750 ms after the depolarization. Ca2'-activated currents and other currents contaminated the site and nature of the interaction is not clear. In this study, we have created an a- However, measures of we therefore defined as the late current blocked STX but not blocked subunit chimera containing the third domain and adjacent cytoplasmic loops of the rat skeletal INaL INaL by by DIDS, and Nifedipine. In the maximum at -20 mV from a muscle sodium channel (rSkMI) in the background of the human heart (hH1I) channel. When this Ni2+, dogs, density (pA/pF) holding potential of -140 for IN.T was 18 ± 7 and 12 + 5, and for IN.L was 0.3 A 0.2 and 1.0 4- 0.6 in NH chimera is in a mammalian cell line (tsA201), its biophysical properties vary from cell expressed (n= II) and HF relative to was increased in HF to cell, exhibiting for 12 cells of 24, properties corresponding to rSkMl: steady-state inactivation (n=6), respectively. IN.L IN.T significantly (from 1.4 ± 1.0 % in NH to 5.3 ± 3.4%/o in HF, p< 0.001), and did not decay after 750 ms depolarization (h.): V1l=-80.9 X 1.6 mV, k,= 5.4 ± 0.3 mV; T,X,= 5.3 ± 0.5; while for the 12 other cells the for in both NH and FH. The midpoint of steady state inactivation for IN.T (I s conditioning step) parameters correspond to those of hHll: h.,,: V,2=-93.6 ± 1.6 mV, kL= 6.0 ± 0.2 mV; [NC= 11.4 ± was similar in both groups (-90 mV). IN.L, however, did not inactivate after 1 s (n=8 NH, n=5 0.9. However when we this chimera with the coexpressed P-subunit, the currents recorded showed HF) or 5 s (n=2, NH) prepulses. In humans, IN.L relative to IN.T was significantly increased in HF electrophysiological properties that correspond to the phenotypes of rSkMI: hI,,: V112=-82.3 ± 2.1 (n=15 cells) compared with NH (n=6 cells). We conclude that increased density of IN L in failing mV, k,=5.1 ± 0.4 mV; Tr,,= 6.2 ± 0.4 ms (n=12). Since this chimera contains domain IH ofrSkMl, hearts may play a role in action potential prolongation and the generation of arrhythmia. our results suggest that the P-subunit preferably interacts with this domain of rSkMl to modulate its electrophysiological properties.

524- Pos 525- Pos A SINGLE SODIUM CHANNEL UNDERLIES UNUSUAL PHARMACOLOGIC TWO MYOTONIA-CAUSING MUTANTS OF THE HUMAN SKELETAL MUSCLE Na+ SENSITIVITY OF IN. IN MOUSE AT-I CELLS. CHANNEL SHOW DIFFERENT SENSITIVITY TO MEXILETINE AND ITS Tao Yang, S. Kupershmidt, G. L. Hemontolor, J. R. Balser, D. M. Roden, Vanderbilt University DERIVATIVE, Me7 School of Medicine, 23rd Avenue South, Nashville, TN 37232-6602 Jean-Francob Dessphy', Annanaria De Luca', Diana Conte Camerino', Alfred L. George, Jr.2, We have previously shown that IN. in mouse atrial myocytes (AT- I cells) is unusually sensitive to 'University of Bari, 2Vanderbilt University TTX (IC50so00nM). In the present study, we compared IN. sensitivity to TTX and lidocaine in We tested the drug mexiletine (Mex) and a new derivative (Me7, a-[(2-methylphenoxy)methyl]- AT-I cells and tsA-201 cells transfected with the human heart sodium channel cDNA (hHll). The benzenemethaamine) on the R1448C and G1306E Nae channel mutants expressed in tsA2OI cells were studied under identical conditions, with a holding potential of -120mV (to entirely cells. Tonic block (TB) ofNa+ channels was evaluated 3 minutes after drug exposure by applying relieve inactivation). hHI was, as expected, less sensitive to TTX (1.5±0.5gM, n=4). The IC50 for a single test-pulse from -120 to -2OmV while use-dependent block (UDB) was evaluated by lidocaine block of hHI was 99±l6pM, compared to 4.1±l.±OPM in AT-I cells. Lidocaine block applying a train of test-pulses at 10 Hz. For TB and UDB of wild-type (WT) channels, the EC50s was unaffected by anti-13I antisense in AT-I cells or by co-transfection ofhHl with P13 subunit. To of Mex were 234 and 60,uM and the EC50s of Me7 were 103 and 14pM, respectively. R1448C address the possibility that multiple sodium channels genes are expressed in AT-I cells, we first currents had a longer decay time constant, and steady-state fast inactivation was shifted to the left. identified regions of nucleotide homology among rat and human brain, heart, and skeletal muscle R1448C channels were 6-fold and 9-fold more sensitive to Mex and Me7 than WT channels, sodium channel a-subunits. Degenerate oligonucleotides, based on these sequences, were then respectively. By contrast, G1306E current, which also had a slower decay but a right-shifted used to amplify a 430bp PCR fragment encompassing the region encoding 373cys, a putative TTX steady-state fast inactivation, was 2.5-fold less sensitive to both Mex and Me7. Although both binding residue in hHll. Sequencing 20 separate subclones revealed only a single hHll-like cDNA, drugs did not change the current decay kinetics of any channel type, the stronger potency of Me7 with a cys at position 373. We conclude that a single sodium channel displaying unique compared to Mex makes this compound of interest in the treatment of myotonia. (Telethon Italy pharmacologic properties is expressed in AT-1 cells. 1208).

526- Pos 527- Pos BLOCK OF HUMAN HEART hHI SODIUM CHANNELS BY AMITRIPTYLINE. AGE AND GENE DOSAGE-RELATED Na CHANNEL ABNORMALITIES IN DMPK- Carla Nau', Ging Kuo Wang', Margaret Seaver2, Sho-Ya Wang2, 'Brigham & Women's Hospital, DEFICIENT MICE 2SUNY at Albany Dilaawar J Mistry', Joseph Randall Moorman', Sita Reddy9, John Paul Mounsey', 'University of Virginia, 2University ofSouthem Califomia, Los Angeles Amitriptyline is a tricyclic antidepressant used to treat major depression and various neuropathic pain syndromes. This drug causes cardiac toxicity in overdosed patients. We characterized the Myotonic dystrophy (DM), an autosomal dominant disorder symptomatic in later life, is tonic and use-dependent amitriptyline block of human cardiac (hHll) and mutant Nae channels characterized by late bursts of skeletal muscle Na channel openings. The genetic abnormality is (hHl-F1760K and hHI-Y1767K) expressed in Hek293t cells under voltage-clamp conditions. trinucleotide repeats near the dmpk gene, encoding a serine-threonine protein kinase DMPK. The Amitriptyline at I pM is an effective use-dependent blocker ofhHI Nae channels during repetitive Na channel abnomiality is recapitulated in young (<30 week) DMPK-deficient mice (dmpk-/-, pulses (-55% block at 5 Hz)..The tonic block measured as a 50% inhibitory concentration (IC50) submitted). We hypothesized that this would worsen with age, and that heterozygous mice for resting and for inactivated hHl channels is 25 and 0.6 FIM, respectively. Substitution of (dmpk+/-) would have a similar defect. We measured Na currents at 0 mV from cell-attached phenylalanine with lysine at the hHI-F1760K position eliminates the use-dependent block by patches in enzymatically-dissociated skeletal myocytes from DMPK-deficient and age-matched amitriptyline at I pM. The time constants of recovery from the inactivated-state amitriptyline wild-type mice. Late bursts were defined as an opening probability >10% measured from 10 to block in hHIl wild-type and hHI-F1760K mutant channels are 8.0 s and 0.45 s, respectively. A 1 10 msec after depolarization. Intermediate (30-60 week) and old (>60 week) homozygous dmpk- substitution at either hHI-F1760K or hHI-Y1767K significantly increases amitriptyline's IC5s /- muscle each had more late bursts than young (10.7 and 25.3%, vs. 8.5%, p<0.001), with values for resting and inactivated states but the increase is much more pronounced with the hH- persistent Na currents of 1.6%, 3% and 4.2% of peak in young, intermediate and old muscle. F1760K mutation. Since these two residues were proposed to form a part of local anesthetic There was no large effect of age on Na channels wild-type muscle. Muscle from heterozygous binding site, we conclude that amitriptyline and local anesthetics interact with a common binding dpmk+/- mice had a Na channel gating abnormality equivalent to dmpk -/- mice (18.6% vs. 10.7% site. Furthermore, the ability of amnitriptyline as a potent use-dependent blocker of Nae channels late bursts, p= NS). We conclude that the mouse models ofpartial and complete DMPK deficiency may, in part, explain its analgesic action. reproduce the Na channel gating abnormality found in the human disease, and that increasing age worsens the abnormality, as observed clinically.

89A 528-533 VOLTAGE-GATED NA CHANNELS SUNDAY

528- Pos 529- Pos ANTI-ACHR ANTIBODIES SELECTIVELY KNOCKOUT ENDPLATE Na+ CHANNELS. ROLE OF INTRACELLULAR CA2+ IN THE CELLULAR PHENOTYPE OF AN LQT-3 Robert Louis Ruff, Cleveland VAMC, 10701 East Blvd., Cleveland, Ohio 44106-1702 MUTATION OF THE HUMAN HEART NA+ CHANNEL a SUBUNIT (SCN5A): A Na+ channels are concentrated at the endplate, which increases the safety factor by reducing the COMPUTATIONAL ANALYSIS. AP threshold. Myasthenia Gravis (MG) is a neuromuscular transmission disease caused by anti- Xander H. Webrens', Hugues Abriel', Candido Cabo', Jesaia Benhorin2, Robert S. Kass', AChR antibodies. I examined if the endplate autoimmune attack involves Na+ channels as well as 'Columbia University, 630W 168th Str, PH 7W 318, New York, NY 10032, 2Bikur Cholim AChRs. A loose-patch voltage clamp measured macroscopic IN. and a giga-ohm seal patch clamp Hospital, Jerusalem, Israel studied single channel properties. Passively transferred MG (PTMG) was induced in rats using a D1790G, an SCN5A mutation linked to one form of the congenital heart disease LQT-3, does not monoclonal antibody directed against AChR. I measured IN, and AP properties on the endplate confer functional properties upon mutant channels that, by themselves, are expected to delay border (EB) and on extrajunctional membrane (EJ). In control rat fibers, AP threshold was 9-10 cellular repolarization. We used computational analysis to determine whether the mutant channel mV lower at the EB compared with EJ membrane. In PTMG, endplate IN. was reduced by 60% properties, instead, might prolong cellular action potentials through effects on other electrogenic and endplate AP thresholds were 8 mV higher compared with controls. IN, and APs on EJ pathways. We modeled the human cardiac ventricular action potential as described by Luo & membrane were similar for PTMG and control fibers. Nae channel conductance, single channel Rudy (1994) as our control. Upon this background, we modified only the properties ofthe voltage- open time and Nae channel inactivation were normal for channels recorded from EB of PTMG gated Na+ channel according to our patch clamp analysis of D1790G channels. Our results muscle or muscle incubated with anti-AChR Abs. CONCLUSIONS: In PTMG, endplate IN. is indicate that, consistent with patch clamp data, D1790G channels do not carry additional selectively reduced, which increases the endplate AP threshold. Endplate IN. is probably reduced (maintained) inward current through Na+channels during the plateau phase. Instead, the mutation because endplate membranes containing Na+ channels are lost in MG. [Support: Dept. Veterans indirectly causes an increase in L-type calcium channel activity during the initial phases of the Affairs, Office of Research and Development of the Medical Research Service] action potential which results in an increased Ca, transient and, subsequently, an increase of inward current through the Na/Ca exchanger and a decrease in (outward current) Iv,,. The net effect is action potential prolongation. Consistent with clinical data, APD prolongation is enhanced at low heart rates (HRs) in this model, and at sufficiently slow HR, action potential prolongation generates arrhythmogenic early after depolarizations.

530- Pos 531- Pos IONIC MECHANISMS RESPONSIBLE FOR THE ELECTROCARDIOGRAPHIC ELECTROPHYSIOLOGICAL CHARACTERISTICS OF WILD TYPE AND MUTANT PHENOTYPE OF THE BRUGADA SYNDROME ARE TEMPERATURE DEPENDENT SODIUM CHANNELS IN A NON-MAMMALIAN AND MAMMALIAN EXPRESSION Robert Dumaine', Jeffrey A Towbin2, Pedro Brugada3, Matteo Vatta2, Dmitri V Nesterenko', SYSTEM Vladislav V Nesterenko, Josep Brugadc4, Ramon Brugada2, Charles Antzelevitchl, 'Masonic Marieke W Veldkampl, Connie Alshinawi', Antonius Baartscheer', Martin Rook2, Maarten van Medical Research Lab., 2150 Bleecker, Utica, NY 13501, 2Baylor College of Medicine, 3OLV den Berg3, Jan Willem Viersma3, Irene van Langen', Arthur Wilde', 'Academic Medical Center, Hospital, Aalst Belgium, 4University of Barcelona, Barcelona Spain Meibergdreef 9, Amsterdam, 1105 AZ Netherlands, 2Utrecht University Hospital, 3Groningen Brugada syndrome is characterized electrocardiographically by an ST-segment elevation in VI to University Hospital V3 and a rapid polymorphic ventricular tachycardia that can degenerate into ventricular We recently described a large family with a highincidence of nocturnal sudden death, QT-interval fibrillation. Our group recently linked the disease to mutations in SCN5A, the gene encoding for prolongation and the 'Brugada ECG', linked to an insertion of aspartic acid in the C-terminal the a subunit of the cardiac sodium channel. When heterologously expressed in frog oocytes, the domain of the cardiac sodium channel protein (1795insD). Expression of wild type (WT) and electrophysiological data from the T1620M mutant failed to adequately explain the ECG mutant Na+channels in Xenopus oocytes revealed that the 1795insD mutation gave rise to a 7.3 phenotype. We hypothesized that at more physiological temperatures, the missense mutation may mV negative shiRt of the steady-state voltage-dependence of inactivation and a 8.1 mV positive change the gating of the sodium channel such that the net outward current is dramatically shift of the voltage-dependence of activation. In addition, 1795insD Na currents had five-fold augmented during the early phases of the right ventricular action potential. In the present study smaller amplitudes than wild type currents. Expression of the channels in a mammalian cell line we test this hypothesis by expressing T1620M in a mammalian cell line, using the patch clamp (HEK-293) similarly showed smaller peak current amplitudes (3-fold) and a 11.5 mV negative technique to study the currents at 32 °C. Our results indicate that T1620M inactivation kinetics shift of the steady-state voltage-dependence of inactivation for the 1795insD mutant. There was, are faster when compared to the wild type at 320C. Recovery from inactivation was slower for however, no significant shift in the voltage dependence of activation. The functional consequences T1620M at 32 °C and steady state activation was significantly shifted towards positive potentials. ofthe observed changes are most likely a reduced sodium current during the upstroke and phase I Our findings explain the features of the electrocardiogram of Brugada patients, illustrate for the of the action potential, which may account for the Brugada phenotype. Interestingly, the 3- to 5- first time a cardiac sodium channel mutation whose arrhythmogenicity is revealed only at fold difference in peak current amplitudes is more than expected on ground of the shifts in temperatures approaching the physiological range, and suggest that some patients may be more at activation- and inactivation curves alone, and suggests the presence of additional differences like a risk during febrile states. reduced sodium channel density or conductance.

532 - Pos 533 - Pos TOWARDS AN UNDERSTANDING OF THE EPITHELIAL SODIUM CHANNEL PORE DIFFERENTIAL RESPONSE OF LQT3 MUTANT SODIUM CHANNELS TO Stephan Kellenberger, Ivan Gautschi, Estelle Schneeberger, Laurent Schild, University of ADRENERGIC STIMULATION Lausanne, Bugnon 27, Lausanne, 1005 Switzerland Todd RIchard, Robert Dumaine, Masonic Medical Research Lab. The epithelial Na channel is of the class of ion channels (ENaC) part that consist of a tetramer of Long QT syndrome (LQTS) associated to the cardiac fast sodium channel (LQT3), results from a homologous subunits with two transmembrane segments each. We have recently shown that a larger late inward current that persists during the ventricular action potential (AP) plateau. LQT3 domain N-terminal of the second transmembrane segment M2 forms the outer pore with the patients are more likely to develop arrhythmias during sleep and exhibit shorter QT intervals selectivity filter and binding site for the diuretic amiloride. To address the role of the M2 during sympathetic stimulation. segments in the formation of the channel pore, we have replaced one at a time, amino acid To address this question we studied PKA regulation of LQT3 channels N1325S and AKPQ as residues in the M2 segment of the a subunit residues. These mutations did not by cys change ion expressed in Xenopus oocytes and challenged by extracellular IBMX (100 FM) and 8Br-cAMP selectivity or amiloride block, indicating that the M2 segment is not involved in the formation of the outer (400liM). Two electrode voltage clamp recordings showed 18.40 ±0.04%, 18.60±0.02 and pore or the selectivity filter. To test whether the M2 segments are part of the inner pore, 19.4±0.03 increases in currents for wild we applied sulfhiydryl reactive methanethiosulfonate (MTS) reagents from the cytoplasmic side peak type(WT), N1325Sand AKPQ respectively. Slope conductance was increased using the cut-open oocyte system. The amiloride-sensitive current of wt ENaC was completely by 12±2%, 11±1%, and 22±5% for WT, NS and AKPQ respectively blocked within 2 minutes by intracellular (but not extracellular) application of 0.1 mM MTSEA. without changes in steady-state activation or inactivation. Co-expression with 02-adrenergic To identify the cys residue(s) involved we have mutated intracellular cys residues of a, 1Band y receptors produced similar effects. PKA stimulation increased WT late conductance by 31±6% subunits to ser. The location of the cys residues modified by intracellular application of MTS after 400 msat OmV while N1325S and AKPQ were not significantly changed. These results reagents and their relation to the internal pore will be discussed. suggest that N1325S and AKPQ mutations disrupt PKA phosphorylation. LQT3 channels are therefore less sensitive and may not as efficiently as WT counterbalance adrenergic increases in outward currents during phase 2 of the ventricular AP. Conversely, a reduction of adrenergic tone may lead to over compensation of smaller outward currents. These findings may provide a basis for understanding the complex sympathetic regulation of LQT3 channels.

90A