880-881 PLATFORM AD - E-C COUPLING & CALCIUM SPARKS I MONDAY

880 - Plat 881 - Plat APOCALMODULIN ENHANCES THE Ca2+ SENSITIVITY OF RYR1 ACTIVATION REPOLARIZATION-INDUCED INHIBITION OF CALCIUM-INDUCED CALCIUM George G. Rodney, Susan L Hamilton, Baylor College of Medicine, One Baylor Plaza, Houstom, RELEASE (CICR) IN SKELETAL MYOTUBES Texas 77030 Norio Suda', Kurt G Beam2, 'Dept. of Physiology, The Jikei University School of Medicine, 3- 25-8 Nishishinbashi, Minato-ku, Tokyo 105-8461, JAPAN., 2Dept. of Anatomy & Neurobiology, The skeletal muscle calcium release channel (RYRI) is a Ca2' binding protein and, in addition, Colorado State University, Fort Collins, CO 80523, USA. binds and is regulated by a Ca2+ binding protein, calmodulin (CaM). RYRI binds four CaM Following a prior depolarization (e.g. -20- -10 mV for >10 s or >0 mV for 0.5-1.0 s), Ca2+ molecules at low and high Ca2+. CaM's effect on the functional state of RYRI is Ca2+ dependent; release induced by 5-20 mM caffeine (CafICR) is terminated by repolarization (RISC; Ca2+-free CaM (apoCaM) activates RYRI while Ca2+-bound CaM inhibits RYRI. RYRI is also repolarization-induced stop of CaRCR) in mammalian skeletal myotubes. To investigate whether directly modulated by Ca2+. We have previously shown that Ca2+ binding to CaM, not Ca2+ repolarization indeed causes inhibition of CICR, caffeine application was started immediately after binding to RYRI, converts CaM to an inhibitor of RYRI at high Ca2+. To determine the repolarization in (whole-cell) patch-clamped mouse skeletal myotubes dialyzed with an internal mechanism by which apoCaM activates RYRI we assessed the effects of both a mutant CaM that cannot bind Ca2+ (B1234Q) and caffeine on the Ca2+ dependence of RYRI activity. At sattrating solution containing 0.5 mM Fluo-3 as a Ca2+ indicator. Caffeine failed to induce regenerative concentrations, caffeine (10mM) alone shifts the Ca2+ dependence of [3H]ryanodine binding from Ca2+ release for 5-10 s regardless of the level of cytoplasmic Ca2+ concentration at the onset of an ECso of 9.0±2 sM (n=5) to 0.3±0.1 FM (n=2) whereas B1234Q (ltM) shifts the EC50 to caffeine application. By contrast, depolarization was able to evoke Ca2+ release during this 5-10 s 3.0±0.8 pM (n=5). effects of The caffeine and B1234Q on the Ca 2+ dependence of [3H]ryanodine period and caffeine was able to evoke Ca2+ release during depolarization or in the resting binding are additive, shifting the EC5o to less than 100nM. The Ca2" dependent decreases in state. [3H]ryanodine binding detected at high (mM) Ca2+ concentrations are not altered by caffeine or Because caffeine is thought to act by enhancing CICR, these results suggest that CICR becomes B1234Q. We propose that apoCaM increases the Ca2+ sensitivity of RYRI activation while Ca2+ refractory more easily than voltage-gated Ca2+ release. Supported by HFSP fellowship to N.S. binding to CaM inhibits channel activity. and NIH grants (NS24444 & AR44750) to K.G.B.

882-885 POSTERS: PROTEIN-LIGAND INTERACTIONS II MONDAY

882 - Pos 883- Pos A NOVEL SE3 LIGAND CONTAINED WITHIN AN SH2 DOMAIN THERMODYNAMIC INVESTIGATION OF PHOSPHOTYROSINE RECOGNITION BY Krlstine N Brazin, D. B. Fulton, A. H. Andreotti, Iowa State University, 4288 Molecular Biology SH2 DOMAINS Building, Ames, IA 50011 J. Michael Bradshaw, Vesselin Mitaxov, Gabriel Waksman, Washington Univ. Src homology 2 (SH2) and Src homology 3 (SH3) domains are small adaptor modules which SH2 domains are protein modules which bind tyrosine phosphorylated sequences within the cell. mediate protein-protein interactions through well characterized binding mechanisms. It has been To evaluate the importance of the phosphotyrosine (pTyr) residue in SH2 domain binding, demonstrated that SH2 domains bind to phosphotyrosine-containing sequences while SH3 titration calorimetry was used to investigate the binding of the Src SH2 domain to a domains interact with proline-rich regions in proteins. Interleukin-2 tyrosine kinase (Itk), a dephosphorylated peptide and the amino acid pTyr. pTyr binds with a surprisingly large AG (4.7 member of the Tec family kinases, contains both an SH2 and SH3 domain. We have observed a kcal/mol), which suggests that the process of specific recognition of tyrosine phosphorylated direct intermolecular interaction between the SH2 and SH3 domains of Itk. Through NMR targets occurs within a narrow range of free energy. The dephosphorylated peptide binds with a chemical shift mapping we have defined the interaction surfaces on both the SH2 and SH3 AG of -3.6 kcaVmol, indicating that the phosphate of tyrosyl phosphopeptides is a critical domains. Unlike most previously characterized ligands for SH3 domains, the region of the SH2 determinant of high affinity binding. Alanine mutagenesis was also used to energetically map domain involved in this binding event is a contiguous nonproline-rich loop that interacts with the residues of the Src SH2 domain required for binding of pTyr. Mutation of the strictly conserved SH3 binding pocket. Therefore, we have identified a novel SH3 ligand within the SH2 domain of Arg PB5 resulted in the large loss in binding free energy while elimination of all other examined Itk. Analogous binding studies between the Itk SH2 domain and SH3 domains from several other residues resulted in very little reduction in protein tyrosine affinity, indicating that Arg PB5 is the "hot spot" of kinases showed no significant chemical shift perturbations in two-dimensional 15N pTyr recognition. In addition, mutation of Cys surprisingly enhanced the phosphopeptide HSQC experiments. Thus, the observed interaction between the SH2 and SH3 domains of Itk is PC3 selective. A detailed NMR binding free energy by 1.1 kcal/mol. These results highlight the importance of a single critical study of the molecular recognition event between the Itk SH2 and SH3 the domains will be presented. Additionally, we propose a functional role for this interaction in interaction, buried ionic bond between the phosphate of the pTyr and Arg 13B5 of the SH2 regulating Itk activity. domain, driving the binding of SH2 domains to their targets.

884 - Pos 885- Pos DESIGN OF FLUORESCENT INDICATORS FOR cGMP EXPRESSION, PURIFICATION AND LIGAND BLOT ANALYSIS OF THE LDL-LIKE Akira Honda', Carolyn L Ellenberger', Stephen R Adams2, Charles Y Cho2, Roger Y Tsien2, DOMAINS OF THE LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN Wolfgang R.G. Dostmann', 'University of Vermont, 2University of California, San Diego (LRP) Johnny Eugene Croy, William Shin, Elizabeth A Komives, University of California, San Diego, To investigate the spatial and temporal dynamics of cGMP in live cells, we designed novel cGMP- 9500 Gilman Drive, La Jolla, Califomia 92037 indicators by fusion of green fluorescent protein (GFP) color variants to deletion fragments of cGMP-dependent protein kinase Ia (cGPK). Exploiting the fact that cGPK undergoes a Low density lipoprotein receptor-related protein has been implicated in the uptake of a number of conformational change upon ligand binding and based on a structural model of cGPK, cyan diverse ligands, ranging from proteinase/inhibitor complexes to 3-VLDL molecules enriched with fluorescence protein (CFP) and yellow fluorescence protein (YFP) were fused to cGMP-binding apoE. Many LRP ligands have also been found in the proteinaceous plaques associated with portions of cGPK at their N- and C-termini. Consequently, cGMP binding to the receptor portion Alzheimers Disease. Classified as a member of the LDL-receptor family, LRP morphologically of the hybrid-protein induced a decrease in fluorescence resonance energy transfer (FRET) resembles the fusion of four LDL receptors in tandem (N terminal LDL-like domain is incomplete between the fluorophores. Thereby, FRET is a direct measure of changes in cGMP. A series of and will not be studied). LRP is expressed as a 600 kDa protein and is processed to a non- CFP-AcGPK-YFP cGMP-indicators were initially expressed and purified from E. coli, excited at covalently bound 515 kDa extracellular domain and an 85 kDa transmembrane domain. Each 432 nm and the fluorescence emission determined at 475 nm (CFP) and 525 nm (YFP). The cGPK LDL like domain contains EGF and Complement type repeats. Due to the high number of deletions Al-77, At-109, A352-670 and A357-670 displayed A475 nm/A525 nm ratio changes of cystiene disulfide bonds, expression and purification has proven to be elusive. 1.15 - 1.3 upon adding cGMP. When expressed in mammalian cell lines these constructs showed predominantly cytosolic fluorescence (by confocal microscopy) with ratio changes of 1.2 - 1.5 We have after addition of the cell-permeable successfully expressed and purified each LDL-like repeat (sLRP 2, 3, 4) from the LRP analog 8-Br-cGMP. We propose that the cGMP-indicators extracellular domain in Pichia pastoris. Each sLRP has been shown here are valuable tools to probe the dynamics of intracellular cGMP by non-invasive purified to homogeneity, as means. demonstrated by N-terminal sequencing and western blot analysis. Binding of each sLRP to the receptor related protein (RAP) was demonstrated using a sandwich ligand blot of a GST-RAP fusion protein followed by rabbit anti-GST arllsera and finally HRP-goat anti rabbit antisera. Results from ligand competition experiments w also be presented.

150A MONDAY POSTERS: PROTEIN-LIGAND INTERACTIONS II 886-891

886 - Pos 887- Pos THE CONTRIBUTION TO HEMOGLOBIN COOPERATIVITY BY THE ASYMMETRIC THERMODYNAMIC LINKAGE ANALYSIS REVEALS NON-COOPERATIVE HALF-OXYGENATED INTERMEDIATE SUPPORTS THE SYMMETRY RULE MODEL BEHAVIOR IN HUMAN HEMOGLOBIN LIGATION INTERMEDIATES OF ALLOSTERIC REGULATION. Alexandra L Klinger, Jo M. Holt, Gary K. Ackers, Wash. Univ. Sch. Medicine, 660 S. Euclid Gary K. Ackers, Jo M. Holt, Yingwen Huang, Alexandra L. Klinger, Wash. Univ. Sch. Medicine, Ave., St. Louis, MO 63110 Biochem. & Molec. Biophysics, 660S. Euclid Ave., St. Louis, MO 63110 Cooperative binding of 02 by human hemoglobin (Hb) is manifested by the classic sigmoidal The contribution to hemoglobin cooperativity by the "asymmetric" half-oxygenated tetramer [a,B: binding curve and equilibrium Adair binding constants K, (i = 1,2,3,4). Resolution of unique ao21o2] was investigated using cryogenic isoelectric focusing. Tetramers were comprised of a,B values for these four parameters that describe the highly cooperative binding curve was shown to be possible using a thermodynamic linkage approach (see Adv. Prot. Chem. dimers containing Fe2'O2 hemesites, in combination with dimers containing Zn2+-protoporphyrin Ackers, 1998, 51, 185) in which sets of concentration-dependent binding isotherms are analyzed globally. This IX sites. The equilibrium abundances and hybridization timecourses were determined for this approach has been extended to the determination of sub-stoichiometric binding constants for two tetramer and its unligated counterpart, yielding their free energies of quatemnary assembly and 02 stable intermediates of doubly-oxygenated human hemoglobin (Hb), [deoxy Zn a2:FeO2 and binding. These constants, together with those determined for the other seven HbO2 intermediates 12] (Huang et al.,1996, Biophys. J. 71, 2094), yield the cooperativity contributions by all eight [FeO2 a2:deoxy Zn 12]. Multiwavelength binding isotherms, obtained over a range of Hb concentrations, were fit globally as a function of total [Hb] using constraints from independent partially oxygenated intermediates. These free energy distributions showed that: (a) The two 02 binding steps leading to the asymmetric tetramer have distinctly favorable cooperativity; (b) By detenninations of dimer-tetramer assembly constants for the unligated and fully oxygenated Zn/Fe contrast, the binding steps leading to the symmetric isomer showed no favorable cooperativity, as species. In both cases (deoxy a:oxy or oxy a:deoxy 1), the two successive binding steps to form was the case for the other doubly-oxygenated intermediates [23] and [24]. These features of the the doubly-ligated species exhibited no favorable cooperativity. This finding is consistent with HbO2 intermediates are not compatible with traditional allosteric models, i.e., MWC or KNF, but previous measurements of dimer to tetramer assembly free energies of the Zn/FeO2 microstates are fully consistent with the Symmetry Rule model (Ackers et al., 1992, Science 255, 54), in and with the Symmetry Rule model of Hb cooperativity (Ackers, et al., 1992, Science 255, 54). By which both concerted and sequential effects play specific roles. contrast, these findings are incompatible with the traditional MWC model. Supported by NIH and NSF. Supported by NIH & NSF.

888- Pos 889 - Pos CHARACTERIZATION OF INDIVIDUAL ALLOSTERIC INTERACTIONS WITHIN ALLOSTERISM WITHOUT SUBUNITS: "ACCELERATED" TITRATION OF A PHOSPHOFRUCTOKINASE FROM BACILLUS STEAROTHERMOPHILUS. MONOMERIC ION CHANNEL. Jennifer L. Kimmel, Gregory D. Reinhart, Texas A&M University, Mail Stop 2128, College Tatlana K Rostovtsevai, T.-T. Liu2, M. Colombini2, V. A. Parsegian', S. M. Bezrukov', 'NIH, Station, Texas 77843-2128 Bethesda, MD 20892, 2University of Maryland, College Park, MD 20742 Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a model system in which the We have been compelled to re-examine the widely accepted connection between cooperativity and interactions between substrates and allosteric effectors have been extensively studied. However, allosterism. The monomeric VDAC channel shows an accelerated pH titration of its transport the oligomeric nature of BsPFK has made it difficult to determine the molecular basis of the properties with a Hill coefficient of about 2. This manifests itself as a sharp peak in conductance allosteric regulation because within the native structure, four separate heterotropic allosteric noise as well as a fast change in channel selectivity with pH. Rather than look at this accelerated interactions are possible between the enzyme's four active and allosteric sites. In an attempt to titration in the traditional but impossible framework that would involve two functional subunits, alleviate the complexity of the system, site-directed mutagenesis has been coupled with a hybrid- we propose that cooperativity derives from a mechanically-linked mobile pair of ionizable groups. forming scheme to create and isolate a tetramer of BsPFK in which a single active and a single Concerted movement of these groups between two states changes the distance from nearby allosteric site are capable of interacting with each other. Characterization of this single allosteric electrostatic charge to influence the pK of the groups. This model of pH-dependent motion interaction indicates that the free energy involved in the inhibition by the allosteric effector produces "cooperative" behavior that fits the observations. Based on this behavior, we claim that, phosphoenolpyruvate is -1.05 + 0.14 kcal/mol compared to the -2.00 i 0.03 kcal/mol measured for quite generally, high phenomenological cooperativity does not necessarily require a large number the native tetramer. This result suggests that the concerted two-state model fails to adequately of interacting subunits, or, in fact, any subunits in the protein structure at all. Compared to large- describe the allosteric regulation of BsPFK. This approach has also been used to characterize a scale conformational transitions, these subtle cooperative structural changes may allow proteins to single interaction between the substrate fructose-6-phosphate and the allosteric activator MgADP. adapt, with high sensitivity, to changes in their environment. To detennine whether each of the four individual interactions contribute equally to the allosteric regulation of the native tetramer, the free energies associated with either inhibition or activation of two separate interactions will be reported and compared. Supported by grants from NIH (GM33216) and the Welch Foundation (A1368).

890 - Pos 891 - Pos MUTATIONS BETWEEN CALCIUM BINDING SITES I & II ALTER INTERDOMAIN CALMODULIN-TARGET INTERACTIONS CRYSTAL STRUCTURES OF ANTICOOPERATIVITY OF CALMODULIN. COMPLEXES OF CALMODULIN WITH TARGET PEPTIDES OF PHOSPHO- 0. Jaren, L. Coffeen, M.A. Shea, University of Iowa College of Medicine, Iowa City, IA 52242- FRUCTOKINASE AND SKELETAL MUSCLE MYOSIN LIGHT CHAIN KINASE. 1109 Peter M Bayley', Emanuel Skordalakes', Lesley Haire', Steve Gamblin', Steve Smerdon', Franco Calmodulin (CaM) is a small protein ( 148 residues) with four Ca2' binding sites. Calcium binding Zanini2, Pete Browne', Murray Skinner', Stephen Martin', Guy Dodson', 'National Institute for to CaM regulates many kinds of protein activities. A pair of Ca2' binding sites lies in both the N- Medical research, Mill Hill, London, NW7 IAA United Kingdom, 2Elletra, Trieste and C-domains of CaM (sites I and II are in the N-domain, residues 1-75, sites III and IV are in The 1.7A resolution crystal structure is reported of the complex of seleno-methionine calmodulin the C-domain, residues 76-148). Each pair of sites binds two Ca2' ions in a positively cooperative with a 21 residue peptide sequence of mammalian phosphofructokinase manner. Negative cooperativity between the pairs of sites makes the calcium affinity of the N- (KLRGRSFMNNWEVYKLLAHIR), which has only low homology with typical basic domain sites lower than that of the C-domain sites. In order to study the linked binding of Ca2' amphipathic helical sequences. Residues 5-78 and 80-148 of calmodulin and 8-21 of the peptide and target proteins, mutant Paramecium CaMs were used. These were first identified in cells are well resolved. Both domains interact with the peptide, of which residues WEVYKLL are defective in chemotaxis due to faulty ion channel regulation (Kung et al., Cell Calcium 13: 425- alpha-helical, with minor distortion. The complex differs from other X-ray structures ICDL and 37). Our hypothesis is that mutations between sites I and II disrupt negative cooperativity 1CDM in the relative disposition of the two domains, although these substantially retain features between the pairs of sites, while mutations within sites III and IV disrupt cooperativity and the of their typical structure. It is closest in structure to ICDM, two prominent hydrophobic residues Ca2+ affinity of those sites. Heteronuclear NMR and proteolytic footprinting were used to monitor of the peptide WI I and L20 being separated by 8 intervening residues. However, the detailed Ca2+-dependent conformational changes in wild-type and mutant Paramecium CaMs. Results structure of the peptide in the complex and 1CDM differ significantly in the orientation relative to indicate that mutations between sites I and II increase the the N-domain of calmodulin, and the nature of a substantial number of the side-chain interactions. Ca2' affinity of these sites to a level similar to that of sites Thrombin Footprinting: Also, the structure of the complex of calmodulin with an 18-residue skeletal muscle myosin light III and IV. This suggests that anticooperativity between R106 near site II chain kinase peptide was solved to 1.7A resolution, and allows detailed comparison to be made the two domains of CaM is disrupted by these mutations. wr with the published nmr structure, (2BBM) and with the corresponding X-ray structure of the Mutations within Ca2' binding sites (e.g. H135R) decrease related complex with the smooth muscle MLCK target peptide, (I CDL). Cas2 affinity and sometimes alter cooperativity between the paired sites. Understanding the effects of these mutations will allow us to dissect how interactions H13SR between the four Ca2+ binding sites of calmodulin correlate with defects in regulation.

151A 892-897 POSTERS: PROTEIN-LIGAND INTERACTIONS II11 MONDAY

892 - Pos 893 - Pos STRUCTURAL CHARACTERIZATION OF THE ULTRASENSITIVE ALLOSTERIC CLUSTERIN BINDS THROUGH THE ACTION OF NATIVELY DISORDERED REGULATION OF CYANOBACTERIAL ADP-GLUCOSE PYROPHOSPHORYLASE. REGIONS Alberto A. Iglesias, Diego F. Gomez Casati, Miguel A. Aon, Instituto Tecnologico de Robert W Bailey, Jian Yang, A. Keith Dunker, Michael D. Griswold, Washington State Chascomus, Camino Circunv. Laguna km6, CC164, Chascomus, Buenos Aires B7130WCA University, School of Molecular Bioscience, Pullman, WA 99164-4660 Argentina Clusterin is associated with cellular injury, lipid transport and apoptosis and may be involved in ADPglucose pyrophosphorylase (AGPase; EC 2.7.7.27) is the key regulatory enzyme in the the clearance of cellular debris caused by cell injury or death. Consistent with this idea clusterin pathway for glycogen and starch biosynthesis in bacteria and plants, respectively. The enzyme binds to a variety of molecules including lipids, peptides, proteins, and the hydrophobic probe 1- from organisms performing oxygenic photosynthesis is allosterically regulated by 3P-glycerate anilino-8-naphthalene sulfonate (ANS). Clusterin's binding action is unknown. We show that (activator) and Pi (inhibitor). AGPase from Anabaena PCC 7120 exhibited an ultrasensitive clusterin contains three regions of natively disordered structure containing putative amphipathic response towards 3P-glycerate, elicited by Pi and the crowding inducing agent polyethylenglycol helices. These regions may be involved in clusterin binding. To test this hypothesis we studied (PEG). Ultrasensitivity was observed either at zero- or first-order region respect to the the effects of denaturation on the fluorescence of the clusterin-ANS complex compared to physiological substrates. Kinetic and regulatory parameters of the enzyme were significantly proteins with structured binding pockets and molten globular forms of proteins. Protein-ANS affected by the presence of PEG in the medium. Spectrofluorometric studies showed that AGPase complexes were titrated with increasing urea concentration and the ANS fluorescence profiles undergoes conformational changes when exposed to molecular crowding conditions. The different (F/Fo) were compared. Clusterin has a F/Fo profile more similar to molten globular form proteins conformational states of the enzyme were related with the changes observed in kinetic and than proteins with well structured binding pockets. Clusterin's disordered regions may bind ANS regulatory properties, including those exhibiting the ultrasensitive response. Fourth derivative in a fashion similar to molten globular proteins. We propose that natively disordered regions may spectra analysis of the protein under ultrasensitivity conditions evidenced the existence of changes form a dynamic binding site and provide clusterin the ability to bind to a variety of molecules. in the environment of tryptophan residues. Studies of molecular exclusion chromatography showed no changes in the quatemary structure of the enzyme. Results suggest that the ultrasensitive response exhibited by AGPase is consequence of intramolecular conformational changes undergone by the enzyme. These structural changes may be operative in vivo, rendering an accurated mechanism for the fine regulation of the enzyme activity, mediated by a cross-talk between 3PGA and Pi.

894 - Pos 895- Pos ASSESSMENT OF A HOMOCYSTEINE: COPPER INTERACTION USING THE BINDING OF ANESTHETIC ALKANES AND AN ALCOHOL TO A DESIGNED FOUR- OXYGEN RADICAL ABSORBANCE CAPACITANCE ASSAY. a-HELIX BUNDLE. Tericka M. Smith', Maria Medina2, 'Barry University, 2University of California, Davis K Solt, JS Johansson, Dept. of Anesthesia, and the Johnson Res. Foundation, Univ. of Homocysteine is an intermediate amino acid that results from the metabolism of methionine, an Pennsylvania, Philadelphia, PA 19104. es-sential amino acid. Many scientific findings have shown elevated levels of homocysteine to be The structural characteristics of general anesthetic binding sites on proteins are being determined associated with cardiovascular disease. However, the mechanism by which homocysteine exerts using a model system consisting of a four-a-helix bundle scaffold with a hydrophobic core. The its atherogenic effect remains unknown. One hypothesis is that homo-cysteine interacts with core can be modified using synthetic chemistry, allowing direct testing of the impact of cavity size copper in the blood plasma. We will utilize the Oxygen Radical Absorbance Capacitance Assay and polar contributions to anesthetic binding. These synthetic peptide bundles serve as scaled- (O.R.A.C.) to access the interaction of homo-cysteine with copper along with a number of other down models for the lipid-spanning domains of several membrane proteins. A four-a-helix Sulfur-containing compounds. bundle variant of (Aa2)2 which contains a binding pocket for halothane (Biochemistry 37:1421, 1998) was synthesized that features a methionine for leucine substitution - (Aa2-L38M)2. (Supported by the MARC-NIH, Grant GM08021-16). Halothane binds to the hydrophobic core of (Aa2-L38M)2 with a Kd of 0.20 i 0.01 mM, as monitored by tryptophan fluorescence quenching, approximating its clinical EC50 of 250 PM. Similarly, chloroform binds with a Kd of 1.4 ± 0.2 mM, quite comparable to its EC50 in dogs of 1.2 ± 0.2 mM (Anesthesiology 30:129, 1969). Finally, 2,2,2-trichloroethanol binds with a Kd = 19.5 ± 1.2 mM, again approximating its EC50 in mice of 27 mM (Biochem Pharmacol 29:3011, 1980). The results indicate that different classes of anesthetic molecules are able to interact with the same site on a protein, suggesting that similar domains may exist on the central nervous system macromolecules that are the targets of general anesthetics. The relative simplicity of the protein bundles allows the structural and dynp"'ic consequences of anesthetic binding to be probed using detailed biophysical approaches, prt ing insight into how anesthetics might alter protein function. Supported by NIH GM55876.

896- Pos 897- Pos THE STRUCTURAL STABILITY OF BINDING SITES PROTEIN-LIGAND INTERACTONS: INSIGHTS FROM VOLUMETRIC STUDIES Irene Luque, Emesto Freire, The Johns Hopkins University, Baltimore, MD 21218 Tigran V. Chalikian, David N. Dubins, Rana Filfil, Robert B. Macgregor, Jr., University of Toronto, 19 Russell Street, Toronto, Ontario M5S 2S2 Canada The structural stability of the binding sites of 16 different proteins (Arabinose-Binding-Protein, Biotin-Repressor, Glycerol-Kinase, HIV-I-Protease, Lysozyme-G, Methionine-Aminopeptidase, We have used ultrasonic velocimetry, high precision densimetry, UV absorbance and CD p38-Mitogen-Activated-Kinase, N-Myristoyl-Transferase, RAB3-GTPase, RAS, Retinoic-Acid- spectroscopy, in conjunction with high pressure UV melting to detect and characterize the binding Receptor, Spectrin-SH3-Domain, Thymidilate-Synthase, TEM-l-,-Lactamase, Tumor-Necrosis- of ribonuclease A (RNase A) to cytidine 2'-monophosphate (2'-CMP) and cytidine 3'- Factor-a-Converting-Enzyme, Tumor-Suppressor-pl9Iik4A) has been explored by a structure-based monophosphate (3'-CMP). We report the changes in volume, AV, and adiabatic compressibility, thermodynamic analysis using the COREX algorithm. The results of these studies indicate that AKs, that accompany the association of 2'-CMP and 3'-CMP with RNase A over a wide the binding sites of many proteins have a dual character: they are simultaneously formed by temperature range. In general, the magnitudes of AV and AKs are small. This results from regions of high structural stability and regions of low structural stability. There are important compensation effects between the intrinsic and hydration terms. Specifically, a significant increase consequences to this structural arrangement. It has been shown before (Freire, E. (1999) Proc. in the interaction volume, V,, compensates decreases in the intrinsic volume, VM, and the thermal Natl. Acad. Sci. USA 96 10118-10122) that the presence of a significant fraction of residues with volume, VT, while an increase in the hydration term, AKh, compensates a decrease in the intrinsic low structural stability in the uncomplexed binding site is important for the transmission of compressibility, KM. The volume and compressibility results suggest that 210 ± 40 water binding effects to distal regions. In those cases, the interactions with ligands will cause a molecules are released to the bulk state upon the binding of 2-CMP or 3'-CMP to RNase A. The redistribution in the native state ensemble and a concomitant change in the residue stability binding of these ligands leads to a -15 % decrease in the configurational entropy of the protein, constants of residues distal to the binding site. If the binding sites were formed by binding- suggesting a decrease in the conformational dynamics of the protein upon ligand binding. The competent, high stability residues only, all the states in the native ensemble would be binding- putative decrease in protein dynamics is consistent with the measured decrease in the intrinsic competent and ligand binding will only induce an energy shift without an internal reordering in the volume, VM, (1.4 to 2.1 %) and the intrinsic compressibility, KM, (5 %) of RNase A upon the ensemble. A ligand induced redistribution in the probabilities of conformational states requires binding to 2'-CMP or 3'-CMP. Thus, RNase A becomes "smaller", "tighter", and "less mobile" that only a subset of the native ensemble is binding-competent. This condition is satisfied when upon binding either ligand. part of the residues that define the binding site exhibit low structural stability or exist in a non binding-competent conformation in the unligated protein.

152A MONDAY POSTERS: PROTEIN-LIGAND INTERACTIONS II 898-03

898 - Pos 899 - Peo EVALUATING PROTEIN pK'S BASED ON TITRATION CURVES AND DESIGN OF BIOSENSORS INTERACTING WITM THE ATP RECOGNITION SITE OF ELECTROSTATICS. DNA TOPOISOMERASE I Leonard J. BanaDsak, Edward Hoeffner, Judy Bratt, University of Minnesota, 321 Church St, LilUane Asairl, Institut de G6n6tique et Microbiologie, CNRS, Universite de Paris-Sud, Centre d, Minneapolis, MN 55455 91405 Orsay C6dex, France. DNA topoisomerase II which is an essential enzyme involved in the segregation of chromosomes, To test a method for assigning pK values to ionizable sidechains, an experimental titration curve is phosphorylated by a specific kinase which thus, regulates its activity. The topography of the was determined for the adipocyte lipid binding protein (131 aa). The implied pK's of the resulting ATP recognition site has evolved during the evolution of living organisms from bacteria to hydrogen ion equilibrium trace were determined using an evolutionary algorithm for curve fitting eukaryotes. Structural changes usually occur upon phosphorylation of the enzyme. In order to by calculating the sum of de-protonation curves with simple Henderson-Hasselbach behavior. measure the variation and to quantify the active and inactive forms of the enzyme, biosensors are Random populations of curves were generated and the least difference between the titration curve designed and will be synthesised. The molecules proposed are based either upon the substrate and the calculated curve was used as the selection criterion. New curves were generated from old structure (1) either the competitive inhibitors structure (2) or upon the peptidomimetics through ones by mutating the underlying pK values with a normal probability distribution centered at the protein-protein interactions. A biosensor like (pyrene-ATP) complex has been used for measuring current pK value. Once the difference between the selection criteria evaluated for the best and interactions between ATP and the myosin ATPase subunit (3). Such a model will be used for worst of the parent population was less than a specified amount, the process was terminated. A designing biosensors recognising the ATP binding site of DNA topoisomerase II and especially list of "calculated" pK's was developed using the computed electrostatic field (DELPHI) of the specific biosensors able to distinguish active and inactive forms of DNA topoisomerase II. protein and the standard pK's. Individual pK's were then assigned by comparing the evolutionarily - Assairi, L. ( 1997) Letters in Peptide Science 4, 403-406. determined list of "observed" pK's with the calculated list. (Supported by a grant from the NIH- 2 - Assairi, L. (1992) Letters in Peptide Science 2, 169-171. GM13925 and the Minnesota Supercomputer Institute.) 3 - Hiratsuka, T. (1997) Biophys. J. 72, 843-849.

900- Pos 901 - Pos RANDOM SEQUENTIAL ADSORPTION - RANDOM SITE MODEL RESOLVES ALLOSTERIC REGULATION OF S-100 DEPENDENT MOLECULES WITH CALCIUM. INCONSISTENCY BETWEEN KINETIC PARAMETERS OBTAINED IN SOLUTION hiroshi none mizukami, wayne.state university, biological sciences, 5047 gullen mall, detroit, (STOPPED-FLOW) AND AT SOLID-LIQUID INTERFACE (BIOSENSOR) michigan 48202 Gabor Csacs', H.R. Bosshard2, 'Lab. for Biomechanics, ETH Zurich, Wagistrasse 4, Schlieren, A model is proposed to describe quantitatively how various concentrations of calcium CH-8952 Switzerland, 2Dept. of Biochemistry, University of Zurich, Zurich CH-8953, allosterically regulate the function of the target molecules of S-100 proteins. S-100 proteins, like Switzerland calmodulin, have many target molecules to which they bind upon activation with calcium. Optical biosensors measure reactions between surface-bound and soluble molecules at a solid- However, it is not well understood how the activity of the target molecules depends on the liquid interface. Kinetic parameters deduced from biosensor measurements may differ from those concentration of calcium and if their activity is allosteric. In this proposed model, calcium ligand for freely diffusible reactants in solution. Discrepancies have been assigned to steric effects at the binds to an S-100 protein allosterically and changes its conformation to bind the target molecule, solid-liquid interface. We have studied antigen-antibody binding by the biosensor technique and phosphodiesterase. Activated phosphodiesterase breaks down the substrate, cAMP, to AMP. by stopped-flow measurements in solution. The apparent rate of association between a single- Using the model proposed by Monod-Wyman-Changeux, the fraction of conformational change of chain antibody fragment (scFv) and the leucine zipper domain of yeast transcription factor GCN4 S-100 is determined under varying concentrations of calcium. Conformationally altered S-100 was 20 times slower when measured with optical light-mode spectroscopy (OWLS) at a solid- binds and activates phosphodiesterase. The activity of enzyme may be expressed with the liquid interface (Weber-Bomhauser et al. Biochemistry 37: 13011, 1998). The kinetic analysis was conventional Michaeles-Menton parameters. The model suggests that the activity of the enzyme is based on a simple Langmuir binding model, which adequately describes the antigen-antibody sigmoidal to the concentration of calcium and is dependent on the dissociation constants between reaction in solution but cannot account for steric effects at the solid-liquid interface, e.g., for the activated S-100 and the target enzymes. The activity of target molecules in signal transduction heterogeneity in surface-bound reactants. Here we show that the inconsistency between biosensor is, in general, to facilitate the process of cascade, but it may also be to scavenge unwanted and stopped-flow measurements disappear when the biosensor data are analyzed by the (RSA-RS). The model was developed some time ago reactions in cells. (Jin et al. AIChE Joumal 40:1658, 1994) but has not yet been used to analyze biosensor data. The model can account, at least in part, for steric effects at the solid-liquid interface.

902 - Pos 903 - Pos NON-COOPERATIVE PATHWAYS IN THE HEMOGLOBIN 02 BINDING CASCADE A MICROSCOPIC VIEW ON COOPERATIVITY Alexandra L. Klinger, Jo M. Holt, Gary K. Ackers, Wash. Univ. Sch. Medicine, 660 S. Euclid Nadja Hellmano, Heinz Decker, Institute for Molecular Biophysics University of Mainz Ave., St. Louis, MO 63110 HEMOGLOBIN BINDING CASCADE. 02 Cooperativity in ligand binding or enzymatic activity is a widespread phenomena. If the number of Understanding the molecular mechanisms by which human hemoglobin (Hb) binds 02 subunits is rather large, the number of models for cooperativity, which can be applied to a cooperatively is contingent upon determining properties of the eight partially oxygenated particular set of data, increases due to the increasing number of free parameters. Recent intermediates (Ackers, 1998, Adv. Prot. Chem. 51, 185). Traditional 02 binding studies, however, developments in the field of single molecule detection seems to open new possibilities to can only yield information on average properties of the intermediates that comprise each distinguish between different models for cooperativity. We want to adress the question, under stoichiometric degree of oxygenation: i.e., species with the same number of 02 molecules bound which circumstances a decision between concerted and sequential mechanism for cooperativity in different site configurations are indistinguishable. To resolve thermodynamic parameters of can be made. We simulated data, for which the ligand binding curve can equally well be described such "ligation microstate" analogs, 02 binding parameters have been measured for Zn2+/Fe2+ by a concerted and a sequential model. The distribution of molecules containing 1,2..n occupied hybrid Hbs in which Fe2+ protopoiphyrin is replaced by inert, but essentially isostructural, Zn2+ binding sites is not suitable to distinguish between the two models. The observation of the protoporphyrin; either in both a or both 15 subunits. Oxygen binding curves for the two hybrid Hbs fluctuations in the saturation level of a single molecule can yield information about the occupation were measured as a function of total protein concentration in order to vary the relative abundance level which is capable of conformational transitions. Under certain circumstances a sequential of dimers and tetramers. The resulting 02 binding curves were found to contain sufficient mechanism leads to different fluctuations compared to a concerted model. However, the best information to extract both dimeric and tetrameric binding parameters for the hybrids using global approach would be to monitor both occupation level and conformation of the molecule as non-linear least squares fitting. The two binding steps that form these symmetric doubly-ligated indicated by an appropiate fluorescence label. The influence of the size of the allosteric unit on the intermediates were found to be essentially non-cooperative. Mechanistic implications of the kinetic behaviour of a single molecule and the consequences for molecular regulation will be presence of these non-cooperative pathways in the HbAo oxygen binding cascade will be discussed. discussed with respect to both the MWC and the Symmetry Rule models. Supported by NIH & NSF.

153A 904-909 POSTERS: PROTEIN-LIGAND INTERACTIONS II MONDAY

904 - Pos 905 - Pos CALORIMETRIC STUDIES OF SMALL LIGAND BINDING TO FLEXIBLE LOOP ANESTHETIC BINDING TO A G PROTEIN COUPLED RECEPTOR DEMONSTRATED VARIANTS OF THE MULTIFUNCTIONAL BIOTIN REPRESSOR USING TRYPTOPHAN FLUORESCENCE. Keehwan Kwon, Dorothy Beckett, University of Maryland, Dept. of Chem and Biochem, College Yumiko Ishizawa, Paul A Liebman, Roderic G Eckenhoff, University of Pennsylvania, Park, MD 20742 Department of Anesthesia, 3400 Spruce Street, Philadelphia, PA 19104 The multifunctional protein, BirA, regulates transcription of the biotin biosynthetic genes via binding to the biotin operator sequence (bioO), and catalyzes biotin linkage to a biotin-dependent Binding of the volatile anesthetic halothane to rhodopsin, a G protein coupled receptor, in the rod carboxylase. The protein is composed of three domains with the central or catalytic domain disk membranes (RDM) has been studied using tryptophan fluorescence spectroscopy. The RDM containing sequences conserved in all biotin holoenzyme synthetases. One of these conserved is exposed to the light, and G proteins are stripped from the membranes. Halothane quenches the segments, "IGRGRRGI0, is part a surface loop that results of thermodynamic studies implicate in steady-state tryptophan fluorescence of the RDM by 80% in a concentration-dependent, saturable both small ligand and DNA binding. Recent X-ray crystallographic studies indicate that this loop manner with a Kd of 1.9mM (95%CI 1.7-2.2mM). The emission maximum is significantly shifted segment directly contacts the ligand in the BirAbiotin complex. In this work isothermal titration from 329.8nm without halothane to 332.6nm with 10mM halothane (red shift). The residual calorimetry has been used to dissect the thermodynamics of ligand binding to three single-site emission intensity further decreases to 10% of the control in the presence of 0.8M iodide, a water- mutants in this loop. Results of these studies indicate that defects in biotin binding in the mutants soluble quencher. The ability of halothane to quench tryptophan fluorescence of the RDM is not are manifested primarily in changes in the enthalpy of binding. Mutants that are defective in bio- affected in the presence of isoflurane, 1-chloro-l, 2, 2-trifluorocyclobutane, or ethanol. These 5'-AMP binding exhibit changes in either the enthalpy or entropy of the process. Finally, a loop results suggest that halothane binds in a hydrophobic domain in rhodopsin, which contains 3 or 4 mutant that is defective in only DNA binding exhibits a thermodynamic profile similar to that of tryptophans of the 5 tryptophan residues in this receptor according to a recently proposed model (J the wild type protein in small ligand binding. Results of these studies allow correlation of the Mol Biol 272:144,1997). The results also suggest that the halothane binding site is selective and thermodynamics with the recently obtained structural data. The results furthermore suggest a specific. direct role of the small ligand binding loop in sequence-specific DNA binding. Supported by GM51595 and EYOOO12.

906- Pos 907- Pos ENERGY OF SALT BRIDGES BETWEEN ARGININES AND PHOSPHATE ARE MORE ANALYSIS OF VAN'T HOFF AND CALORIMETRIC ENTHALPIES FROM ITC THAN ADDITIVE AND SUPPORT A MODEL IN WHICH ANY DEVIATION FROM Kenaeth P. Murphy', James R. Hom', Donald J. Russell2, Edwin A. Lewis2, 'University of Iowa IDEALITY INCREASES QUADRATICALLY. College of Medicine, 4-403 Bowen Science Bldg., Iowa City, IA 52242, 2Calorimetry Sciences Robert M Stroud, Janet S. Finer-Moore, University of California in San Francisco, San Francisco, Corp. California 94143-448 Significant discrepancies have been noted between the binding enthalpy determined In thynmidylate synthaae, four arginines each provide two hydrogen bonds to the oxygens calorimetrically ( H,,w) from isothermal titration calorimetry (ITC) experiments, and those of the phosphate group of the substrate, dUMP. Kinetics and crystal structures of dUMP determined from analysis of the temperature dependence of the binding constant, the so-called complexes of single mutants, and double mutants and are analyzed. The free energy for van't Hoff enthalpy ( H,H), determined from the same ITC experiments. We have studied the productive dUMP binding, deltaGs increases by at least 1 keal/mol for each mutant, even when binding of 2'-CMP to ribonuclease A, and the binding of Ba+to 18-crown-6 as a function of dUMP orientation and mobility in the crystal structure is the same as in wild-type enzyme. Thus, temperature using ITC in order to investigate possible sources for this discrepancy. The role of the third and fourth arginine do not contribute excess positive charge to the P042 binding site; heat capacity changes, error in data analysis, and linked equilibria will be discussed. rather the four positive charges ideally complement the two negative charges and geometry of the phosphate anion. More-than-additive increases in deltaGs seen in the double mutants are consistent with quadratic increases in deltaGs predicted for deviations from ideal electrostatic balance of charge, and may also reflect cooperative binding of the arginines to the phosphate oxygens. These results therefore support an electrostatic model in which the second shell and the protein buffers the arginine shell around the phosphate, making it effectively balance the two negative charges. And they support the case that any deviation from such ideal balance increases as the square of the difference in charge from ideal.

908 - Pos 909 - Pos BIND - A DATABASE FOR STORING BIOMOLECULAR INTERACTIONS, INSECTICIDAL ACTIVITIES OF SEVERAL TRIORGANOTIN COMPOUNDS BIOCHEMISTRY AND KINETICS. AGAINST ADULT ANOPHELESSTEPHENSIMOSQUITO. Gary D Bader', Ian Donaldson2, Berivan Baskin2, Tony Pawson2, Christopher W.V. Hogue2, QUYEN DIEU DUONG, Nwaka Ogwuru, George Eng, UDC, CHEMISTRY, 4200 Connecticut 'University of Toronto, Dept. of Biochem., 2Samuel Lunenfeld Research Inst. Ave., N.W., Washington, DC 20008 Each protein expressed in a cell can interact with various different proteins and other molecules in the course of its function. interactions are Protein-protein often mediated by modular protein The insecticidal activities of fourteen triorganotin compounds (R3SnX) against the adult female domains. One example is the SH3 domain which binds a proline rich motif. These "interaction networks" form conventional Anopheles stephensi mosquito were investigated using a contact studies protocol. The female signaling cascades, transcription activation complexes, vesicle Anopheles stephensi mosquito is the vector of the parasite causing human malaria. The controlling mechanisms, cellular growth and differentiation systems, among other cellular Known cellular interactions will preliminary results indicated that the potency of the triorganotin compounds was depended on the machinery. protein eventually comprise more information than anionic X group atached to the tin atom. The contact studies indicated that the most effective the Human Genome Project. We present a new public submission database called BIND Interaction Network database compound after a 24h exposure was triphenyltin fluoride with a mean LC55 value of 0.40ppm. (Biomolecular Database). This spans the complexity of interaction The other triphenyltin compounds were less effective but they still exhibited a rapid knock-down information gathered through experimental studies of biomolecular interactions. Interaction effect (still alive but not flying) against the adult female Anopheles stephensi information will come from the literature, submitters and other databases. BIND contains mosquitoes. interaction, molecular complex and pathway records. An interaction record is based on the interaction between two objects. An object can be a protein, DNA, RNA, ligand or molecular complex. Description of an interaction encompasses cellular location, experimental conditions used to observe the interaction, conserved sequence, molecular location of interaction, chemical action, kinetics, thermodynamics, and chemical state. Molecular complexes are defined as collections of more than two interactions that form a complex, with extra descriptive information such as complex topology. Pathways are defined as collections of more than two interactions that form apathway, with extra descriptive information such as cell cycle stage.

154A MONDAY PHYSICAL CHEMISTRY 910-915

910- Pos 911 - Pos CONFORMATIONAL CHANGES OF PROTEINS AT SOLID/WATER INTERFACES NEPHROCALCIN AFFECTS KIDNEY STONE MORPHOLOGY BY BINDING Maarten F.M. Engel, C.P.M. van Mierlo, A.J.W.G. Visser, Wageningen University, Dreyenlaan CALCIUM OXALATE CRYSTAL SURFACES 3, Wageningen, 6703 HA Josh W. Kurutz, Mauricio Carbalho, Sanjeev Shroff, Yasushi Nakagawa, University of Chicago, Knowledge about the conformation of proteins at interfaces is important in a large number of food 5841 S. Maryland Ave., MC-5084, Chicago, IL 60637 and non-food applications. Lysozyme (hen egg white) and a-lactalbumin (bovine) are adsorbed at hydrophilic silica and hydrophobic polystyrene nanoparticles that form a suspension in water. $ephrocalcin is a human urine protein that prevents microscopic calcium oxalate crystals, which These two proteins have large similarity in their three- dimensional structure but their adsorption are present in everyone, from developing into kidney stones. There are four isoforns of behaviour is completely different. Lysozyme is a structurally stable protein, while a-lactalbumin nephrocalcin: A and B, the predominant forms in the urine of people not likely to form stones; and is a flexible protein that shows significant conformational changes when adsorbed. The effect of C and D, the major forms in people who suffer from calcium oxalate kidney stones. To investigate changes in concentration, pH and ionic strength on the conformational changes of the adsorbed the mechanism of nephrocalcin's inhibitory effect, calcium oxalate crystals were grown in the proteins is studied with far and near UV Circular Dichroism and intrinsic steady state and time presence of each isoform and in the absence of protein. Resultant crystal size was determined resolved fluorescence spectscopy. In the near future asnide proton exchange and NMR using a Coulter counter. Crystal morphology was assessed with electron microscopy. Topography experiments will be used to investigate the conformation of lysozyme adsorbed at a solid/water of the crystal surface was explored using Scanning Force Microscopy (SFM). We observed interface. significant structural differences between crystals grown in the presence of nephrocalcin and those grown with no protein. Moreover, we found that isoforms C and D, the less beneficial proteins, promoted microcrystalline aggregation, whereas isoform A was found to reduce crystal size. SFM reveals a protein coat over the crystals, strongly suggesting that nephrocalcin acts by coating crystals to prevent growth. We are currently involved in filrther high-resolution SFM aimed at discerning the mechanism by which nephrocalcin binds to calcium oxalate surfaces. This represents the first investigation of kidney stone surfaces incorporating molecular-level resolution and stone-inhibiting proteins.

912 - Pos 913 - Pos ANOMALOUS DIELECTRIC BEHAVIOUR OF SINGLE CRYSTAL GLYCINE NEAR OSMOTIC STRESS AND HOFMEISTER SOLUTES IN ROOM TEMPERATURE HYDROXYPROPYLCELLULOSE SELF-ASSEMBLY Benno P. Schoenboms', Terry C. Chilcott2, Wayne D. Cooke', Hans G.L. Coster2, 'Los Alamos John K Cbik', S Mizrahi2, Donald C Rau', Adrian Parsegian', 'NIH, Bg 12A Rm 2041, Bethesda, National Laboratory, M888, Los Alamos, New Mexico 87545, 2University of NSW MD 20892-5626, 2Technion Reversible heat precipitation of hydroxypropylcellulose (HPC), an uncharged water soluble Preliminary impedance measurements of glycine crystals with a novel high resistance dielectric derivative of cellulose, from dilute solution and x-ray scattering from carefully concentrated HPC impedance device have revealed that the electrical conductance of these crystals exhibit an films allow us to explore the solvation of neutral surfaces under two extreme density regimes. apparent anomalous diphasic dependence on temperature. On cooling the crystal from 50 °C, the Solute effects on the precipitation temperature (as measured by optical turbidity) are consistent electrical conductance first decreased with decreasing temperature (by a factor of >1.3 for a -10 with their ordering in the ubiquitous Hofineister series. Combined solutes sometimes act °C change in temperature). Further cooling below 31 °C produced a three thousand fold increase additively, but often produce unexpected enhancements. For example KF, a "salting-out" agent, in conductance for a -10 °C change in temperature. The dependence of the capacitance on drops the precipitation tenmperature faster in the presence of KSCN, on its own a "salting-in" temperature was small for temperatures above 31 °C and was generally negatively correlated with solute. Osmotically stressing concentrated HPC films reveals an exponentially decaying repulsive that of the conductance for temperatures below 31 °C but decreases precipitously below this force, a signature of the hydration force and, to our surprise, a sharp jump in x-ray spacing over a temperature. This unusual dielectric behaviour is not explained by normal conduction mechanism small osmotic pressure range which we attribute to a conformational transition of HPC. This jump but is consistent with the onset of pyroelectric properties at 310 C. These dielectric measurements and the repulsive force depend strongly on temperature. Added salts modify these forces in ways are now being augmented with neutron diffraction studies below and above the transition parallel to their effect on the heat precipitation of dilute HPC. Solutes that salt out HPC compact temperature. the concentrated film. In both cases, this effect is explained by the solute's net exclusion from HPC. For salting-in agents, the x-ray spacing of the HPC film expands which we interpreted as inclusion of these solutes with HPC.

914 - Pos 915- Pos HYDRATION OR DEHYDRATION, WHICH IS THE MORE SENSITIVE TO OSMOTIC THE MECHANISM OF HYDROPHOBIC SOLVATION DEPENDS ON SOLUTE STRESS? RADIUS Adrian Parsegian', Sergey M. Bezrukov2, Tatiana K RostovtsevaW, Donald C Rau', Nina Y Noel T Southall, Ken A Dill, University of California at San Francisco, Graduate Group in Sidoroval, 'NIH, NIH, Bethesda, MD 20892-5626, 2NICHD/NIH, Bldg.9, Rm/IE-122, Bethesda, Biophysics, 3333 California St., San Francisco, CA 94118 MD 20892

We study how the size of a nonpolar solute affects the thermodynamics of its solvation. We use a Hemoglobin, hexokinase, aspartate transcarbamylase, cytochrome C, DNA/protein complexes, simplified statistical mechanical model of water, the Mercedes Benz (MB) model, in NPT Monte ionic channels, and many other molecular assemblies in fact, probably all the macromolecules in Carlo simulations. This model qualitatively predicts the temperature dependence of the volume cells -- exquisitely sense the chemical potential of water. This is because changes in conformation anomalies of pure water and the transfer free energy, enthalpy, and entropy for inserting a or association incur large changes in the number of associated waters. Each water molecule acts as nonpolar solute into water. Inserting small solutes is opposed by entropy due to ordering the a ligand to create the significant collective power of all waters. Thermodynamic measurements by surrounding waters, while insertion into hot water is opposed by enthalpy due to breaking our group and by others have now evolved to see the action of osmotic stress on kinetics. So far hydrogen bonds among the surrounding waters. But the mechanism for inserting a large nonpolar we have found that when a dehydrated form is stabilized by osmotic stress, that stress lengthens solute into water is temperature independent and driven by enthalpy: a large surface drives water the life of the dry state (closed alamethicin or porin channel; specific DNA-protein complex) to break hydrogen bonds at all temperatures. Thus the traditional "iceberg" explanation for the rather than shorten the life of the wet (open channel or non-specifically bound complex). For large heat capacity does not apply to large solutes; i.e., the first-shell water structure does not melt the lifetime of example, dry, specifically associated protein/DNA changes with the same out with temperature. This model also explains why the free energy of creating an oil/water sensitivity to osmotic stress as does the between specific and non-specific association. Ergo, K,. surface at planar interfaces (75 cal A-2 molI ) is three times that at interfaces of small osmotic sensitivity resides in the dry/closed/specific rather than the wet/open/non-specific state. molecules(25 cal A-2 mol1 ). A key conclusion is that hydrophobic solvation depends not only on the surface area of a solute, but also on its shape and curvature.

lSSA 916-921 PHYSICAL CHEMISTRY MONDAY

916 - Pos 917- Pos VARIETIES OF KNOTS AND HINGES-THE MACHINERY OF CATALYSIS CONFORMATIONAL DYNAMICS STUDIES OF CO BINDING TO WATER SOLUBLE rufus lumry, university of minnesota, 207 Pleasant St.,SE, Minneapolis,, MN 55455 IRON PROPHRYINS USING PHOTOTHERMAL BEAM DEFLECTION Most familiar proteins have substructures: knots for folded stability and genetic stability; Randy William Larsen, Beverly D. Barker, University of Hawaii-Manoa, Chemistry, 2545 The matrices for physiological function and surfaces for communication with the environment. Some Mall, Honolulu, Hawaii 96822 single protein units such as protease inhibitors appear to depend for function on dynamic The focus of this study is to examine volume and thermodynamic profiles of ligand binding matching to their targets but no common relationship between function and structure for the entire associated with CO-Fe(II) tetrakis-(4-sulphonatophenyl)porphyrin (COFe(II)4SP) in aqueous class has yet emerged and may not exist. On the other hand those enzymes that do not utilize solution. Temperature dependent photothermal beam deflection was employed to probe the overall cofactors directly in function have a single sophisticated construction principle dependent on the enthalpy and volume changes associated with CO-photolysis and recombination. The analysis pairing of two single units in a dyad arrangement. The dyad axis is non-crystallographic and demonstrates that ligand recombination occurs with a pseudo first-order rate constant of passes between the two chemically-functional groups and the point at which the units are (2.1+0.2)x105 s-l (at 260C) with a corresponding volume increase of 14.5+4 ml/mole. The connected, often called the hinge. The two single units required per catalytic function are matched to oscillate like a fork so oriented as to transient enthalpy of activation (AH+) and activation volume (AV+) for CO recombination (determined dynamically tuning raise by compression from temperature/pressure dependent transient absorption spectroscopy) are found to be 6.9 the potential energy of the transition state. This ubiquitous catalytic mechanism is a result of evolution of B-factor In each unit the atoms with lowest kcal/mole and -7.1 mL/mole, respectively. Overall the data show a single step volume change near-perfect palindromic patterns. B associated with CO binding to iron porphyrins. These results provide a complete volume factors fit together to form the rigid knot. The two knots of a catalytic dyad are palindromically profile related but have little if residue cores" as described for the CO-rebinding reaction and demonstrate the utility of coupling photothermal techniques any similarity. "Slow-exchange by their with variable pressure/temperature transient absorption residues are not quite identical with knots described by atoms. The many knots so far described spectroscopy. demonstrate that the strict symmetry requirements can be met by a wide variety of different constructions. Thus four families of ribonuclease-A enzymes have totally different detailed structures and furthermore very different hinges. Representative examples from the embryonic catalog of knots and hinges illustrate some of the variety of successful enzyme evolution. (cf. Lumry, Methods Enzymology, vol 259, chap. 29)

918 - Pos 919- Pos EFFECT OF CALCIUM BINDING ON THE STABILITY AND BIOLOGICAL STABILITY OF MYELIN STRUCTURE AS A RESULT OF VARYING MEMBRANE ACTIVITY OF m-AMYLASE. ADHESION AND LIPID PACKING Ali Akbar Saboury, F. Karbassi, A. A. Moosavi-Movahedi, University of Tehran, Tehran, Iran Yufang Ha', Cynthia Husted', Jacob N Israelachvili2, 'Univ. of California, Santa Barbara, The interaction of a-amylase from Bacillus amyloliquefaciens with divalent calcium ion was Chemical Engineering, Santa Barbara, CA 93106, 2University of California, Santa Barbara investigated by different techniques including equilibrium dialysis, isothermal titration calorimetry and UV spectrophotometry in 10 mM Tris buffer at pH=7.5 and 298 K. There is a set of 17 The stability of myelin membrane is key to the understanding of Multiple Sclerosis, which is binding sites for calcium binding on the enzyme with weak positive cooperativity (Hill coefficient believed to be an auto-immune disease characterized by demylination of neuron axons. Recent equal to 1.135). The binding of calcium is exothermic (AH=-16 kJ/mol) with mean dissociation study has shown vesiculation of myelin membranes as a possible precursor of demylination binding constant of 0.55 mM. The interaction of calcium caused the more stability of the enzyme (Gerain, C.P., Cannella, B., Hauser, S. L., Raine, C.S., Nature Medicine 5, 170 (1999]). Viewing against surfactant denaturation resulting in an increase both of the surfactant concentration for this vesiculation as a consequence of myelin membrane instability triggered by immunological transition and AG' (H2O). Thermal denaturation study of ca-amylase showed that calcium binding events, one can hypothesize that this instability is caused by a combination of two parameters: The leads to thermal stability enhancement. The transition temperature (T,) and AG' (298 K) of the change in packing condition of lipid in myelin membrane and the reduction of adhesion between enzyme, which are obtained from the sigmoidal thermal denaturation curves, raised by increasing the membranes. This hypothesis will be examined in this study using dynamic light scattering and concentration of calcium ions. The presence of calcium ions prevents from the spontaneous force measurements techniques. decrease in biological activity of a-amylase. The spontaneous denaturation of a-amylase, due to its spontaneous deactivation, has been studied by monitoring the heat exchange and residual activity of an enzyme solution with respect to time. The kinetics of denaturation was found to obey a first order law, depending on the concentration of ax-amylase. The rate constants of denaturation decreased by increasing the concentration of calcium; however, the heat of necessary for enzyme denaturation increased.

920- Pos 921 - Pos BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF A NEW OSMOTICALLY INDUCED SHAPE CHANGES TO LARGE UNILAMELLAR CONSTRUCT OF DIPHTHERIA TOXIN REPRESSOR PROTEIN, SGH-DtxR(C102D). VESICLES: EFFECTS OF LIPID AND SOLUTE COMPOSITION G. Parthasarathy, P.D. Twigg, L. Guerrero, T.M. Logan, D.L.D. Caspar, Institute in Molecular Jeremy Pencer, Erin Bamett, Gisele White, F. Ross Hallett, University of Guelph, Guelph, Biophysics, Florida State University, Tallahassee, FL 32306 Ontario NIG 2WI Canada DtxR regulates expression of diphtheria toxin. It is the best-characterized element of a family of regulators controlling the iron-responsive genes. The repressor is activated by Fe+2 induced We have used static (SLS) and dynamic (DLS) light scattering to investigate the effects of solute dimerization. The dimeric form of DtxR in apo, metal and DNA-bound form has been crystallized and lipid composition on osmotically induced shape changes to large unilamellar vesicles (LUVs) and well characterized. However, the characterization of the monomer form of DtxR has been prepared by extrusion. We induce shape changes by diluting vesicles into media of higher difficult because of dimerization in the absence of metal at [DtxR]>l0 M. Here, we report the osmolalities. For the vesicles used here, the volume of LUVs will decrease to balance the lumenal biochemical and the biophysical properties of a new construct of the repressor, sghC102D, which osmolality with that of the medium while the surface area of the membrane remains constant (1,3). has three additional residues at the N-terminus of the protein. These three residues apparently Based on these volume changes, we can compare changes in the radius of gyration (measured by block the dimerization of sghCI02D under the conditions the native protein shows dimerization SLS) and hydrodynamic radius (measured by DLS) to those predicted for a variety of shapes (as shown by the non-denaturing gels). Also, the sghC102D construct lacks the DNA binding including prolate and oblate ellipsoids of constant surface area. We also examine the apparent activity, as shown by the gel shift assay. However, the sghCI02D construct binds activating metal elongation ratio using the method of Jin et al (2) and compare it to that for prolate and oblate ions, as assayed by fluorescence quenching. The addition of metal has different effects on the local ellipsoids. We find that the categories of shapes and shape changes exhibited vary with both tryptophan environment in the sghC102D construct as compared to the native protein. However, solute and lipid composition. the CD measurements indicate no significant difference in the overall secondary structure of the metal-free and the metal-bound forms. The construct and sghC102D the wild-type protein show L. different Tm's, around 55°C and 63°C respectively, as characterized by CD measurements. 1. Beney, et al. (1998), Eur. Biophys. J., 27: 567 - 574. Preliminary DSC measurements also show similar melting temperatures. We also report the 2. A. J. Jin, et al. (1999), Eur. Biophys. J., 28: 187-199. determination of thermodynamic parameters associated with protein-DNA interactions. 3. G. White, et al. (1996), Biophys. J., 71: 2701 - 2715. (Supported by NSERC of Canada)

156A MONDAY MEMBRANE PROTEINS 922-927

922 - Pos 923 - Pos CRYSTALLOGRAPHIC STRUCTURE AND FUNCTIONAL INTERPRETATION OF PEPTIDES WITH THE SEQUENCE OF LOOPS IN BACTERIORHODOPSIN ADOPT THE CYTOPLASMIC DOMAIN OF ERYTHROCYTE MEMBRANE BAND 3. THE SAME CONFORMATION IN SOLUTION AS IN THE CRYSTAL STRUCTURE Philip S. Low, D. Zhang, A. Kiyatkin, J. Johnson, J. T. Bolin, Purdue University, 1393 Brown Philip L Yeagle, Madan Katragadda, Univ. Connecticut, U-3125, Storrs, CT 06269 Bldg., West Lafayette, IN 47907-1393 We have recently been investigating the degree to which peptides containing the sequence of turns The red cell membrane is depicted in virtually all modern biochemistry textbooks as a model of (between tranamembrane helices) of integral membrane proteins retain in solution the same animal cell plasma membranes. A major organizing center of this membrane is the cytoplasmic structure as in the intact protein. Bacteriorhodopsin offers an excellent opportunity to test this domain of band 3 (cdb3) which links 8 distinct proteins to the membrane, including ankyrin (the hypothesis.since several crystal structures are available for this transmembrane protein. In these major bridge to the membrane skeleton), protein 4.1, protein 4.2, glyceraldehyde-3-phosphate crystal structures, tbree of the loops are reasonably well defined: loops CD, DE and FG. We dehydrogenase, aldolase, phosphofructokinase, hemoglobin, hemichromes, and the protein synthesized individual peptides containing the sequence of each of these loops. The sequences are tyrosine kinase, p72s'k. We report here the crystallographic structure of cdb3 at 2.6A resolution. relatively hydrophobic and these peptides were not soluble in water. However, they were soluble CDB3 is shown to be a dimer of two tight dimers, each stabilized by interlocking arms. The in DMSO. Their structures were determined in DMSO by homonuclear two dimensional H-1 monomers are in turn comprised of two domains, a small interdigitating dimerization domain and NMR. In each case, the peptides formed a defined structure in solution and the results showed a a much larger peripheal protein binding domain. The latter domain is constructed of a central turn as part of that structure. When these structures were superimposed on the appropriate loops eight-stranded 13-pleated sheet sandwiched between multiple a-helices. Binding sites of several in the structures available from crystallography, remarkable fidelity was observed. peripheral protein ligands can be approximated in the structure, as can a possible source of the major conformational change that regulates membrane-skeletal interactions. Mutations that result in altered erythrocyte morphology are also easily localized. An better defined structure of the protein network at the inner surface of the red blood cell membrane can now be presented.

924- Pos 925 - Pos DEUTERIUM NMR REVEALS HELLX PACKING INTERACTIONS IN RESOLUTION STRUCTURE OF THE M2 TRANSMEMBRANE PEPTIDE (TMP) H+ PHOSPHOLAMBAN CHANNEL FROM SOLID STATE NMR Weiwen Ylng, Steven 0 Smith, SUNY Stony Brook, Biochemistry and Cell Biology, Nicholls Junfeng Wang', Jeffery Denny2, Sanguk Kim', Yiming Mo', Katsuyuki Nishimura', Jack Quine2, Rd, Stony Brook, NY 11794 Tim A Cross', 'National High Magnetic Field Laboratory, Tallahassee, FL, 1800E Dirac Dr., Phospholamban is a 52 residue membrane protein involved in the regulation of calcium levels Tallahassee, FL 32310, 2Florida State University across sarcoplasmic reticulum membranes in cardiac muscle cells. The C-terminal 22 amino acids The transmembrane ion channel formed by a tetramer of M2 protein from influenza A plays an are largely hydrophobic, adopt a-helical secondary structure and associate non-covalently to form important role in viral infection. Recent observations of "helical wheel" resonance patterns in 2D a pentameric complex. A novel deuterium NMR approach is presented for establishing the 15N PISEMA spectra in which 15N chemical shift is displayed versus 15N-lH dipolar interaction rotational orientation of the phospholamban monomer within the pentamer. We targeted three provide a unique method for determining the global orientation of alpha helices in an oriented consecutive leucine residues (Leu42, 43 and 44) in the transmembrane domain of phospholamban lamellar phase lipid environment Our studies on M2 TMP indicate that the helical tilt from the to establish the relative motion of the side chains. A distinct difference in lineshapes is observed channel axis (tau = 38±3'), and the rotational orientation about the helical axis (rho = -105±100), between Leu42 and Leu43, 44. The spectra of -d3-Leu43 and Leu44 are similar and exhibit a can be determined from uniformly and/or amino acid specifically labeled protein without characteristic Pake pattern with a quadrupole splitting of 33 kHz. The deuterium lineshape of 8-dr knowledge of resonance assignments. The comparison of simulated "PISEMA wheels" from Leu42 has lost the distinctive 33 kHz quadrupole splitting due to increased rotational motion about various M2 TMP models in the literature with the experimental results will be shown. A high the Ca-Coi and CO-Cy bonds. The observed lineshapes of the three consecutive leucine residues in resolution M2 TMP backbone structure is determined and refined based on the orientational phospholamban are consistent with Leu42 being oriented towards the lipids where it exhibits constraints from 2D PISEMA experiments. The "PISEMA wheel" pattern and the derived tau and fewer steric contacts and Leu43 and Leu44 being oriented towards helix interfaces which restrict rho play a critical role in addressing the possible orientational ambiguities associated with their motion. This orientation of the three leucine residues is in agreement with two recent models structural determination from orientational constraints for generating a unique structure. of the phospholamban pentamer based on mutational data.

926- Pos 927- Pos STRUCTURAL STUDIES OF PHOSPHOLAMBAN USING SOUID-STATE MAGIC- THREE-DIMENSIONAL MAP OF AQUAPORIN I AT 4 A RESOLUTION ANGLE SPINNING (MAS) NMR METHODS. DETERMINED BY ELECTRON CRYO-CRYSTALLOGRAPHY OF FROZEN- Zareen Ahmed, Clemens Glaubitz, Anthony Watts, David A Middleton, Biomembrane Structure HYDRATED 2-DCRYSTAL Unit, Biochemistry Department, South Parks Road, Oxford, Oxon OXI 3QU United Kingdom Gang Ren, Peter Melnyk, Anchi Cheng, The Scripps Research Institute, 10550 N. Torrey Pines Phospholamban (PLB) is a 52 amino-acid membrane spanning peptide found in the sarcoplasmic Road, MB2 1, La Jolla, CA 92037 reticulum of cardiac myocytes. Its role is to regulate the activity of the resident Ca2'pump (Ca2+- ATPase) via an inhibitory association. of in to Phosphorylation PLB, response 3-adrenergic A three-dimensional (3D) density map of deglycosylated, human aquaporin 1 (AQP1) determined stimulation, causes it to dissociate from the Ca2+-ATPase and restore pump the Ca2'pumping at 4A resolution in-plane and -8.5A resolution perpendicular to the bilayer is presented. The map activity. The inhibitory site is thought to lie in the transmembrane region of PLB, with long-range was calculated by analyzing images and electron diffraction patterns recorded from tilted (up to modulation occurring through cytoplasmic interactions. 60'), ice-embedded 2-D crystals of AQPI in lipid bilayer membranes. This map significantly The structure of the transmembrane domain, PLB29.52, was investigated in reconstituted extends the findings related to the folding of the AQPIpolypeptide chain determined earlier by us membranes using rotational resonance 13C solid-state NMR. The local secondary structure of at a lower 7A by -20A resolution. The solvent-accessible volume within a monomer has a PLB2,.52 between "3CO(Phe-32) and '3Ccx(Ala-36), both in the absence and presence of Ca2'- vestibular shape with a narrow, -6A diameter constriction near the center of the bilayer where the ATPase, was found to lie be between 0.4 0.57nm. Computer modeling shows that this is location of the water-selective channel is postulated. Clearly resolved densities for the consistent with a a-helical conformation. transmembrane helices display protrusions expected for bulky side chains which allowed the A new technique, magic angle sample spinning of uniformly oriented membranes (MAOSS) was positioning of a polyalanine model. Stretches of density within the helix barrel suggest the spatial applied to full lengti PLB,.52 (containing 5N, '3C and 2H labels in both the cytoplasmic and dispositions of the functionally important Asp-Pro-Ala (NPA) loops. In addition, bands of density transmembrane region). Analysis of sideband intensities from '5N, '3C and 2H -MAS was used to positioned near the extremities of the transmembrane helices suggest the locations of some of the measure orientations of immobile molecular groups within PLB,.52 and orientations of the inter-helix loops. Based on the improved map, a model for the threading of the AQPI polypeptide proposed transmembrane helix with respect to the membrane plane. Phosphorylation induced chain is presented. conformational changes was also investigated.

157A 928-933 MEMBRANE PROTEINS MONDAY

928- Pos 929 - Pos IN CUBO MEMBRANE PROTEIN CRYSTALLIZATION. EFFECT OF DODECYL STRUCTURAL STUDIES OF FECA, THE FERRIC CITRATE TRANSPORTER OF MALTOSIDE ON THE PHASE PROPERTIES OF HYDRATED MONOOLEIN. ESCHERICHN colU OUTER MEMBRANE. Xi Ai, M. Caffrey, The Ohio State University, Columbus, OH 43210 Herve Celia', Volkmar Braun2, Franc Pattus', 'CNRS UPR9050, Bd Sebastien Brandt, Illkirch, F- Because of their mild nature, the alkyl glycosides have found extensive use in solubilizing 67400 France, 2Universtat Tuebingen membrane proteins for subsequent structure charcterization, reconstitution and crystallization FecA is an E. coli outer membrane receptor and is responsible of the binding and active transport studies. Dodecyl maltoside (DM) has been used in such applications and is currently being of iron citrate into the periplasm. The structures of two closely related iron transporters, FhuA and evaluated for its effects on the system used to induce crystallization of membrane proteins in cubo. FepA, have been solved by Xray crystallography (Locher et al., Ferguson et al., 1998; Buchanan The latter method uses a multicomponent system where hydrated monoolein in the cubic et aL, 1999). Despite this high resolution structural information, the precise mechanism on how mesophase figures prominently. While the exact role of the cubic phase in the crystallization the iron is transported remains unknown. process is still a mystery, it exists as the major phase prior to and subsequent to protein crystal Using an overexpressing strain, we have purified FecA to homogeneity. Three-dimensional formation. Accordingly, adventitious materials, have the potential for destabilizing the host cubic crystals have been obtained in the presence of different detergents, and will be used to solve the phase. We have used low-angle x-ray diffraction to characterize the phases formed when hydrated structure of the protein by Xray crystallography. As for FhuA (Lambert et al., 1999) the monoolein in the cubic phase is combined with DM. Interestingly, the cubic phase tolerates high reconstitution of FecA into liposomes has permitted to grow two-dimensional crystals of this concentrations of the detergent but eventually destabilizes in favor of the lanellar phase. The protein, suitable for electron crystallographic studies. By combining these two approaches, we transformation makes good sense given the complementary shapes of the detergent and monoolein wish to determine what are the different conformational changes of FecA that lead to the molecules. The T-dependence of the DM-induced cubic-cubic and cubic-lamellar transitions translocation of ferric citrate. determined under both equilibrium and metastable conditions shows that the cubic phase can be Locher et al. (1998) Cell 95: 771-778 accessed and worked with down to 0°C with a DM concn. up to ca. 0.1M. Ferguson et al. (1998) Science 282: 2215-2220 Buchanan et al. (1999) Nat. Struct. Biol. 6: 56-63 Lambert etaaL (1999) J. Struct. Biol. 126: 145-155

930 - Pos 931 - Pos MPEx & MPtopo: TOOLS FOR MEMBRANE PROTEIN TOPOLOGY ANALYSIS. ANALYSIS OF A TRANSMEMBRANE PEPTIDE: PRION(110-137) INBICELLES Sajlth Jaysslnghe, Kalina Hristova, Stephen H. White, Univ. of California, Dept. of Physiology Kerney Jebrell Glover, Matthew J. Wood, Regitze R. Vold, Elizabeth A. Komives, University of and Biophysics, Irvine, CA 92697 California, San Diego, 9500 Gilman Dr Mail Code 0359, La Jolla, CA 92093 Prediction tools continue to be important in the analysis of membrane protein topology. We Transmembrane domains are involved in a variety of cellular processes including introduce two tools that are useful for structural analysis: Membrane Protein Explorer (MPEx), a receptor dimerization and channel formation. Specifically, the bicelle has emerged as a Java applet available over the WWW for computing protein hydropathy profiles, and MPtopo, a particularly powerful medium for the examination and characterization of these domains due to its sequence database of proteins with experimentally determined transmembrane segments. MPEx "native-like" characteristics. Bicelles are composed of two types of phospholipids: computes hydropathy profiles using the experimentally determined, whole-residue dimyristoylphosphatidylcholine (DMPC) and dihexonylphosphatidylcholine (DHPC). The long hydrophobicity scales of Wimlcy and White'. It is highly interactive and provides several useful chain lipids (DMPC) form a true planar bilayer in the center of the bicelle while the short chain capabilities such as the ability to protonate/deprotonate Asp, Glu and His residues selectively. lipids (DHPC) occupy the rim where they shield the long chain lipid tails from water. MPEx also contains two modules: Totalizer, for the calculation of partition free energies and Furthermore, the unique phase behavior of bicelles allows for studies under both isotropic and Wheeler, for the generation of helical wheels that facilitates the rapid investigation of peptides or anisotropic conditions. short protein segments. We have carried out detailed examinations of the hydropathy profiles of We are focusing on the twenty-seven amino acid ER membrane-spanning domain of an proteins in MPtopo of known 3D structure. The transmembrane segments predicted by MPEx are isoform of the prion protein which is thought to confer severe neurodegenerative disease. Circular in excellent agreement with those observed experimentally. MPtopo, freely available and Dichroism analysis reveals that although the transmembrane region is not highly structured, it accessible via MPEx, contains over 70 proteins of kmown and unknown 3D structure, with a total does contain a significant degree of I-strand conformation. In addition, high resolution NMR data of over 300 experimentally verified TM segments. MPEx and MPtopo are available for use from shows predominately random coil chemical shifts. Solid state 2H NMR reveals that the bicelles our website at http://blanco.biomol.uci.edu/mpex. Supported by GM-46823 retain their structural integrity in the presence of peptide, and the quadrupolar splittings of the 'White, S.H., and Wimley, W.C., 1999. Annu. Rev. Biophys. Biomol. Struct. 28:319-65. lipids are those expected for an ideal bicelle.

932 - Pos 933- Pos FREQUENCY DEPENDENCE OF MEMBRANE CAPACITANCE AND DETERMINATION OF ACTIVATION PARAMETERS OF THE PROTON TRANSFER CONDUCTANCE CHANGES BY ACTION OF THE Na,K-ATPase AND OF THE RETINAL RE-ISOMERIZATION IN THE BACTERIORHODOPSIN Valerij S Sokolov', A. G. Ayuyana', Hans-Juergen Apell2, 'Frumkin Institute of PHOTOCYCLE BY USE OF TIME-RESOLVED VIBRATIONAL SPECTROSCOPY Electrochemistry, Moscow, Russian Federation, 2Department of Biology, University of Konstanz, Colin D Heyes, Jianping Wang, Mostafa A El-Sayed, Georgia Institute of Technology, 770 State Konstanz, Germany street, Atlanta, GA 30332-0400 Na,K-ATPase-enriched membrane fragments adsorbed to a BLM aie a convenient system to study non-steady-state electrogenic ion transport induced by ATP-concentration jumps or by extenally Abstract applied alternating voltage. Earlier the ATP-induced capacitance change was measured at different frequencies and fitted by one Lorentzian function to determine the rate constant of the transport The kinetic parameters of process (Sokolov et al, 1998, Eur. Biophys. J. 27:605). However, this constant was much higher the protonation/deprotonation of Asp85 and of the retinal re- isomerization in are step scan than that obtained by voltage-clamp experiments on cells. A possibly reason is that the bacteriorhodopsin (bR) determined using time resolved FT-IR investigated partial reaction includes several steps. To prove it the complex admittance spectroscopy by following the peak at 1750cm'1 and the bleach peak at 1528cm" respectively. A versus (capacitance and conductance) was measured in a wide frequency range. The results were fitted by plot of the peak intensity time at various temperatures enabled us to determine the rate constants and the activation parameters for each of these processes. The values obtained for the sum of two Lorentzians, one with a characteristic rate constant of 50-100s at 24°C the kinetic parameters for each process are discussed. (comparable to measurements on cells), the other one with -1000s-' (in agreement with our previous data). The rate constants were decreased at low temperatures and in solutions containing high concentrations of choline salts with different anions. Qualitatively, their effect was the same as in experiments with short circuit currents measuring after photorelease of ATP, both findings in conforsnity with the Hofmeister series of chaotropic ions (Cl'

158A MONDAY MEMBRANE PROTEINS 934-939934-939

934 - Pos 935 - Pos OF THE PROTEIN CONFORMATION CHANGE ON SIMULATING PACKING: DEVELOPMENT OF ALGORITHMS FOR PREDICTION OF TRANSMEMBRANE REGIONS OF ALPHA-HELICAL MEMBRANE PROTEINS Jianping Wang, El-Sayed, Georgia Institute of Technology, 770 State street, Atlanta, Jonathan N Sachs', Thomas B Woolf, 'The Johns Hopkins University School of Medicine, 30332-0400 Biomedical Engineering, 725 N. Wolfe St., Baltimore, MD 21205, 2Johns Hopkins Medical photocycle of bacteriorhodopsin (bR) was followed by use of time-resolved Fourier-transform School infrared (FTIR) spectroscopy in the temperature range of 15°C to 85°C, during which the a,, -4 ct, conformational transition occurs in bR. The photocycle rate increases with increasing temperature, A complete description of the function of membrane bound proteins will ultimately rely upon an efficiency is found to be drastically reduced as the transition takes place. This is shown to understanding of helix packing within the membrane. This has been clearly demonstrated with the result in a large shift of the all-trans *+13-cis equilibrium due to the increased stability of the 13- structure of the KcsA channel (Doyle et al., Science 280:69-77, 1998), which suggests that cis a, form. This, together with the increase in the rate of dark adaptation as the changes in the arrangement of the helical-domains underlies gating. With the structure of the temperature increases, leads to a large increase in the 13-cis isomer concentration in the a, form transmembrane homodimer of glycophorin now available (MacKenzie et al., Science 276:131- bR. 13-cis retinal has a much reduced absorption cross section and has no proton 133, 1997), our lab has begun to probe the basis of helix-helix association. Our approach uses a pump ability leads to an observed large reduction in the concentration of the observed photocycle reduced representation of the helices. Following the model of Richmond and Richards (J. Mol. intermediates, well as the proton gradient at a given light intensity. These results suggest that Biol. 119:537-555, 1978), each amino acid in a helix is represented as a sphere with an empirically might have selected the a,, rather than the a, form as the helical conformation in bR to assigned volume to capture important packing attributes. This ball-helix model is used in a first the all-trans retinal isomer that is a better light absorber and is capable of pumping stage of structure prediction, in which association pairs with 'impossible' interactions are protons. eliminated, thus reducing our search space. Utilizing this ball-helix model, we are able to predict the structure of the glycophorin transmembrane domain through a computational minimization algorithm. Predicting packing motifs for membrane proteins containing three or more transmembrane helices requires an efficient global minimization algorithm. To this end we have adapted the branch and bound algorithm of Maranas and Floudas (J. Chem. Phys. 100:1247-126 1, 1994). We believe that the membrane environment plays a crucial role in packing, and thus we minimize on a potential energy function which contains a membrane interaction term.

936 Pos 937 - Pos THE CATALYTIC POCKET OF HUMAN ADENYLYL CYCLASE TYPE-5 REFOLDING OF A MISFOLDED INTEGRAL MEMBRANE PROTEIN. BINDING OF A PURINE (P)-SITE INHIBITOR. Charles R. Sanders', Bonnie Gorzelle', Joanna Nagy', Kirill Oxenoid', Willis Lonzer', David Diana Cherbavaz', Leyton Murray2, Jemes E. Tomlinson', Roger A. Johnson2, David M. Cafiso2, 'Case Western Reserve University, Cleveland, OH 441064970, 2University of Virgina Sedlock', Therapeutics, Inc., 256 East Grand Avenue, South San Francisco, CA 94080, 2SUNY, Stony Brook, 11794 In this presentation, misfolding of the trimeric membrane protein diacylglycerol kinase (DAGK) is Adenylyl cyclase (AC) catalyses conversion of 5'-ATP to 3':5'-cyclic AMP. This reaction is documented and a reliable method for refolding the protein is presented. 29 single-cysteine by 2',5'dideoxy-3'-ATP (dd3'-ATP), a member of the P-site family (D6saubry al., JBC, mutants of DAGK were examined. A majority were found to have lower-than-expected activities 1996). capitalize on its interaction with AC, [ -32P]dd3'-ATP was synthesized and tested in when purified into micellar solutions, with additional losses in activity often being observed type-I binding assay (Napolitano et aL, FASEB J., 1998). We have expressed human type- following membrane reconstitution. Evidence indicates that the low activities observed for most 5 (hAC5) HEK293 cells and used the radioligand to study binding parameters in this system. of these mutants in micelles or vesicles is due to kinetically-based misfolding of the protein, with assay was validated by defining specific binding as compared with null vector misfolding often being manifested by the formation of aberrant oligomeric states. A method for cell membranes, boiled hAC5 membranes, and excess unlabeled dd3'-ATP. refolding is presented and is referred to as "reconstitutive refolding". This method is based upon determined to promote P-site binding were: 5mM MgCI2, lOOnM GTP S-activated reconstituting DAGK into POPC vesicles by exhaustively dialyzing the detergent Gs pyrophosphate, and 50mM HEPES buffer adjusted to pH 7.2. Equilibrium was dodecylphosphocholine out of mixed micellar mixtures of DAGK. For none of the 29 mutants quickly, 1 minute. The half-maximal concentration for dd3'-ATP was 112 inhibitory tested was there a loss of DAGK activity during reconstitutive refolding and in 21 cases there was a greater than 2-fold increase in activity during this process. dd3'-ATP binding: Gs forskolin, pyrophosphate, and a panel of metal ions, were and forskolin, activators of hAC5, increased ligand binding in a dose-dependent manner. Cation species and pyrophosphate concentration also affected ligand binding. Pyrophosphate enhanced the apparent affinity of adenosine analogs for the P-site binding pocket by competition-displacement assays. These data were used to generate a structure- activity profile for inhibition of hAC5.

Pos 939 - Pos OF THE MAJOR HOMOLOGY REGION OF HIV-1 GAG IN MEMBRANE A PROLINE RESIDUE IN THE CORE OF A HYDROPHOBIC PEPTIDE DECREASES BINDING. THE STABILITY OF TRANSMEMBRANE INSERTION Provitera, Dr. Carol Carter, Dr. Suzanne Scarlata, State University of New York at Stony Gregory A Caputo, Erwin London, State University of New York at Stony Brook, Dept. of Brook, Physiology and Biophysics, Stony Brook, NY Biochemistry and Cell Biology, Stony Brook, NY 11794-5215 Assembly proteins and RNA on plasma membranes of host cells is a critical step in the We investigated the effects of inserting a Pro residue near the middle of helix-forming processing infectious particles. The Major Homology Region (MHR) is a highly conserved hydrophobic peptides upon the structure of the peptides in membranes. The molecules studied sequence throughout all retroviruses, including HIV-1. Its role in assembly is crucial but were 25 residue peptides with a 19 residue hydrophobic core, and had the sequences The MHR is a 20 amino acid stretch found in the capsid domain of HIV-I Gag. In KKGL9WL,KKA (pLeu) and KKGL7PLWL9KKA (pLeu(PI1)). The fluorescenceXpax of the Trp determine the role of the MHR in viral assembly, membrane and RNA fluorescence based at the center of the peptide was used to assess peptide structure in a series of bilayers with binding were conducted. As models, LUVs, of and were used. composed POPS, tRNA, different widths (phosphatidylcholine bilayerswith monounsaturated fatty acyl chains that are 14, Type Gag exhibits approximately 3 fold greater binding to lipid bilayers, but there is not any 16, 18, 20, 22, or 24 carbon atoms long). TheApax of fluorescence emission for pLeu and difference in tRNA binding as compared to the AMHR Gag Mutants. These data indicate that the pLeu(PI1) showed a similar overall dependence on acyl chain length. This pattemshows red involved in the interactions that drive may protein-protein assembly. To begin to shifted fluorescence in thin bilayers, probably due to the presence of transmembrane oligomers, a characterize these interactions, accessibility of MHR antibody to Gag bound to RNA and blue shift at intermediate widths, corresponding to a monomeric or nearly monomeric were assessed. These studies show that the MHR remains accessible during tRNA transmembrane orientation, and a red shift in wide bilayers, due to the movement of the peptide to binding, however, it is inaccessible during membrane binding and when both tRNA and a non-transmembrane orientation close to the surface. (The change in position in wide are bilayers membrane present. This indicates that membrane binding induces strong Gag-Gag interactions results from the peptide being too short to span the hydrophobic part of the membrane.) may mediated by the MHR. Further studies to better understand these protein interactions However, behavior was affected was presented. NIH GM53132 by the presence of a Pro. In wide bilayersfluorescence significantly more red shifted for pLeu(P II) than for pLeu. The same pattern was found at a peptide:lipid ratio of 1:250 and 1:1000 (mole/mole). One explanation for the difference between peptide behavior with and without Pro, is that Pro destabilizes the transmembrane state for the more monomeric forms of the peptide, which predominate in wide bilayers. A second is that Pro shortens the end-to-end length of the peptide by inducing a helix bend, and thus destabilizes transmembrane insertion in wide bilayers. This is consistent with previous observations showing that shortening the number of hydrophobic residues in a peptide also destabilizes transmembrane insertion in wide bilayers.

159A 940-945 MEMBRANE PROTEINS MONDAY

940 - Pos 941 - Pos DETERGENTS AND LIPIDS MODULATE ASSOCIATION, BUT NOT HELICITY, OF CONFORMATIONAL PROPERTIES OF A FRAGMENT FROM TM6 OF THE LH THE GLYCOPHORIN A TRANSMEMBRANE DOMAIN RECEPTOR AND OF MUTANTS INVOLVED IN FAMILIAL MALE PRECOCIOUS LIllIan E. Fisher', James N. Sturgis2, Donald M. Engelman', 'Yale University, Dept. of PUBERTY (FMPP) Chemistry, New Haven, CT 06520, 2lnstitut de Biologie Structural et Microbiologie, CNRS, Shirley Schreier', Fabio Casallanovo', Amy N. Abell2, Deborah L. Segaloff2, 'University of Sao France Paulo, Institute of Chemistry, C.P. 26077, Sao Paulo, Sao Paulo 05599-970 Brazil, 2University of Iowa Understanding how the lipid environment influences transmembrane helix association requires Certain mutations (D578G and D578Y) in the 6th TM of the LtH/chorionic gonadotropin receptor, thermodynamic neasurements that can be interpreted in terms of specific chemical interactions. a protein from the GPCR family, lead to receptor constitutive activation and to the disease FMPP. We have used Forster resonance energy transfer to measure dimerization of the Glycophorin A We have examined three undecapeptides corresponding to the N-terminal region of TM6 transmembrane helix in detergent micelles and lipid bilayers. The observed Kd depends containing either D578 (I), or G (II), or Y (III) as their ninth aminoacid. CD (for 1, II, III), and dramatically on the identity and concentration of the environment. In contrast, the helicity of the fluorescence (for III) spectra were obtained in solution, as a function of pH and of %TFE, and in Glycophorin A helix, as measured by far UV circular dichroism, is independent of dimerization the presence of detergents. CD spectra indicated that the peptides acquired some a-helical and of the specific environment. These measurements support a long-standing assumption about conformation in the presence of TFE, but displayed a great tendency to aggregate in aqueous the Glycophorin A transmembrane domain, that detergents and lipids uncouple helix formation solution, with a high content of 3-sheet structure. Addition of detergents caused a small loss of from helix dimerization. The approach is applicable to a variety of systems in diverse agreggation. The extent of aggregation decreased when detergent solutions were added to peptide environments, extending our ability to measure how interactions with complex solvents affect the films. A considerable contribution of f3-sheet structure was still present, but an increase in a- thermodynamics of membrane protein oligomerization. helical content, as well as random coil, was found, in a differential manner, II displaying a greater ability to become monomeric. Thus, short, hydrophobic peptides tend to form aggregated 3-sheets in spite of the prediction of a-helical conformation for their sequence in TM helices. Supported by FAPESP, CNPq, and NIH.

942 - Pos 943 - Pos FhuA, A FERRICHROME-IRON TRANSPORTER AND PHAGE RECEPTOR: ROLES MEMBRANE INTERACTION OF ANNEXIN XII STUDIED BY SITE-DIRECTED SPIN OF THE BETA BARREL, THE "CORK" AND THE EXTERNAL LOOPS IN LIGAND LABELING BINDING AND STABILITY OF THE PROTEIN Jose Mario Isas', Ralf Langen2, Wayne L. Hubbell3, Harry T. Haigler', 'University of California Lucienne Letellier, Pascale Boulanger, Laure Plancon, M6lanie Bonhivers, Michel Desmadril, Irvine, Physiology and Biophysics, 345-D Medical Science 1, Irvine, California 92697-4560, Greg Moeck, CNRS UMR 8619, Laboratoire des Biomembranes, Universite Paris Sud, Bat.430, 2University of Southern Califomia, 3UCLA, 100 Stein Plaza, Los Angeles, CA 90095-7008 Orsay, 91405 France Annexins are soluble proteins that bind to phospholipids in a calcium-dependent manner. FhuA (MM 78,9 kDa) is an E. coli outer membrane protein which transports iron coupled to The biological functions of annexins are not known, however, they have been implicated in a ferrichrome and is the receptor for phage T5. The 3D structure of FhuA was recently detemined: number of membrane-related processes including vesicle trafficking, fusion, and ion-channel it consists of extracellular hydrophilic loops involved in ligand binding, of a 22 beta strands barrel formation. Site-directed spin labeling was employed to investigate the structure of annexin XII in and of a globular domain, the "cork", facing the periplasm. This domain is located within the beta solution and on membranes. Residues 134-163 were selected for nitroxide scanning experiments barrel and occluds it. This unexpected structure raises the question of the connectivity of the in which the native side chains sequentially were replaced with nitroxide spin label. These different domains and of their respective role in the different functions of the protein. To address residues are located in the D and E helix and their connecting loop. EPR studies showed that these questions we have compared the functional and structural properties of the wild type and of a secondary structure and location of the tertiary contact sites in the D and E helices were similar to mutated FhuA of which a large part of the cork had been removed. Immunochemical and those in the crystal structure. Although, the fold of the membrane-bound protein also resembles biophysical approaches (differential scanning calorimetry, fluorescence) showed that the cork and that of that of the crystal structure, there are some dramatic differences in some regions. For the beta barrel behave as autonomous folding domains and that ferrichrome, although binding to example, residues 144 and 145 in the connecting loop were mobile in solution but immobilized the external loop, contributes to the stabilization of the cork domain. when bound to membranes in the presence of calcium. These data and other EPR studies show that pb5, the FhuA-binding protein of phage T5 was purified in a functional form; pb5 bound FhuA 144 and 145 interact directly with the bilayer and begin to define the role of the DE loops in with high affinity and with a pb5/FhuA stochiometry of 1/2. The influence of removing the FhuA calcium-dependent phospholipid binding of annexins. cork on pbS binding was studied. Taken together these studies indicate that a change of conformation upon ligand binding on the external loops of FhuA can be sensed by the cork. Such results are expected to apply for proteins of the same family (like FepA) which share similar structures.

944 - Pos 945- Pos THE FOLDING KINETICS OF OMPA INTO LARGE UNILAMELLAR VESICLES ARE STRUCTURAL STUDAY OF THE HIV-1 ACCESSORY PROTEIN VPU VIA OF SECOND ORDER AND DEPEND ON THE HYDROPHOBIC THICKNESS OF THE SYNCHROTRON X-RAY REFLECTIVITY FROM LANGMUIR MONOLAYERS LIPID BILAYER. Songyan Zheng', Joseph Strzalka', Che Ma', Stanley J Opella', Ben M Ocko2, J Kent Blasiel, J6rg H. Kleinschmidt, Lukas K. Tamm, Dept. of Molec. Physiol. and Biol. Phys., University of 'University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, 5National Synchrotron Virginia, P.O.Box 10011, Charlottesville, Virginia 22906-0011 Light Source, Brookhaven National Laboratory, Upton, New York 11973 Outer membrane protein A (OmpA) of E. coli is an integral membrane protein of the 3-barrel type which becomes completely unfolded in 8 M urea. OmpA is known to spontaneously insert and Vpu is an 81-amino-acid integral membrane protein encoded by the HIV-I genome with two fold into detergent micelles or sonicated unilamellar lipid vesicles. Attempts to refold OmpA into independent biological functions. Vpu's primary sequence coupled with solid-state NMR extruded unilamellar vesicles were so far unsuccessful. experiments on oriented phospholipid multilayers and solution NMR experiments on phospholipid In the present study we have used intrinsic tryptophan fluorescence and the different detergent micelles containing Vpu indicate that Vpu folds into two distinct domains, a electrophoretic mobilities of folded and unfolded species on polyacrylamide gels to study the hydrophobic "transmembrane" single helix domain and a hydrophilic "cytoplasmic" domain that kinetics of folding of OmpA into large unilamellar vesicles as a function of the hydrophobic chain contains two amphipathic helices. The hydrophobic helix is oriented perpendicular to the bilayer length of the phospholipid. At 20 °C, OmpA could be suceessfully refolded into short chain (CIO plane and the amphipathic helices are oriented parallel to bilayer plane. Here we present the - C12), but not longer chain (C14 or C18: 1) phosphatidylcholine bilayers. The initial rates of experimental results of the synchrotron radiation-based X-ray reflectivity from the Langmuir folding and the yields of b-barrel formation decreased with increasing chain lengths. The initial monolayers of pure phospholipid and Vpu/phospholipid mixtures at the air-water interface. kinetics of insertion followed second order rate laws with respect to protein and phospholipid Analysis of the X-ray reflectivity data provides the electron density profile of the monolayer. concentration. Comparison of the electron density profiles as a function of increasing Vpu/phospholipid mole ratio clearly indicates the contribution of the protein to the monolayer profile. Our results show the hydrophobic helix is perpendicular to the plane of monolayer within the phospholipid hydrocarbon chain layer and the amphipathic helices of the hydrophilic domain lie on the surface of the phospholipid headgroups without extending further into the bulk water subphase.

Funded by the NIH under Grand No. GM56538

160A MONDAY MEMBRANE PROTEINS 946-951

946 - Pos 947 - Pos TRANSMEMBRANE AND WATER-SOLUBLE HELIX BUNDLES DISPLAY REVERSE VAL'5-GLU MUTATION WITHIN THE TRANSMEMBRANE DOMAIN OF HUMAN PATTERNS OF SURFACE ROUGHNESS ERBB-2: LOCAL PHYSICAL EFFECTS IN POPC BILAYER MEMBRANES BY 5H NMR Robert Renthal, University of Texas at San Antonio, San Antonio, Texas 78249 Simon J. Sharpe, K. R. Barber, C. W. M. Grant, University of Westem Ontario, Medical During membrane protein folding, part of a transmembrane helix surface remains Sciences Building, London, Ontario N6A 5CI Canada exposed to lipid hydrocarbon chains, and part forms helix-helix contacts. What distinguishes the Point mutations within the tranmmembrane domains of class I receptor tyrosine kinases have been lipid-exposed surface from the helix-helix contact surface? High-resolution structures for associated with oncogenic transformation in vitro and in vivo. An important example is the membrane proteins show tight atomic packing and highly complementary helix-helix contact Val-Glu substitution in human ErbB-2/Neu. The present work describes a local conformational surfaces. Specific protein side chain interactions are generally believed to provide a major change induced by this mutation. A series of 50-residue peptides, ErbB-2Tm, containing the contribution to the thermodynamic stability of transmembrane helix association. Helix-helix transmembrane domain of ErbB-2, were expressed in E. coli. 'Oncogenic' peptides, ErbB- contact surfaces of water-soluble proteins are known to be rough, sometimes described as knobs- 2TmMu, were likewise expressed, differing only in that Val659 was replaced with Glu. In each case, in-holes, ridges in grooves, or zippers. To quantitate surface roughness, I calculated the fractal deuteromethyl alanine was incorporated at specific sites. This pernitted 2H NMR studies of the dimension of the surfaces of twelve helices from three transmembrane a-helix bundles and conformation and dynamics of ErbB-2 peptides incorporated into fluid POPC bilayers. Mutation thirteen helices from seven water-soluble a-helix bundles. I found that transmembrane helix of Val59 to Glu within the hydrophobic domain induced major probe reorientation of nearby bundles have relatively rough surfaces exposed to the lipid bilayer hydrocarbon chains and Ala"7 , but had little effect at more distant sites including positions 648 and 670. These relatively smooth surfaces along helix-helix interfaces. This pattern is the reverse of what occurs observations in fluid lipid bilayers support a local deformation, probably a bend centred around in water soluble helix bundles, where relatively rough surfaces occur at helix-helix interfaces and GluO", with potential significance for the oncogenic activity of mutant ErbB-2. CD spectra of relatively smooth surfaces are exposed to water. The relatively rough exposed surfaces and buried ErbB-2rm and ErbB-2TMMu indicated a-helical structure for 58.3% of the wild-type peptide, but smooth surfaces of transmembrane helices could contribute to the stability of transmembrane only 40.9°/o a-helix in the mutant, supporting a deviation from a standard helical structure in the helical bundles by increasing the entropy of lipid hydrocarbon chains. latter.

948 - Pos 949- Pos APO-PYOVERDIN SIDEROPHORE BINDS TO ITS OUTER MEMBRANE RECEPTOR MEMBRANE PROTEIN-LIGAND INTERACTIONS IN E. COLI VESICLES AND FPVA IN PSEUDOMONAS AERUGINOSA. LIVING CELLS MONITORED VIA A BIOSYNTHETICALLY INCORPORATED Isabelle J Schalk', K Poole2, M A Abdallah', F Pattus', 'ESBS, Bld Sebastien Brant, Illkirch, 67 TRYPTOPHAN ANALOG 400 France, 2Queen's University, Canada Jaap Broos, Frank ter Veld, George T. Robillard, University of Groningen, Nyenborgh 4, The Pseudomonas aeruginosa FpvA receptor, is a TonB-dependent outer membrane transport Groningen, 9714 AG Netherlands protein that catalyzes uptake of ferric pyoverdin across the outer membrane. Recently the complete structure of two siderophore outer membrane receptors of Eschericia coli have been This paper presents a deceptively straightforward experimental approach to monitor membrane published (Ferguson et al., 1998; Locher et al., 1998; Buchanan et al., 1999) protein-ligand interactions in vesicles and in living E. coli cells. This is achieved via the FpvA was purified and characterized by N-terminal sequencing, UV and circular dichroism. biosynthetic incorporation of 7-azatryptophan, a tryptophan analog with a red-shifted absorption Surprisingly, FpvA expressed in P. aenuginosa grown in an iron-deficient medium, copurifies with spectrum, allowing collection of the emission signal of the target protein in a high tryptophan a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron- background via red-edge excitation. The approach is demonstrated for the mannitol permease of E. free pyoverdin (apo-PaA, Schalk et al., 1999). The properties of ligand binding in vitro revealed coli, (EII"), an integral membranejprotein of 637 amino acids, including four tryptophans, and similar affinities of apo-PaA and ferric-PaA to FpvA (Schalk et al., 1999). The binding properties single-tryptophan mutants of EII By using a tryptophan auxotroph, a high level of 7- apo-PaA and ferric-PaA have also been study in vivo, in wild-type cells, protonophores treated azatryptophan incorporation in ElI" was achieved.. Changes in emission signal could be cells and tonB deficient mutants. The obtained results let a much more suggest complicated iron monitored (-5%) when the enzyme was situated in vesicles, although it constituted only 10-15% of transport mechanism than already described in the litterature. The iron transport mechanism in P. the total cytoplasmic membrane fraction. Of the five single-tryptophan mutants, the emission aersginosa via the pyoverdin pathway will be discussed in the light of all these new findings. signal of the mutant with 7-azatryptophan at position 198 was the most sensitive for mannitol Ferguson et al. (1998) Science 282: 2215-2220 binding. Changes in emission signal were not only observed in vesicles (-18%) but could also be et al. (1998) Cell 95: 771-778 monitored in viable cells (-5%). The fact that only modest expression levels and no protein

Buchanan et are needed makes al. (I1999) Nat Struct Biol 6: 56-63 purification this an useful approach for the characterization of numerous protein systems under in vitro and in vivo conditions. (Lit.: Biochemistry (1999) 38, aL (1999) Biochemistry 38: 9357-9365 9798)

950 Pos 951 - Pos STRUCTURE AND INTERACTIONS OF THE OLIGOMERIZATION AND INTERACTION AMONG MYELIN BASIC PROTEIN, ZINC AND LANGMUIR- SCAFFOLDING DOMAINS OF CAVEOLIN IN MEMBRANE BILAYERS. BLODGETT PHOSPHOLIPID LAYERS: A XAS STUDY Omar Bakht, Markus Eilers, Weiwen Ying, Steven 0. Smith, SUNY Stony Brook, Nicolls Road, Slvia Morante', Stefania Nuzzo2, Paolo Cavatorta3, Heinrich Haas4,Carlo Meneghini2, Settimio Stony Brook, NY 11794 Mobilio5, Paolo Riccio', 'University of Rome "Tor Vergata", Via della Ricerca Scientifica,I There is an amount of evidence increasing to show that the lipid bilayer contains well-ordered Roma, 00173 Italy, 2ESRF Grenoble France, 3Dept.of PhysicsUniversity of Parma Italy, 4Dept. of structural domains which have specific cellular functions. Signal transduction across the Chemistry University of Sao Paulo Ribeirao Preto Brazil, 'Dept. of Physics University of Rome membrane is largely mediated, in many cells, by domains formed from a high concentration of Italy, 6DBAF University of Basilicata Italy cholesterol and the protein caveolin. Caveolin interacts with the cell signaling molecules through its "scaffolding" region, residues 82-101, and forms its homo-oligomers through It has been recently observed that the presence of Zinc contributes to the integrity and "oligomerization" region, residues 61-81. How this 41 residue stretch of amino acids is able to compactness of the sheath the cell myelin by inhibiting the release of Myelin Basic Protein during sequester signaling molecules as well as oligomerize has yet to be elucidated. The demyelinization processes. structure and membrane interactions of a peptide corresponding to residues 61-101 are being investigated using ATR-FTIR and MAS NMR on ordered membranes. ATR-FTIR experiments reveal the orientation and secondary structure of the caveolin segment. We have shown that the In order to get a deeper understanding of the interplay among lipids of the myelin sheath, Myelin peptide associates readily with POPC/POPS membranes and adopts a predominantly extended Basic Protein and Zinc, we have perfonned X-ray absorption spectroscopy experiments at the Zinc beta-strand conformation based on a band at 1626 cm -1. MAS NMR experiments are currently in K-edge on a system formed by Langmuir-Blodgett phospholipid multi-layersboth in presence and progress to determine the three dimensional structure of isotope-labeled caveolin associated with in the absence of the Myelin Basic Protein. membranes. The analysis of the Extended X-ray Absorption Fine Structure and Near Edge regions of the X- rayabsorption spectra suggests that Zinc is bound to the phospholipid heads thus probably helping in stabilizing the binding of the Myelin Basic Protein to the Langmuir-Blodgett multi-layer.

161A 952-957 MEMBRANE PROTEINS MONDAY

952 - Pos 953 Pos A DYNAMIC GATED CORRAL MODEL FOR REGULATION OF MEMBRANE IMAGING MYELIN BASIC PROTEIN ADSORPTION TO LIPID BILAYERS WITH PROTEIN MOBILITY THE ATOMIC FORCE MICROSCOPE. Frank L H Brown, David M Leitner, J Andrew McCammon, Kent R Wilson, UCSD Department Hana-JlArgen Butt', Henning Mueller', Emst Bamberg2, 'University of Mainz, Institute of of Chemistry and Biochemistry Physical Chemistr, Mainz, 55099 Germany, 2Max-Planck-Institute of Biophysics We describe a two-state, dynamic gated corral model for the regulation of protein mobility in cell Myelin basic protein (MBP) plays an important role in the formation of functional myelin. For an membranes. An open gate permits a protein that is otherwise trapped inside a corral to escape to understanding of this function, information on the interaction between MBP and the membrane is an adjacent corral in the cytoskeletal network. We solve for the escape rate over a wide range of of interest. We used contact atomic force microscopy in physiological buffer to image MBP parameters, and compare these results with Monte Carlo simulations. Results of our dynamic adsorption (concentration: 0.5-50 ug/ml) to various phospholipid bilayers. On acidic and basic corral model are consistent with available data for Band 3 in erythrocyte membranes. We also bilayers MBP formed monolayer-aggregates which at high concentration covered the entire discuss how the statistical properties of protein transport under a two-state corral model are bilayer. On neutral bilayers, MBP adsorbed irregularily. Investigation of the mechanical properties distinct from those of plausible alternatives. We demonstrate that it may be possible to by spatially resolved force spectroscopy indicated that the MBP/bilayer assembly is more stable in experimentally differentiate between dynamic and static barriers to protein diffusion by examining the lateral dimension than the bare bilayer. Bovine serum albumine (BSA) did not adsorb to any the time-dependent variance of protein population inside corrals of the cytoskeletal network. bilayer type (concentration < 500 jig/ml). Poly-L-lysine (MW: 20,000 30,000) formed irregular Simulations modeling Band 3 diffusion in erythrocyte membranes provide a specific example of monolayers on acidic bilayers. On neutral and basic bilayers it adsorbed in complex dendritic how the study of such statistical properties of transport might prove useful in exploring the structures. Cytochrome c fonned monolayers on acidic bilayers, small monolayer clusters on mechanism by which the cytoskeletal network regulates protein mobility. neutral bilayers and did not adsorb to basic bilayers (concentration < 50 jig/ml). The different adsorption behaviors lead to the conclusion that MBP adsorption to lipid bilayers is not exclusively electrostatically driven and depends on the specific lipid bilayer composition. Adsorption of MBP and poly-L-lysine to lipid bilayers could be induced by penetrating the bilayer with the AFM tip.

954 - Pos 955 - Pos A FLUORESCENT ASSAY PERMITS CONTINUOUS MONITORING OF PLA2 PRESSURE-INDUCED FUSOGENIC CONFORMATION OF VSV G PROTEIN ACTIVITY. Aadre Marco 0 Gomes', Anderson S. Pinheiro', Carlos F.S. Bonafe2, Jerson Lima Silva', David M. GLImer, Emina Stojkovic, M. Walczak, Anne Walter, St. Olaf College, Biology Dept, 'Universidade Federal do Rio de Janeiro, Av. Brigadeiro Trompowski s\n, Rio de Janeiro, Rio de Northfield, MN 55057 Janeiro 21941-590 Brazil, 2Universidade de Campinas PLA2 cleaves phospholipids at the sn2 fatty acyl bond, yielding fatty acids and Vesicular stomatitis virus (VSV) is composed of a ribonucleoprotein core surrounded by a lipid lysophospholipids. Fluorescein-phosphatidylethanolamine (F-PE) incorporated in the lipid envelope presenting an integral glycoprotein (G). The homotrimeric VSV G protein displays a mixture used to prepare LUV was tested for its suitability as a probe. A standard curve of F-PE membrane fusion activity that can be elicited by low pH. The fusion event is important to virus fluorescence intensity as a function of mol% oleic acid+lysoPC incorporation in the vesicles was entry and disassembly in the cells. To understand the conformational changes involved in this prepared. PLA2 was added to vesicles composed of brain phosphatidylcholine (PC) and brain process, the effects of high hydrostatic pressure and urea on VSV G protein were investigated. phosphatidylserine (PS) (1:1) with 1% F-PE and fluorescence was monitored continuously (ex/em High urea produced a large red shift in the tryptophan fluorescence of G protein whereas pressure 459/520 nm). Similar experiments were performed using the fatty acid-sensitive fluorescent promoted a smaller change. Pressure induced equal fluorescence changes in isolated G protein and probe, ADIFAB. Aliquots were taken for fatty acid and lyso-lipid analysis by TLC or HPLC. F- virions, indicating that the pressure inactivation is due to changes in the G protein. We propose PE fluorescence increased with the addition of fatty acid in the bilayer in a linear fashion up to at that pressure elicits a conformational change in the G protein, which maintains the cellular binding least 30 mol%. Fluorescence increased when PLA2 was added to PS/PCIF-PE vesicles and the properties but suppresses the internalization process. Binding of bis-ANS indicates the presence of lag-burst phenomenon, previously reported, was observed. The F-PE and ADIFAB assays agreed hydrophobic cavities in the G protein. Equal to pressure, bis-ANS binding abolishes infectivity of quantitatively but there were details in the progress curve that varied. One concern with the the virion probably acting by a similar mechanism. Pressure also caused a reversible increase in ADIFAB assay is its dependence on the fatty acid partition coefficient which varies with lipid light scattering of VSV G protein, reinforcing the hypothesis that high pressure is increasing the composition. Direct measurement of products also confirmed the F-PE results. Thus we have a fusogenic activity of VSV G protein. This "fusion-intermediate state" induced by pressure has continuous assay of PLA2 activity that is relatively inexpensive, easy to use and reports events in minor changes in secondary structure and is likely the cause of nonproductive infections. the bilayer itself (NSF C-RUI DBI-9710795)

956 - Pos 957 - Pos EVIDENCE FOR A NOVEL SERINE/THREONINE PROTEIN PHOSPHATASE EVALUATION OF LIPID EXPOSURE OF TRYPTOPHAN RESIDUES IN MEMBRANE REGULATING PHOSPHOLAMBAN IN CARDIAC SARCOPLASMIC RETICULUM PROTEINS AND PEPTIDES. (SR). Alexey S. Ladokhin, Univ. of California, Irvine, CA 92697 Ming T. Jiang, H. H. Valdivia, Univ. of Wisconsin-Madison, 1300 Univ Ave, Madison, WI Fluorescence quenching is used to gain information on the exposure of tryptophan residues to lipid 53706 in membrane bound proteins and peptides. A protocol is developed to calculate this exposure, based on a comparison of quenching efficiency and of a fluorescence lifetime (or quantum yield) Phosphorylation of phospholamban (PL) by PKA and CaMKII stimulates Ca2e uptake by cardiac measured for a protein and for a model tryptophan-containing compound. Various methods of SR. Previous studies indicate that the major protein phosphatase (PP) that reverses the effect of analysis of depth-dependent quenching are compared and three universal measures of quenching PL phosphorylation is PPI, an okadaic acid (OA)-sensitive PP. However, we observed that in profile are derived. One of the measures, related to the area under profile, is used to estimate cardiac SR, PL is substantially dephosphorylated despite the presence of OA, indicating the quenching efficiency. The method is applied to single tryptophan mutants of a membrane- presence of a OA-insensitive, SR-associated PP. This study was conducted to test whether a novel anchoring nonpolar peptide of cytochrome b5 and of an outer membrane protein A (OmpA). or a known PP of cardiac muscle is involved in dephosphorylating PL in canine cardiac SR. A Analysis of quenching of cytochrome's nonpolar peptide by a set of four brominated lipids reveals concentration of OA that inhibits both PPI and PP2A (5 iM) enhanced peak PL phosphorylation a temperature-controlled reversible conformational change, resulting in increased exposure of by endogenous CaMKII by -I 10/o, but did not inhibit subsequent dephosphorylation (-65% after tryptophan to lipid and delocalization of its transverse position. Kinetic quenching profiles and 4 min). EGTA and cyclosporin A, inhibitors of PP2B, or EDTA, inhibitor of PP2C, did not fluorescence binding kinetics reported by Kleinschmidt et al. (Biochemistry (1999) 38:5006-5016) prevent PL dephosphorylation, suggesting that they are not involved. In contrast, OA enhanced PL were analyzed to extract information on the relative exposure of tryptophan residues during phosphorylation by exogenous PKA by -60%, and completely inhibited PL dephosphorylation at folding of OmpA. Trp-102, which translocates across the bilayer, was found to be noticeably pCa 7 but only partly at pCa 5 (-50%). Thapsgargin (2 pM), which inhibits SR loading, did not shielded from the lipid environment throughout the folding event as compared to Trp-7, which prevent PL dephosphorylation. These data provide evidence for a novel PP associated with cardiac remains on the cis side. The approach described here provides a new tool for studies of low- SR, which acts preferentially on CaMKII-phosphorylated PL. resolution structure and conformational transitions in membrane proteins and peptides. Supported by GM-46823 (Prof S. H. White, PI).

162A MONDAY MEMBRANE PROTEINS 958

958 - Pos INVESTIGATIONS OF THE MOBILITY OF THE LAMBDA-RECEPTOR IN E. COLI MEMBRANES USING OPTICAL TWEEZERS. Lene B Oddernhede', Sonia Grego', Stanley Brown2, 'Niels Bohr Institute, Blegdamavej 17, Copenhagen, 2100 Denmark, 2Dept of Molecular Cell Biology, University of Copenhagen We have developed a 1064 nm laser based optical tweezer to study the physical properties of membrane proteins, first in prokaryotic cells and later in eucaryotic cells. We are modifying the gene for the lambda-receptor of E. coli by inserting DNA encoding an in vivo biotinylation site. We expect this to place a biotin on the extemal face of the lambda- receptor. This will allow us to attach an avidin-coated latex bead to the lambda-receptor by avidin-biotin binding. By manipulating this bead, measuring the brownian motion and force- distance relations within the pico-Newton and nanometer regimes with an optical tweezer, the mobility of the lambda receptor in the cell membrane of E. coli will be studied.

MONDAY PROTEIN FOLDING & STABILITY: MEMBRANE PROTEINS 959-962

959- Pos 960- Pos THERMODYNAMICS OF THE a-HELIX-COIL TRANSITION OF AMPHIPATHIC STRUCTURAL STUDIES OF THE N- AND C-TERMINAL TRUNCATED HUMAN PEPTIDES IN A MEMBRANE ENVIRONMENT. APOLIPOPROTEIN A-i Torsten Wieprecht, Joachim Seelig, Biocenter of the University of Basel, KlingelbergstraBe 70, YUhllg Fang, Olga Gursky, Vassilis I Zannis, David Atkinson, Boston University School of Basel, Switzerland Medicine, 715 Albany Street, Boston, MA 02118 The amphiphatic a-helix is the membrane binding motif in many proteins. The corresponding peptides are often random coil in solution but are folded into an a-helix upon interaction with the Apolipoprotein A-l (apoA-l, 243 amino acids) is the major protein of high-density lipoprotein membrane. Here we present a new method to quantitatively analyze the thermodynamics of (HDL). In addition to its strucural role, apoA-l mediates reverse cholesterol transport, activates peptide folding at the membrane interface. We have systematically varied the helix content of a lecithin-cholesterol acyltransferase, and is responsible for the recognition of HDL by HDL given amphiphatic peptide when bound to the membrane and have correlated the thermodynamic receptors. Secondary structural models of apoA-1 show that the middle region (residues 60-183) binding parameters determined by isothermal titration calorimetry with the a-helix content contains exclusively amphipathic a-helices formed from a tandem repeat of about 22 a. a. To obtained by CD spectroscopy'. The peptides investigated were the antibacterial peptide magainin analyze the structure and stability of this core domain, we use a series of C- and N-terminally 2 amide and three analogs in which two adjacent amino acids were substituted by their d- truncated mutants of apoA-1. The proteins were expressed in C127 cell line and in baculovirms enantiomers. The thermodynamic parameters controlling the a-helix formation were found to be infected Sf-9 cell line. The proteins from C127 cells were purified on FPLC. The proteins from Sf- linearly related to the helicity of the membrane-bound peptides. Helix formation at the membrane 9 cells were 6xHis-tagged and purified on Ni-NTA resin. So far, we have obtained pure proteins of surface is characterized by an enthalpy change of AHhli,, a -0.7 keal/mol per residue, an entropy the following mutants: residues 1-197, 1-208, 42-243, 60-243, 42-184, and 60-184. The change of ASh,eix, -1.9 cal/molK residue and a free energy change of AGhdi, = -0.14 kcal/mol conformation and thermal unfolding of these mutants were examined by circular dichroism (CD). residue. Helix formation is a strong driving force of peptide insertion into the membrane and Far-LW CD spectra show that, at -0.lmg/ml protein concentration, these mutants have well- accounts for 50%/o of the free energy of binding. defined secondary structure consisting of 40-60%/o a-helix, with melting temperatures from 54-58 'Wieprecht, Apostolov, Beyermann, Seelig, Joumal of Molecular Biology, in press. 'C. Comparison with the wild type apoA-I (60% a-helix, T,=57 °C) suggests that deletion of the N or C terminus may have either stabilizing or destabilizing effects on apoA-1.

961 - Pos 962 - Pos LATTICE MODEL OF MEMBRANE PROTEIN FOLDING: MONTE CARLO COLICIN El CHANNEL PEPTIDE FOLDING AND UNFOLDING STUDIES USING SIMULATION OF HYDROPHOBIC POLYPEPTIDES FLUORINATED TRYPTOPHAN ANALOGUES Chi-Ming Chen, National Taiwan Normal Univ., 88 Sec. 4 Ting-Chou Rd., Taipei, Taiwan Pa.ul M Malinowskil, M J Weller', A R Merrill2, A G Szabo', 'University of Windsor, 401 Sunset Ave, Windsor, Ontario N9B 3P4 Canada, 2University of Guelph Folding of a 18 residue hydrophobic polypeptide chain into unique 3D structures in a membrane is The channel-forming colicins are plasmid-encoded bacterial toxins that kill E.coli and related investigated by Monte Carlo simulation using the bond fluctuation model. Its ground state cells. This mode of action is of interest in studies of related problems of protein import and structure is a helix for bending rigidity (relative to hydrogen bonding energy) e < 0.1 1, while a toxicology. Colicins kill bacterial cells by forming voltage-dependent ion channel in the double helix is the ground state for e > 0.11. The folding pathway of hydrophobic polypeptides in cytoplasmic membrane, which in tum causes the membrane to depolarize and inhibit active a non-polar environment is found to favor the helical structure over the double helix. In the case trasport We have been investigating severd colicin El single tryptophan mutants.' Tryptophan where the double helix is the ground state, the polypeptide chain always folds into a helix before it is strategically placed in different segments of the protein in order to look at changes occurring reaches the ground state. The mean first passage time (MFPT) of the double helix is 100 times upon membrane insertion. Protein unfolding eperiments have been performed to gain insight longer than that of the helix. Folding at low temperatures exhibits an Asrhenius-like behavior. We into that process. Previously we have reported the effect of incorporation of tryptophan analogues, discuss the kinetics of both random folding and channel complex assisted folding of a polypeptide namely 7-azatryptophan (7AW).2 In this work we report the incorporation of fluorinated chain. tryptophan analogues such as 5-fluorotryptophan (SFW) and 6-fluorotryptophan (6FW). Thermally and salt induced protein unfolding experiments using steady state fluorescence and circular dichroism spectral measurements have been performed. New insights into the unfolding processes of the colicin El channel peptide will be presented. Steer BA, Merrill AR Biochemistry (1997), 36(10):3037-46 2 P. Malinowski, A.G. Szabo, M.J. Weller, Merrill A.R. Biophysical Jouwnal (1999), 76:A171

163A 963-968 PROTEIN FOLDING & STABILITY: FOLDING PATHWAYS & KINETICS MONDAY

963 - Pos 964 - Pos EARLY AND LATE EVENTS IN THE FOLDING OF DIHYDROFOLATE REDUCTASE THERMODYNAMIC AND KINETIC STUDIES OF YEAST TRIOSEPHOSPHATE Roxana M Ionescu, C. Robert Matthews, The Pennsylvania State University and Center for ISOMERASE THERMAL-DENATURATION. Biomolecular Structure and Function, 152 Davey Lab, University Park, PA 16802 Claudia Guadalupe Benitez-Cardoza, Arturo Rojo-Dominguez, Andres Hemirndez-Arana, The folding of dihydrofolate reductase (DHFR) from E. coli, a monomeric / protein containing Universidad Aut6noma Metropolitana Iztapalapa, Av. Michoacan y la Purisima s/n, Col. 2 subdomains and 159 residues, is a very complex process that is proposed to occur via four Vicentina, Mexico D.F., Mexico D.F. 09340 Mexico parallel pathways (Jennings et al. 1993, Biochemistry 32, 3783-3789). The C85S/C152E double Thermal denaturation of yeast triosephosphate isomerase (TIM) was studied by means of mutant has a simplified response, folding through two major channels (Iwakura et al. 1993, differential scanning calorimetry, fluorescence and circular dichroism (CD). At all concentrations Biochemistry 32, 13566-13574). Fluorescence studies of the kinetics of folding/unfolding and the tested the endotherms showed a single peak with marked asymmetry. Transitions were found to be equilibrium unfolding of SE-DHFR provide insights about the kinetic mechanism, the rates of irreversible and protein samples showed strong aggregation. Thermal denaturation transitions interconversion between species and some of their structural properties. Additional information followed by CD at 220 nm revealed that the unfolding process is strongly dependent on the for the development of a kinetic scheme was obtained from ligand binding studies. Global heating rate, suggesting that the process is under kinetic control due to an irreversible reaction. analysis reveals that the early organization of the adenosine binding domain has a significant The time course of TIM denaturation was followed by fluorescence spectroscopy and CD at 220 impact on the late stages of the folding process. This result has implications for the folding of a nm at several temperatures. All the kinetic curves got a final value corresponding to the expected large class of proteins containing structural homologues of the NAD(P) binding domain, one of signal for the fully denatured protein. This behavior is characteristic of an irreversible process. the most common motifs in biology. This work was supported by NSF grant MCB-9604678. Nevertheless, the denaturation reaction of TIM is not irreversible in the sense that denatured TIM can fold back and recover its native secondary structure if it is rapidly cooled. This refolding capacity decreases continuously at longer periods of protein exposition to high temperatures. These results support the proposal that TIM thermal-denaturation can be described by the mechanism: N 4 X -+ D; where N is the native protein, X is a form of the protein capable to refold and D is the irreversibly denatured protein.

965 - Pos 966 - Pos FOLDING KINETICS OF THE PARALLEL n-HELIX PROTEIN PECTATE LYASE C. TIME RESOLVED FLUORESCENCE STUDIES OF TRYPTOPHAN ANALOG Douglas Kamen, Robert W Woody, Department of Biochemistry and Molecular Biology, INCORPORATED RAT PARVALBUMIN F102W MUTANT: AN ALTERNATIVE Colorado State University, Fort Collins, Colorado 80523 PROTEIN FOLDING MODEL. Pectate lyase C (pelC) was the first protein observed to adopt a parallel ,-helix fold. This Arthur G Szabo', Mauro Acchione2, 'Univeristy of Windsor, 401 Sunset Ave, Windsor, Ontario molecule was chosen as a model to study the folding and stability of this class of proteins. We N9B 3P4 Canada, 2University of Windsor, 401 Sunset Ave, Windsor, Ontario N9B 3P4 Canada have previously examined the denaturation of this protein under equilibrium conditions and Time resolved fluorescence is an attractive tool in studying protein folding due to both its concluded that this apparently single domain protein is actually composed of two structural blocks sensitivity and its specificity. We had reported earlier circular dichroism and steady state that cooperate very strongly but can behave somewhat independently under proper conditions. We fluorescence measurements on the thermal and chemical denaturation of Rat Parvalbumin F102W have initiated kinetic experiments to understand the actual folding mechanism of this molecule. mutant into which we had incorporated several different tryptophan analogs. In an attempt to We will present results from kinetic CD unfolding experiments under various pH conditions, as obtain more detailed information on the unfolding pathway and intermediates these studies have well as refolding kinetics. Our results so far indicate that the folding and unfolding mechanisms been extended here with the use of Picosecond Time correlated single photon counting are more complex than a simple two-state transition, in agreement with our previous results. fluorescence to monitor several temperature points. At 20°C this protein exists as a single (Supported by USPHS Grant GM-22994) exponential. The experimental data we observe can best be rationalized in terms of a model where the protein expands but retains a constricted volume around the tryptophan. This is followed by a larger unfolding transition. The time resolved results revealed that a two-state unfolding model does not apply in this case.

967 - Pos 968 - Pos DIFFERENCES IN THE PATHWAYS FOR UNFOLDING AND HYDROGEN TRYPTOPHAN SCANNING AND SINGLE/DOUBLE CYSTEINE SCANNING PROTEIN EXCHANGE FOR MUTANTS OF E. COLI ALKALINE PHOSPHATASE FOLDING STUDIES OF PHOSPHOGLYCERATE KINASE. Christopher J Fischer, Joseph A Schauerte, Kathleen C Wisser, Ari Gafni, Duncan G Steel, Joseph M. Beechem', Pilar Lillo2, 'Vanderbilt University Medical Center, 2C.S.I.C Madrid Spain University Of Michigan, 300 N. Ingalls, Rm. 954, Ann Arbor, MI 48109-2007 The inherent information content of any single fluorescence spectroscopic experiment on protein folding reactions is (by necessity) rather limited. However, if fluorescence spectroscopic probes Our initial studies of hydrogen-deuterium (HID) exchange near the site of tryptophan 109 in E. are scanned throughout the tertiary structure of a protein, a much more detailed understanding of coli Alkaline Phosphatase suggest that significant unfolding might occur during the exchange complex protein folding reactions can be reaction. To of this process, we prepared a resolved. In this study, an attempt is made to further our understanding of the microscopic features 2O2~4 |, series of mutants designed to produce cavities around the exchanging residue and compared the ,12combine equilibrium and kinetic studies rates of H/D exchange to the rates of unfolding. The H/D exchange kinetics of these mutants X~~ 1 3St ~z 3s(unfolding and refolding) on yeast varied significantly, however, the thermostability of both wild-type and the mutants were Phosphoglycerate Kinase (PGK) using data from: identical; therefore the differences in H/D exchange rates were not simply a reflection of a general 5 single tryptophan mutants, 6 double-cysteine destabilization of the protein. In order to investigate whether the unfolding and/or "breathing" _ . 40;. fluorescence energy-transfer pairs (6 intra- motions leading to H/D exchange were related to the unfolding pathway of the protein we then 38 molecular distance changes during refolding 8 r ) and compared the H/D exchange rates to the proteins' lability in Gd:HCI. The complex unfolding 36 37 j"/ j shown in figure), 6 single cysteine labeled kinetics of the mutants in the of showed several components with lifetimes that presence Gd:HC1 CD~~9 3fjt" O PGKs. In this manner, one can 'span-the-space' differed substantially among these proteins, but, surprisingly, it was found that none of the rates 0 SAM loan 0 I 40 seeof thoecomplex reaction coordinate of folding induced with Gd:HC1 denaturation was consistently correlated with the H/D exchange rates. reactions much more completely. A new "rapid- Supported by NIA Grant #AGO976 1, NIH Molecular Biophysics Training Grant scan-labeling-approach" is being developed 3j j which will allow fluorescence energy-transfer 36 ; 67toZ12 2 =2distance measurements to be made on hundreds- 3. t e xeoe * ,s-s-. of-pairs, with the goal of making high resolution "intramolecular-distance-movies" of folding time (sec) reactions. Supported by NIH GM45990 to JMB.

164A MONDAY PROTEIN FOLDING & STABIUTY:STABILITY: FOLDING PATHWAYS & KINETICS 969-972

969 - Pos 970 - Pos KINETIC CONSTRUCTION OF A MODULAR ASSEMBLY MODEL FOR HYSTERESIS IN THERMAL UNFOLDING/REFOLDING OF DIMERIC APO- STAPHYLOCOCCAL NUCLEASE (SNakse) FOLDING. ALKALINE PHOSPHATASE INDICATES DISTINCT FOLDING AND UNFOLDING T. Y. Tsong, Z. D. Su, Dept of Biochem, Mol Biol & Biophys, Univ of Minn, St. Paul, MN 55108 PATHWAYS. Folding of SNase and several of its mutants in the time ranges sub-ms to 5,000 s elicits a modular Joseph A Schauerte, K. C. Wisser, P. M. Wolanin, D. G. Steel, A. Gafni, University Of Michigan assembly model. 1) It begins with the accumulation in sub-ms of structural embryos, which E. Coli Alkaline Phosphatase (AP) is a homodimeric Zn/Mg binding protein with high constitute 20% the helical content of SNase (or 5% of the peptide chain). 2) In 10 - 500 ms, the thermostability. However, apo-AP melts at approximately 35C lower temperature (-60C) and hydrophobic side chains collapse to forge the oligonucleotide binding (OB) and the C-terminal (C) demonstrates a pronounced hysteresis in the temperature dependent unfolding and refolding domains, concomitant with the appearance of the ANS binding sites. 3) Within 1.5 s these processes. Hysteresis in the thermal unfolding/ refolding pathways of apo-AP indicates that these hydrophobic clusters undergo compaction. As a result ANS binding sites are sequestered. 4) processes are not in equilibrium during temperature ramping measurements, and therefore a Merging of the OB and the C domains occurs in 30 s. The protein gains 95% of structure by substantial barrier exists that separates the native-> denatured and denatured-> native pathways. CD222,, and protein stability by microcalorimetry (AHF). 5) Despite the near completion of folding Spectroscopic techniques including Circular Dichroism and Room Temperature Phosphorescence by structural and energetic indicators, SNase remains inactive. The nuclease activity generates in a were used to characterize intermediates in the folding process, leading to identification of a separate 36-s reaction. 6) Most surprisingly, the first five folding events do not depend on proline slowly formed intermediate we hypothesize to involve formation of a critical N-terminal salt isomerization. The secondary structure of PallA (all six Pro replaced with Ala) folds in Is, 1,000 bridge. Analysis of the energetics of the conversion between the folding intermediates suggests times faster than the complete folding of the wild type but hydrophobic collapses and active site the unfolding/refolding pathways are distinct and do not represent a reversal of intermediates in a generation are similar for the two proteins. Furthermore, the trans/cis isomerization of Prol 17 is single unfolding/refolding pathway. We utilize crystal structures of apo-AP to describe a folding 50 times slower than the generation of the enzysne activity. 7) Trivial reaction aside, active site sequence involving hydrophobic collapse and subsequent salt bridge formation to explain the pH alignment appears to be the bottleneck in SNase folding. The folding funnel is rugged; the and salt dependence of the equilibrium and kinetic measurements of the folding process. population travails along the least activation path seeking the low ground of free energy. (Supported by NIA grant AG09761) Experiments were done, where necessary, by stopped-flow double-jump method to avoid interference of substrate and ANS on kinetics of protein folding.

971 - Pos 972- Pos THE MULTIPLICITY OF PATHWAYS BY WHICH A PROTEIN MAY FOLD CAN D/H AMIDE KINETIC ISOTOPE EFFECTS REVEAL WHEN HYDROGEN BONDS DETERMINE ITS FOLDING RATE FORM DURING PROTEIN FOLDING Jack Schoabrun, Ken A Dill, University of California at San Francisco, Graduate Group in Tobin R Sosaick', Bryan A Krantz', Liam B. Moran2, Alex Kentsis3, 'University of Chicago, 920 Biophysics, 3333 California St. Suite 415, San Francisco, CA 94122 E. 58th St., Chicago, II 60637, 2lllinois Institute of Technology Research, 3Mount Sinai School of Medicine Navigating the large ensemble of possible structures that they may adopt has generally been cited as a reason for proteins to have specific folding pathways. We present evidence that the large size We have exploited a procedure to measure when H-bonds form under fast, two-state folding of conformational space may instead help speed folding. Using eigenvalue methods to rigorously conditions using the equilibrium and kinetic deuterium/hydrogen isotope effects in backbone average over all possible folding pathways in a simple model, we find that many relaxation amides. Deuteration of backbone amides decreases the stability of equine cytochrome c and the processes are faster than expected. This comes from the fact that they may happen by multiple dimeric and cross-linked versions of the GCN4-pl coiled coil by about 0.5 kcal mol'. For all three routes. As folding proceeds, the number of viable routes goes down, and folding slows. It is systems, the decrease in equilibrium stability is reflected by a decrease in refolding rates and an possible for this slowing down to lead to a separation of time scales, and hence account for the equivalent increase in unfolding rates. This apportionment of the isotope effect indicates that about two-state nature of folding seen in kinetic experiments. 50%/o of the native H-bonds are formed in the transition state of these helical proteins. In contrast, an c/do protein, mammalian ubiquitin, exhibits a small isotope effect only on unfolding rates, suggesting that its folding pathway may be different. These four protein systems recapitulate the general trend that about 50%/o of the surface area buried in the native state is buried in the transition state, leading to the hypothesis that H-bond formation in the transition state is highly cooperative with a-helical proteins forming a number of H-bonds proportional to the amount of surface area buried in the transition state.

MONDAY HEMOGLOBINS 973-975

973 - Pos 975- Pos HEMOGLOBIN HYDRATION IN THE PRESENCE AND ABSENCE OF OSMOLITES HEMOGLOBIN INTRATETRAMERIC COMMUNICATION PATHWAYS OF 193 Herman E. Kwansa, Daniele Arosio, Grzegorz Piszczek, Enrico Bucci, U.Maryland Med. School, CYSTEINE (F-HELIX) PERTURBATIONS. 108N. Greene St., Baltimore, MD 21201 Rhoda Ellaon Hlrisch, Nazim A. Fataliev', Iraj Lalezari2, Celia Bonaventura3, 'Albert Einstein We used static (SLS) and dynamic light scattering (DLS) for comparing the mass (MW) College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, 2Montefiore Medical and hydrodynamic radius (Rh) of several hemoglobin systems, namely human hemoglobin, bovine Center, Bronx, NY, 3Duke University Marine Laboratory, Beaufort, NC hemoglobin , sebacyl crosslinked human hemoglobin and adipyl crosslinked bovine hemoglobin. The regulatory function of the 1393 cysteine residue has again become a focus of hemoglobin Measurements were performed in 0.1 M phosphate buffer at pH 7.0 in the absence and presence of research. It is well known that its covalent modification results in altered oxygen affinity, and, either betaine or glycerol up to 1.7 molal concentrations. We used a photon counting machine with more recently, in vivo nitrosation of this site has been suggested to play a significant role in the a diode laser at 783 nm, where hemoglobin is transparent. For all systems both in liganded and regulation of oxygen delivery to tissues. The interplay of this site with NO and regulatory ions unliganded form the mass increased with increasing concentration of osmolites, to a maximum of such as chloride is a subject of intense investigation (e.g., Bonaventura et al., 1998, 1999; Vasquez 20-25%. The hydrodynamic radii (Rh ) of all investigated systems with and without osmolites were et al., 1999). We explore the intratetrameric communication pathway of this residue by covalent close to Rh = 3.17 nm. Therefore the estimated average hydration volumes of the various systems site-specific labeling with a fluorescent probe. 5-iodoacetamidofluorescein is site-specifically decreased fiom 32.6 L/mole in the absence to 21.2 Umole in the presence of 1.7 molal osmolites. bound to 193 Cys (Hb-AF) as previously described (Hirsch et al., 1986), with verification by The constant value of the Rh implies that the decreased hydration volume was compensated by the mass spectrometry. Front-face fluorometry is used to study perturbation at this site by chloride mass un cases to a increased volume. The thickness of the water shell all remained close single and central cavity allosteric effectors. The central cavity 13-p and a-a clefts, and the A-helix 136 layer of water. The osmolites dependence of oxygen affinity reported by Colombo et al. [Colombo microenvironment may be directly involved in the communication pathway. Notably, (1) 16 M.F., Rau D.C., Parsegian V.A. Science 1992, 256,655-659], suggests a possible role of mass mutants exhibit differential responses to these perturbations, and (2) chloride enhances the as an volume allosteric effector of hemoglobin. mechanical precipitability of oxy HbS (-2-fold) and oxy HbA-AF (4-fold), but not for HbA.

165A 976-981 HEMOGLOBINS MONDAY

976 - Pos 977 - Pos DISTAL CONTROLLED ALLOSTERIC MECHANISM IN A DIMERIC HEMOGLOBIN THE LINKER CHAINS OF THE HEMOGLOBIN OF THE EARTHWORM: FROM MYCOBACTERIUM TUBERCULOSIS SEQUENCES AND STOICHIOMETRY Syun-Ru Yeh', Manon Couture', Yannick Ouellee, Uri Samuni', Joel M. Friedman', Michel Wea-Yen Kao', Kenzo Fushitani2, Claire K. Riggs', Austen F. Riggs', 'University of Texas, Guertin', Denis Rousseau ', 'Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, 'Kawasaki Medical School, 577 Kurashiki, Okayama, 70101, Japan NY 10461, Laval University, Quebec, GIK 7P4, Canada < P The allosteric transition in all cooperative hemoglobins studied to date is The extracellular hemoglobin (Hb) of the earthwonm, Lumbricus terrestris, has four major kinds initiated by a structural movement along the iron-histidine bond on the of 02-binding chains, a, b, c and d, that are present in equimolar proportions. Assembly of the full- proximal side of the heme in response to ligand coordination. Here we sized 4.1 MDa molecule requires participation of additional non-heme, non-globin structural 02 report a new allosteric mechanism discovered in a homodimeric chains, called "linkers". Determination of the amino acid sequences of the four different linkers ~jI~?, hemoglobin, HbN, from Mycobacterium tuberculosis. In this hemoglobin, via cDNA reveals that each linker has a highly conserved cysteine-rich segment of -38 residues heme-heme communication is manifested via conformational changes on that is homologous with the ligand-binding domains of the low-density lipoprotein (LDL) receptor the distal side of the heme induced by a strong hydrogen bond between the in humans. These domains have an absolutely conserved calcium-binding site comprising three r2 bound oxygen and a tyrosine residue at the BIO position. This novel aspartyl and one glutamyl residues which are also conserved in the linkers. New measurements of communication mechanism represents the first example of an allosteric heme and asnino acid content provide a linker-globin stoichiometry that is fully consistent with ~< 3 transition that is triggered exclusively by distal interactions. prior results of Zhu et al. (1996). The results suggest that the intact Hb has 24 linkers each with a high affinity calcium-binding site. Additional calcium participates in assembly and allosteric control of ligand binding. Although superoxide dismutase activity has been reported to be present in Lumbricus Hb (Liochev et al., 1996), sequence analysis of the linkers fails to show the expected motif of a Zn-Cu active site. Supported by NSF grants MCB 951179, 972385 and NIH grant GM 35847.

978 - Pos 979 - Pos HEMOGLOBIN ENCAPSULATED IN POROUS SOL-GELS. ISOLATION, CHARACTERIZATION AND KINETIC PROPERTIES OF Joel M. Friedman', Imran Khan', David Dansker', Laura Juszczak', Uri Samuni', Adam POLYMORPHIC HEMOGLOBIN FROM THE BLUE TREVALLY (CARANX Friedman', Roman Shinder3, 'Albert Einstein College of Medicine, Physiology and Biophysics, 1300 Morris Park New York 10461, of 3Comell Univ MELAMPYGUS). Ave., Bronx, 2University Penna, Randy Wiliam Larsen', James R. Bell', William McAulliffe2, 'University of Hawaii-Manoa, The exciting prospect exists of using sol-gel encapsulation both for biosensors and as a means of Chemistry, 2545 The Mall, Honolulu, Hawaii 96822, 2University of Guam trapping nonequilibrium protein structures. Despite the widespread interest in using this no of to Two hemoglobins from the predatory blue trevally (Caranx melampygus) have been technology, in-depth biophysical study protein response sol-gel encapsulation has been isolated and anion carried out. The of on the and purified using exchange chromatography. The two hemoglobins exhibit spectral impact encapsulation protocols conformation, dynamics ligand a of has been studied. A new is properties similar to human hemoglobin (HbA). In addition, both hemoglobins exhibit binding properties hemoglobin (Hb) systematically protocol significant Root effect below described for encapsulation that locks in the initial tertiary and quatemary protein structure and pH 6.5 and demonstrate significantly lower affinities towards CO, conformational both the relative to HbA, as judged by the rate of ligand recombination subsequent to photolysis. At pH 7.7 prevents subsequent change. Ligand rebinding kinetics, including the first-order constants of CO recombination and solvent ns have been pseudo rate to HbTFI and HbTF2 (first and second geminate phase rebinding following photodissociation (7 pulse), fractions, respectively, from a DE-52 column equilibrated with 50 mM Tris obtained for a variety of equilibrium and nonequilibrium forms of encapsulated COHb. The buffer, pH 7.7) accessibility and binding of allosteric effectors to encapsulated Hb were studied using a subsequent to photolysis are found to be (1.44+0.2)x103 s-l and (2.03+0.04)x103 s-l, combination of effectors including chloride, IHP, L35 and PyTS, a fluorescent analogue of DPG. respectively, as compared to (1.03_0.4)x104 s-l for HbA (PCO=I atm). At lower pH (< 6.5) both UV and visible resonance Raman spectroscopy as well as near IR absorption measurements were used to characterize the conformation and conformational dynamics of encapsulated Hb's. The HbTs (hemoglobin, trevally) exhibit biphasic kinetics with rate constants of (I. 1+0.07)x103 s-1 / results indicate that the encapsulation technique minimally perturbs the initial protein 5.44+1.2 s-l for HbTFI and (1.33+0.1)x103 s-I / 105+2 s-I for HbTF2. These data are consistent conformation but increases, in a temperature dependent fashion, tertiary and quatemary relaxation with a model in which each of the HbTs exhibit two populations of heme groups with dramatically times by many orders of magnitude while minimally impacting accessibility of added small different ligand binding affinities. The overall functional similarities between the two HbTs bring substrate molecules. into question their physiological role in the respiratory system of the fish.

980- Pos 981 - Pos QUATERNARY STRUCTURE OF NONSYMBIOTIC PLANT HEMOGLOBIN PREPARATION OF MULTIPLE DIASPIRIN-CROSS-LINKED HEMOGLOBINS AND Matthew D Goodman, D. B. Fulton, Mark S. Hargrove, Iowa State University SIMULATION OF THE CROSS-LINKER BINDING PATHWAY A large number of nonvertebrate hemoglobins have been charterized which exist in a number of Kevin M Bobofcbak, Barb Swedo, John Blatz, Kim Caswell, Kenneth W. Olsen, Loyola different quatemary states, some of which bind ligands cooperatively. There are two classes of University Chicago, Chemistry, 6525 N SheridanRd, Chicago, IL 60626 hemoglobins in plants; the symbiotic hemoglobins and nonsymbiotic hemoglobins. This second A series of cross-linking reagents, similar to the reagent bis (3,5-dibromosalicyl) fumarate (DBSF) class of plant hemoglobins is found in many plant species where expression occurs in a variety of have been synthesized and reacted with human hemoglobin A (HbA). The reagents differ only in tissues. While the function of this class of proteins is unknown, it is unlikely that they function in number of methylene spacers, resulting in a series of varied-length cross-linkers. As the length oxygen storage and/or transport due to their extremely low oxygen dissociation rate constants. increased, the reaction yield decreased, resulting in an optimal reaction with the fumarate/succinate reagents. HbA cross-linked under deoxy conditions exhibited decreased The crystal structure of a nonsymbiotic hemoglobin from rice (riceHbl) has recently been solved. oxygen affinity, and cross-linking under oxy conditions resulted in higher affinity, as expected. The asymmetric unit in this structure contains two molecules that contact in a phylogenetically The products were also analyzed for thennal stability and rate of auto-oxidation. Analysis of the conserved domain. Solution experiments had previously suggested that this protein is monomeric, binding pathway has been investigated using the Conjugate Peak Refinement Method (Fischer & in contrast with the crystal structure. The goal of this research is to eliminate this discrepancy and Karplus, Chem Phys Let. (1992) 194:252). Initial results show the DBSF fitting inside the central determine the conditions under which riceHbI is monomeric or dimeric. cavity without distortion of the HbA tetramer.

Our hypothesis is that riceHbl exists as a low affnity self associating dimer. We have used analytical size exclusion chromatography, analytical ultracentrifugation, and NMR spectrometry to characterize this system. Our results indicate that riceHbI exists as a dimer only at relatively high concentrations. In addition we have used site directed mutagenesis to probe the effects of quatemary structure on ligand binding.

166A 982-986 MONDAY111.10.11- HEMOGLOBINS

982 - Pos 983 - Pos SYNECHOCYSTIS CYANOGLOBIN IS A CHLOROPLAST HEMOGLOBIN. FOUR-CHAIN FUNCTIONAL SUBUNIT OF LUMBRICUS HEMOGLOBIN: Angela Noel Hvitved, Scott Premer, Mark S. Hargrove, Iowa State University, Biochemistry & MOLECULAR MODELING AND SPECTROSCOPIC APPROACHES. Biophysics, Ames, Iowa Jeffil D. Madura', Roda Elison Hirsch2, John P. Harrsnton3, 'Duquesne University, Pittsburgh, Novel hemoglobins and flavohemoglobins have recently been discovered in a number of PA, Albert Einstein College of Medicine, Bronx, N.Y, University of South Alabama, Mobile, organisms including plants, nonvertebrate animals, protozoans, fungi and bacteria. The functions AL of many of these hemoglobins are still unknown. In many cases, low in vivo concentrations rule The hemoglobin of the earthworm (L. terrestris) is one of the most studied acellular hemoglobins. out traditional oxygen storage and transport. Many hypotheses have been proposed as possible This protein is composed of more than 200 polypeptide chains. Four heme-containing chains functions. A hemoglobin from the nitrogen fixing cyanobacterium Nostoc commune has been (designated a, b, c, d or IV, II, Im, I) have been identified along with at least four linker chains that attributed a role in nitrogen fixation. A homologous hemoglobin is present in synechocystis, a play an essential role in the overall molecular architecture (-4 x 106 Da) of this oxygen transport cyanobacterium which cannot fix nitrogen. Both of these proteins share homology with a group of protein. Chains a, b, and c form an intra and intermolecular disulfide-linked trimer (abc) that is hemoglobins from diverse phylogenetic backgrounds including chlamydomonas, which has been coupled to the d-chain to form a functional tetrameric subunit (abed). Although a low resolution attributed with a function in chloroplasts. Biophysical characterization of the chiamydomonas crystallographic study of this hemoglobin was carried out earlier (Royer and Hendrickson, 1990), hemoglobin has shown that this protein is very different in its ability to bind oxygen than that from the high resolution x-ray structure is presently unavailable for either the intact LtHb or the N. commune. The goal of the research presented here is to determine whether the hemoglobin functional subunits. Two modeling efforts to establish the structure of the abcd unit have been from synechocystis is related to that from N. commune or chlamydomonas, or if it represents a new initiated (Madura et al, 1996; Viana et al.1998). Our effort employed a homology model approach biophysical class within this homologous group of proteins. Our hypothesis is that synechocystis using two different methods of analysis and modeling(LOOK and QUANTA). Although different hemoglobin is likely related to the chlamydomonas protein. We have tested this hypothesis by in their approach to predicting the structures of the heme-containing chains, both analyses expressing and measuring ligand binding kinetics for recombinant synechocystis hemoglobin and indicated myoglobin-like folding for the chains and both methods produced similar C. backbone several site directed mutants. Our results indicate that synechocystis hemoglobin is related structures. In each case the disulfide linkages of the abc trimer were considered. These studies biophysically to chlamydomonas hemoglobin. address several questions that have been the focus of our attention spectroscopically: a) the nature of the subunit interactions, exposure of aromatic amino acid residues, b) Cae2 interactions between subunits, and c) possible H-bonding residues associated with previous acid/alkaline dissociation studies.

984- Pos 985 - Pos EXTENDED MONOD-WYMAN-CHANGEUX (MWC) ALLOSTERIC MODEL OF THE CONFORMATIONAL STABILITY AND OXYGEN AFFINITY FOR HUMAN ADULT HEMOGLOBIN (HbA) IN THE PRESENCE AND ABSENCE OF HEMOGLOBINS IN HIGH ALTITUDE AND LOW LAND ALLOSTERIC Davood Ajloo, M. R. Dayer, A. A. Saboury, A. A. Moosavi-Movabedi, Institute of Biochemistry Takashi Yonetani, Sunglck Park, Kiyohiro Imai, Antonio Tsuneshige, Dept. of Biochemistry & and Biophysics, University of Tehran, Iran Biophysics, Univ. of Pennsylvania Medical Ctr., Philadelphia, PA 19104-6089, and Dept. of Physiology & Biosignaling, Osaka Univ. School of Medicine, Suita, Osaka 564-0083, Japan Oxygen dissociation curves for eight hemoglobin (Hb) samples (hemolysate form) were obtained We have measured oxygen binding equilibria of human adult hemoglobin (HbA) at 15'C by an by means of interaction of sodium dithionite (Na2S204 c = 8 mM-cnm' at X=3 14 nm) using UV- improved Imai method (Imai, K. and Yonetani, T. (1977) Biochim. Biophs. Acta 420 164-170)] Vis spectrophotometry in 50 mM Tris buffer pH 7.2. The value of oxygen affinity, [Dithionite],02 in a wide range of pH (pH 6.6 to 10.6) and in the presence and absence of allosteric effectors, C1, for Human, Bovine, Sheep, Chicken, Long-legged Buzzard (buteo buteo), Goose, Pigeon and 2,3-biphosphoglycerate (BPG), inositol hexaphosphate (IHP), and bezafibrate (BZF), individually Duck were obtained 0.99, 0.63, 0.58, 0.45, 0.43, 0.39, 0.30 and 0.23 mM, respectively. or in combination. Adair parameters (K,, K2, K3, and K4) and MWC parameters (KT, KR, and L) The difference between oxygen affinity of high altitude and low land samples relating to intrinsic analyses of the equilibrium data as a whole indicate that KT (u K,) and KR (a1K4) as well as L must oxygen affinity and the anionic effectors such as, 2,3-diphosphoglycyrate (2,3-DPG), be varied in order to describe these data consistently in the framework of the linear energy adenosinetriphosphate (ATP) and inositolpentaphosphate (IP5). The oxygen affinity decreases in ligation, the basic tenet of the MWC model. When HbA is moderately constraint by allosteric the presence of cited effectors. effectors (Phase 1; cf. Imai, K (1983) J. Mol. Biol. 167. 741-749), L. reaches a maximum of .-07 The unfolding of Hb was made by DTAB (Dodecyl trimethylammonium bromide), to obtain and KT decreases to -0.05mmHg', whereas L4 and KR remain constant at ~10-3 and -10mmHg-', [DTAB],0. as a stability parameter for Hb. The values of [DTAB]Jt are equal to 1.32, 1.29, 1.26, respectively. However, under increasingly stronger allosteric constraints (Phase 2), KT and KR 1.25, 1.10, 0.8, 0.4 and 0.38mM in 274 nm and 5.4, 5.0, 4.9, 4.7, 4.2, 3.6, 3.4 and 3.1 mM in 414 converge to a minimal value of -0.006mmHg-' and both LO and L4 approach unity, taking the nm for corresponding Hb,s, respectively. The data is shown the less stability for avian Hb related functional state that corresponds to the low-affinity extreme state [Yonetani el al (1998) J. Biol. to others and also confirmed by previous thermodynamic data. Chem. 273, 20323-20333]. To explain Phase I and newly found Phase 2 simultaneously it is The partial specific volume ( v ) is also measured by densimetry.The v values are equal to 0.752, needed to extend the original MWC model to accommodate variations of KT and KR caused by 0.785, 0.792, 0.814, 0.815, 0.832, 0.843 and 0.854 cm3/mol for corresponding Hbs respectively. preferential and substantial binding of allosteric effectors both to the T state and to the R state. The densimetric and previous thermodynamic data are shown the more flexible and larger volume in NIH HL14508. Supported part by for avian Hb s It can be concluded that the less stability of avian Hb structure corresponds to the lower oxygen affinity (high oxygen releasing).

986 - Pos S-NITROSYLATION OF HEMOGLOBIN AND OTHER SULFHYDRYL COMPOUNDS IN AQUEOUS SOLUTION BY REACTION OF NITRIC OXIDE UNDER AEROBIC CONDITIONS Antonio Tsuneshige, Takashi Yonetani, Dept. of Biochemistry & Biophysics, Univ. of Pennsylvania Medical Ctr., Philadelphia, PA 19104-6089, Philadelphia, PA 19104-6089 The S-nitrosylation in aqueous solution of sulfhydryl compounds, such as cysteine, glutathione, and hemoglobin (Hb), by reaction of nitric oxide (NO) in the presence of oxygen has been studied by monitoring optical spectral changes in the 300 - 400 nm region. The formation of S-nitroso compounds was detected by an increase in the absorbance at 337 nm. NO in the presence of oxygen forms NO2-, which can be easily recognized by a characteristic absorbance band around 372 nm, with a fine structure that increases in intensity as pH is decreased and N02- becomes protonated (pKa- 3.2). The S-nitrosylation reaction followed a first order scheme with respect to the concentration of sulfhsydryl compound, but was not affected by the concentration of NO in the solution. This reaction was found to be pH dependent and the rate decreased about 100 folds as pH was brought from 1.8 (tl, 11 sec) to 7.4 (t5, - 1200 sec). The reaction rate increased progressively as pH was lowered below 3.8, suggesting that the species responsible for the S- nitrosylation reaction might be HNO2 (or N203). Removal of oxygen from the solution prior the addition of NO, or decomposition of N02- by treatnent with ammonium sulfamate greatly impaired this reaction. To avoid the progressive oxidation of hemes in Hb, the cyanmet derivative, [Fe3'(CN)Hb], was used as an R-state model to study the S-nitrosylation process of the CysP93 residues. The reaction followed a behavior similar to those exhibited by the above-mentioned compounds, suggesting that under physiological conditions the S-nitrosylation process does not reach a favorable course (tl5 - 700 sec). Supported in part by NIH HL14508.

167A 987-992 SICKLE CELL HEMOGLOBINS MONDAY

987- Pos 988 - Pos PERSISTENCE LENGTH AND YOUNG'S MODULUS OF SICKLE HEMOGLOBIN A SPECTROSCOPIC STUDY OF SICKLE CELL HEMOGLOBIN POLYMER FIBERS. MELTING USING LASER PHOTOLYSIS OF CARBON MONOXIDE Robin W. Briehl', M. S. Turner2, G. Agarwal', S. Kwong', F. A. Ferrone3, 'Albert Einstein Joseph G. Louderbackl, Samir K. Ballas2, Daniel Benjamin Kim-Shapiro', 'Wake Forest College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, 2Rockefeller University, University, Department of Physics, 2Cardeza Foundation, Jefferson Medical College 3Drexel University We have previously reported time-resolved, wavelength dependent absorption and scattering Pathogenesis in sickle cell disease results from rigidification of red cells that contain a solid-like measurements of sickle hemoglobin (HbS) polymer melting in high concentration (1.8 M) gel consisting of non-covalently cross-linked fibers of sickle cell deoxyhemoglobin (HbS). phosphate buffer. Our results indicated the possibility of polymer melting via a mechanism that Although data exist on macroscopic rheology of the gel, there is no information on its basis in included carbon monoxide (CO) binding directly to the polymer. In this study, CO-ligated HbS in microscopic rheology. Therefore, we examined the spatial fluctuations of single fibers of phosphate buffered saline is photolysed with an argon ion laser to induce polymerization. Once deoxyHbS by video-enhanced differential interference contrast (DIC) microscopy. The bending polymerization has occurred, the laser beam is interrupted, permitting rebinding of the CO to modulus, ic, can be expressed as, occur causing the polymers to melt. Time-resolved absorption spectra are recorded during rebinding. These results are compared to those from normal adult hemoglobin (HbA). K = k_TL3/48 This work is supported by the Heart Lung and Blood Institute of the National Institutes of Health (grant No.HL58091 awarded to DBK-S). where is the mean squared displacement of the center of a fiber segment of length L, k3 is the Boltzmann constant and T is temperature. The persistence length is lp = c/kBT and for a rod of radius r, Young's modulus Y = 4ic/sr4. Single fibers were isolated from gels by selective local polymerization and depolymerization employing photolysis of CO HbS. We find a persistence length of 13 mm, corresponding to Y = 7 GPa, showing the fiber to be very stiff, with Y 3% that of steel. Since the fiber displacements are small and any random error increases the calculated value, our value represents a maximum displacement and hence minimum values for Ip and Y.

989- Pos 99 - Pos CLASSICAL AND POLARIZED LIGHT SCATTERING FROM SICKLE CELL NEW ATOMIC CONTACTS IN THE SICKLE HEMOGLOBIN POLYMER HEMOGLOBIN Frank Ferrone, Adam Roufberg, Drexel Univ., Physics, 32nd & chestnut streets, Philadelphia, PA Kejilg Chen', Roy R Hantgan2, Daniel Benjamin Kim-Shapiro', 'Wake Forest University, 19104 Department of Physics, Box 7507, Reynolda Station,, Winston-Salem, NC 27109-7507, 2Wake Sickle hemoglobin (36 Glu-Val) forms twisted 14 stranded polymers, whose structure has been Forest University, Department of Biochemistry, Medical Center Boulevard, Winston-Salem, NC reconstructed by electron microscopy. Crystals of deoxyHbS form linear Wishner-Love double 27157-1017 strands whose structure has been resolved to 2.0 A atomic resolution. Considerable evidence has Sickle cell polymers are formed in patients with sickle cell anemia by the aggregation of a mutant established that the 14 straned fiber is comprised of 7 Wishner-Love double strands. Within the form of hemoglobin (HbS). The polymerization of HbS in the red blood cells causes these cells to double strands, only 1 136 val is involved in an intermolecular contact, and model building by become rigid which contributes to microvascular occlusion characteristic of the disease. A good Watowich and Josephs has shown how such double strands can wrap into the observed fiber understanding of HbS polymerization requires a way to quantify the degree of polymerization. As structure. We have created a refinement of that model in which 4 of the 7 strand-pairs employ our preliminary calculations show, total intensity (classical) light scattering is not always linearly the second 136 in a contact almost identical with the primary P6 contact geometry. The paired dependent on the amount of polymer. However, previous measurements of polarized light double strands, which we call a "tetrad", appear as the outer strands of the fiber rather than those scattering from sickle red blood cells found that some types of polarized light scattering may be at its core. Support for this model also comes from co-polymerization studies of HbC or HbA more linearly dependent on the amount of polymer present. We have developed an instrument to with HbS. The averaged experimental value is found to be 0.37 which is inconsistent with measure the Mueller scattering matrix, which completely describes how the polarization state of models with only half the 16 sites employed (which implies a copolymerization probability of light is altered upon scattering. We have made measurements of all the Mueller matrix elements 0.5) of but are very consistent with the tetrad model proposed here which predicts a copolymerization both spheres, of which the precise results can be exactly calculated, and HbS samples. probability of 0.357. This work is supported by NIH (HL58091).

991 - Pos 992 - Pos NONIDEALITY AND THE NUCLEATION OF SICKLE HEMOGLOBIN NUCLEATION OF SICKLE HEMOGLOBIN POLYMERS MAY REQUIRE THAT THEY Frank Ferrone, Maria A. Ivanova, Drexel Univ., Physics, 32nd & chestnut streets, Philadelphia, TWIST PA 19104 Robert Josephs', Zhiping Wang', Frank ferrone2, Maria A. Ivanova2, Ravi Jasuja2, Lada Sickle hemoglobin polymerization occurs in highly concentrated solutions, which necessarily Krassnaiosselska2, Min Ding3, Kazumi Horiuchi3, Kazuhiko Adachi3, 'University of Chicago, entails the use of large activity coefficients. The theory for nucleation kinetics also uses large Molecular Genetics & Cell Bio, Chicago, IL 60637, 2Drexel Univ, 3The children's Hospital of activity coefficients for the nucleus, based on scaled particle theory and quasi-spherical shape of Philadelphia the nucleus. In order to test the accuracy of this theory we have used stochastic methods to Hemoglobin C Harlem is a naturally occurring mutant Hb in which the sickle mutation (136 Glu measure nucleation rates when cross-linked HbA is added to solutions of HbS. The crosslinked -Val) is augmented by loss of a surface charge at 173: Asp-+Asn. Polymerization of this HbA will not reproportionate with HbS, and has the same size as HbS. The homogeneous occurs at elevated nucleation hemoglobin slightly solubilities relative to HbS (24 g/dl vs 17 g/dl) indicating rates decrease by about 104 when half of the HbS is replaced by HbA. With no that the mutation creates a slight destabilization of the polymer. However, although exponential additional parameters, the present models of nucleation accurately predict this change and its high concentration kineticsare observed, delay times increase bi about 10,000 and homogeneous nucleation rates are dependence. Heterogeneous nucleation is not so well predicted, and improved dramatically slowed by a factor of about 10 Polymer domains show radiating crystallites rather models for that process are presently under investigation. Accurate understanding of non-ideality is essential for than conventional radial domains, and DIC images also show straight rather than bending fibers. understanding polymerization in the presence of oxygen, or other hemoglobins. Viewed by electron microscopy, no 14 strand fibers are observed, but abundant crystals are seen that are indistinguishable from needle-like crystals of HbS. Taken together these results imply that (1) most of the stabilization of the fiber occurs in the Wishner Love double strand, and not in contacts between strands, (2) heterogeneous nucleation can still occur, implying in turn that the process is a function of the double stand geometry, not that of the fiber and (3) without a twist, nucleation is frustrated despite similar thermodynamics.

168A MONDAY SICKLE CELL HEMOGLOBINS 993

993 - Pos A PLAUSIBLE PATHWAY FOR NUCLEATION IN SICKLE HEMOGLOBIN POLYMERIZATION Ravi Jasuja', Frank Ferrone', Maria Ivanova', Lada Krasanaiosselska', Rossen Mirchev2, Robin Briehl3, Gunjan Aggarwal3, Suzanna Kwong3, 'Drexel Univ, Physics, 32nd and Chestnut streets, Philadelphia, PA 19104, 2Harvard University, 3Albert Einstein College of Medicine Sickle hemoglobin (HbS) differs from normal human hemoglobin (HbA) by the replacement of Glu by a Val at the P-6 position. HbS at high concentrations will form polymers of 14 strands in 7 pairs. In HbS crystals, only one of the 2 P-6 Val sites stabilizes the double strand. In fibers, most strands will also have a single 136 contact, though we have proposed (Roufberg and Ferrone, these abstracts) that in some cases a second P6 is employed. Thus, AS hybrids may polymerize with not much if any disruption of significant fiber contacts. We have prepared such hybrids, cross linked at the N-termini of the alpha chains or at a 99 to prevent their disproportionation. The solubility of such cross-linked AS hemoglobin is slightly elevated relative to HbS. DIC microscopy reveals formation of fibers similar in appearance to HbS polymerization. However, the homogeneous nucleation rate measured by stochastic fluctuations is dramatically slowed, by a factor of approximately 104. The small change in solubility is consistent with added contacts as proposed by Roufberg and Ferrone (these abstracts). The great impact on homogeneous nucleation can be understood if the added contacts in the tetrad are essential for the nucleus.

MONDAY TRANSLATION 94-996

994- Pos 995- Pos WHEAT GERM TRANSLATION INITIATION FACTOR EIF4B AFFECTS EIF4A AND STUDIES OF THE MECHANISM OF UV LASER INDUCED PHOTOCROSSLINKING EIFISO4F HELICASE ACTIVITY BY INCREASING THE ATP BINDING AFFINITY OF IN PURINES AS RELATED TO THE STRUCTURE OF THE RIBOSOME EIF4A Stefan Frauzen, Simon Lapp[, Tatyana Shapkina, Paul Wollenzien, North Carolina State Xiping Bi, Jianhua Ren, Hunter College, City Univ. of New York, 695 Park Av., New York, NY University, Chemistry, P.O. Box 8204, Raleigh, NC 27695 10021 It has been proposed that during translational initiation, structures in the 5' untranslated region of Since the 1970s many groups have crystallized the ribosome and yet there is still no high- mRNA are unwound. eIF4A, a member of the DEAD box family of proteins, separately or in resolution structure due the large size of the protein (2.5 Mdaltons). We are developing laser- conjunction with other eucaryotic initiation factors, utilizes the energy from ATP hydrolysis to induced UV photocrosslinking experiments as a tool for determination of structure, and more unwind these structures. We have utilized direct fluroescence measurements, ATPase assays, and importandy dynamics during ribosomal function. Since UV photocrosslinking provides points of helicase assays to elucidate the mechanism of this reaction. The helicase activity of wheat germ contact (similar to NOEs in NMR spectoscopy) time-resolved UV laser photocrosslinking eIF4A is similar to other mammalian systems, however eIF4F or elF(iso)4F is required, probably experiments can be used to deternine the changes in structure that occur during translation. because of the low binding affinity of wheat germ eIF4A for mRNA. Direct ATP binding Crosslinking is possible between the protein coat, mRNA and tRNA and the ribosomal RNA. The measurements showed that eIF4A had a higher binding affinity for ADP than ATP (Kd'2.8 sM exact location of crosslinks can be pinpointed using gel electrophoresis and LC/MS analysis of the for ADP; Kr-=14.2 3M for AMP-PNP), resulting in a limited hydrolysis and procession along the digested ribosome. Crosslinks found in these experiments involve purine-purine and pyrimidine- RNA in the helicase assay. The addition of eIF-4B resulted in a change in binding affinity of pyrimidine crosslinks, whereas purine-pyrimidine crosslinks are rare. While pyrimidine- eIF4A for ATP, increasing it almost ten-fold (Ke=1.6 pM for AMP-PNP in the presence of pyrimidine crosslinks are well known (e.g. thymine dimers), crosslinks between purines have been eIF4B), while the ADP binding affinity was approximately the same. The data presented in this much less studied. However, the combined use of pulses of various time duration and frequency paper suggest that eIF4F or eIF(iso)4F act to position the eIF4A and stabilize the interaction with possible with tunable UV lasers combined with LCIMS studies of isotopically labeled nucleosides mRNA. ATP produces a conformational change which allows a limited unwinding of the RNA provide insight into the mechanism of crosslinking. Using a large number of constraints duplex. The binding of eIF4B either prior to or subsequent to hydrolysis allows for increased introduced by this and other crosslinking methods a model of the three-dimensional structure can affinity for ATP and for the cycle of conformational changes to proceed, resulting in fisrther be constructed. Ultimately, these techniques can be used on actively translating ribosomes. unwinding and processive movement along the mRNA.

996 - Pos LOCAL SWITCH AND GLOBAL TUNING OF THE DECODING RIBOSOME: A CRYO- EM STORY. Irene Gabashvll', Rajendra Agrawal', Robert Grassucci2, Catherine S Squires3, Albert E. Dahlberg4, Joachim Frank2, 'HRI at the Wadsworth Center, Empire State Plaza, PO Box 509, Albany, NY 12201-0509, 2Howard Hughes Medical Institute, HRI at the Wadsworth Center, 3Dept. of Mol. Biol. and Microbiol., Tufts University School of Medicine, Boston, MA, 4Dept. of Mol. and Cell Biology and Biochemistry, Brown University, Probidence, RI

Conformational changes in the Ecoli 70S ribosomes imduced by altemative base-pairing in the 16S RNA have been studied by three-dimensional cryo-clectron microscopy. Thermodynamically unstable base-pairing of the 912-910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885-887 (GGG) and 888-890 (GAG) was stabilized in either of the two states by point mutations at the 912 (C912G) and 885 (G885U) positions. A wave of rearrangements can be traced arising fiom the area of the switch in the three base-pairs involving functionally important regions in both subunits of the ribosome. This affects the topography of the A-site tRNA binding region on the 30S subunit significantly and explains changes in tRNA affinity for the ribosome and in fidelity of decoding mRNA.

169A 997-1001 DNA REPAIR MONDAY

997- Pos 998- Pos PECULIARITIES OF LONG-RANGE INTERACTION BETWEEN BROKEN DNA THEORY OF DEPOLYMERISATION AND AUTOREPAIRING OF DNA AT SEPARATE FRAGMENTS AND COMBINED ACTION OF IONIZING RADIATION Anatoliy 0. Pinchuk, Vladimir Vysotskii, Kyiv T. Shevchenko University, Volodymyrska 64, Vladimir L. Vysotskil, Anatoliy 0. Pinchuk, Kyiv T. Shevchenko University, Volodymyrska 64, Kyiv, 252033 Kyiv, 252017

In this work the energy of long-range intermolecular interaction between nucleotide pairs situated The time-dependent dynamics of formation, relaxation and auto-reparation of DNA double breaks on the opposite ends of a broken DNA helix in the intracellular milieu was considered. The total at combine radiation action and non-tradition processes of degradation was considered. The auto- long-range interaction energy corresponds to the sum of electrostatic interactions between charges repairing of DNA double breaks is related with the peculiarities of long-range interaction of of atoms involved in the structure of nucleotides and electrodynamics interaction between these nucleotide charges, atoms and molecules in the intracellular milieu. The properties of intracellular nucleotide pairs. The accounting of ion solution on Coulomb interaction was made based on liquid and the characteristics of force interaction between end pairs of nucleotides in the area of Debye - Hunkel theory. The indispensable dispersion characteristics for the calculation of van-der- DNA break in response to radiation are changed. Each kind of radiation is characterized by the Waals interaction between nucleotide pairs were found based on the Kramers - Kronig analysis of certain effectiveness of double DNA beaks formation but simultaneously one creates the the absorption spectra of the nucleotides in IR and UV range. conditions for their liquidation. On the base of the analysis and correlation of these processes the The numerical calculations of the total interaction energy have shown that during interaction time-dependent theory for DNA degradation was created, including hormesis phenomenon, between nucleotide pairs CG-CG and AT-AT a potential repulsive barrier arises at a distance R radiation antagonism, the validity of anomaly influence of low and large doses at sharp and chronic radiation and others effects. The qualitative and quantitative correspondence of the theory 7 A with the amplitude U(R) z 2 kT. This barrier can prevent DNA from selfrepairing after and experimental results of radiation biology was obtained. a double-strand break. The barrier vanishes at increasing of ionic force solution of intracellular medium. However, the remaining possible configurations of nucleotide pairs do not have a such barrier U(R)(O.

999 - Pos 1000- Pos MOLECULAR DYNAMICS SIMULATIONS OF DNA WITH AN ABASIC SITE. ROLE OF DNA BENDING IN DNA DAMAGE RECOGNITION BY UDG Gintaras Deikus, J.B. Alexander Ross, Roman Osman, Mount Sinai School of Medicine, One Eleanore Seibert', Edward L. Rachofskyl, Ning Luo', James T. Stivers2, Roman Osman', J.B. Gustave L. Levy Place, New York, NY 10029 Alexander Ross', 'Mount Sinai School of Medicine, I Gustave L. Levy Place, New York, New The first glycosylase-specific step of DNA excision repair creates an abasic site with a York 10029, 2Center for Advanced Research in Biotechnology, Rockville, MD 20850 deoxyribose that is a mixture of a- and P-hemiacetals. NMR studies have demonstrated that the The spontaneous deamination of cytosine to uracil in DNA or misincorporation of uracil during a and 3 forms of the sugar exist in dynamic equilibrium (Beger & Bolton, 1998, J. Biol. Chem. replication results in a potentially mutagenic basepair mismatch. Uracil DNA glycosylase (UDG) 273:15565). This equilibrium will have an impact on DNA structure, which could alter is a DNA base-excision repair enzyme that removes U from DNA, leaving an abasic sugar residue recognition of the abasic site by the apurinic/apyrimidinic endonuclease, the enzyme that cleaves that is processed subsequently by an apurinic/apyrmidinic endonuclease. Binding of UDG to the abasic site 3'-phosphodiester bond. Evidence suggests that monovalent and bivalent cations DNA induces bending, which facilitates base flipping into an extrahelical position and is a key affect DNA structure in a sequence-dependent manner, causing bending of DNA towards either step in specific damage recognition (Parikh et al. (1998) EMBOJ. 17:5214-26). We use the major or the minor groove. To assess the effects of cations on the dynamics of DNA with an fluorescence resonance energy transfer and molecular dynamics simulations to study the effects of abasic site, we carried out nanosecond molecular dynamics simulations of the DNA bending on recognition of the damaged base and binding by UDG. The oligos are labeled at deoxyoligonucleotide (5'-CGCGABACGCC-3'/5'-GGCGTATCGCG-3'), where B represents the the 5' phosphate of each of the complementary strands with a donor on one and an acceptor on the abasic site containing either the a or 3 form. The simulations were done in the presence of other. Analysis of molecular dynamics simulations of the unbound DNA with a U-G mismatch different concentrations of Na' and Mg2+. The results show that both forms bend towards the and of the complex with UDG, provides a distance distribution between the donor and the major groove, but the a form is more distorted allowing access of ions and water to the abasic site acceptor, which is compared with that obtained from energy transfer. The distributions are then sugar. The results of the simulations are used to analyze the dynamic properties of the DNA as used to evaluate global effects on DNA conformation and dynamics due to UDG binding to DNA well as the distribution of ions and water. that contains either U or an abasic site. Supported by NIH grant ROt CA-63317. Supported by NIH Training Grant GM 08553, and PHS grants CA 63317 and GM 56834

1001 - Pos QUANTITATION OF DNA DAMAGE BY INTACT BAND DEPLETION FOR HETEROGENEOUS DNA POPULATIONS. John C. Sutherland, Betsy M. Sutherland, Brookhaven National Laboratory, Biology Department, Upton, NY 11973 One method used to quantify DNA damage produced by agents that produce either strand breaks or lesions that can be converted to strand breaks is to measure the number of the initial population of NLO molecules of length L base pairs that remain unbroken after dose D. Assuming Poisson statistics, NL D = NLO e- where q1D is the probability of forming a lesion per base pair. Suppose we have a population of DNA molecules of heterogeneous lengths between two limiting values of L, and L2 that are sufficiently similar that they form an isolated band on a gel and that band is well resolved with any other DNA on the gel. Multiplying each side of the above equation by L and summing gives LNN = LNeLN L . Ethidium bromide produces a signal L- LI, proportional to the total number of base pairs at any point on a gel. Thus the products LNL.D and LNL.o are proportional to the fluorescence generated by molecules of length L. Converting to gel coordinates gives f f5(x)dx f= .f(x)ee ,L(,) dx, wheref(x) is the fluorescence from point x, x] and x2 are the distances of migration of molecules of length L, and L2 and L(x) is the gel dispersion function. From this equation and measured values of fD(x), fo(x) and L(x), we determine OD by numerical methods. Supported by OBER, USDOE.

170A MONDAYMONDAY VIRUS STRUCTURE, ASSEMBLY, ACTIVITY 1002-1007

1002 - Pos 1003 - Pos STRUCTURAL PROPERTIES OF THE PORTAL PROTEIN SUBUNIT OF CHARACTERIZATION OF THE ASSEMBLY OF P22 PORTAL RINGS IN VITRO BACTERIOPHAGE P22 IN RELATION TO QUATERNARY ASSEMBLY Peter E Prevelige Jr., Seas D Moore, Univ. of Alabama at Birmingham, 845 19th St. South, Arantxa Rodriguez-Casado', Sean Moore2, Peter E. Prevelige, Jr.2, George J. Thomas, Jr.', Birmingham, AL 35294 'University of Missouri - Kansas City, 2University of Alabama - Birmingham, 3School of Biological Sciences, University of Missouri - Kansas City, 5100 Rockhill Road, Kansas City, MO Double-stranded bacteriophage contain a portal vertex through which the genome is translocated. 64110 Structural studies on the portal proteins of various phage have revealed that they form ring-like structures composed of 11 to 13 identical subunits depending on the phage. To date, little is The portal protein subunit (gp 1, 84 kDa) of bacteriophage P22 is a member of a class of proteins known about the mechanism by which the protein subunits assemble into higher-order structures. that form a conically-shaped oligomeric ring through which viral dsDNA passes during the To characterize the oligomerization process of the bacteriophage P22 portal protein, the 84 kDa genome encapsidation process [1,2]. The portal assembly is essential not only for DNA subunit was cloned with a C-tenninal hia-tag, expressed to very high levels and purified. The translocation, but also for attachment of the virion tail that is required for host infection. Using a protein was isolated almost entirely in a highly soluble, un-polymerized form, but readily and recently developed expression system, recombinant His-tagged gpl has been produced in irreversibly fonned rings upon storage. The temperature-dependence of the in vitro quantities appropriate for biophysical analyses. The protein has been examined in both polymerization process was monitored over the temperature range of 15 to 30 degrees C, and an monomeric and assembled forms by Raman and CD spectroscopies. A preliminary analysis of activation energy of 12 Kcal per mole was calculated using an Arrhenius plot. The un- Raman marker bands of specific residues of the portal subunit, including the prominent Raman polymerized and ring forms were further characterized using fluorescence and circular dichroism S-H stretching band from the four cysteine sulfhydryls per subunit (C153, C173, C283, C516), spectroscopy. These studies revealed that the portal subunit is highly (>40%) alpha-helical and indicates structural sensitivity to the state of gpl assembly. The results provide an indication of undergoes a conformational change upon polymerization in vitro. These findings are s. F arted by the molecular basis of subunit recognition in portal ring formation, and constitute an important recent Raman spectroscopic data collected on the two forms.' This information will serve as a foundation for subsequent spectroscopic examination of portal interactions with other constituensts guide in our efforts to understand the mechanism of portal polymerization during viral of the P22 virion and viral precursor particles. [Supported by NIH grant GM50776.] morphogenesis. 1. Bazinet, C., et al. (1988) Biochemistry 27, 1849-56; Bazinet, C. & King, J. (1985) Annu. Arantxa Rodriguez-Casado, Sean D. Moore, Peter E. Prevelige, and George J. Thomas, Jr., Rev. Microbiol. 39,109-29. "Structural Properties of the Portal Protein Subunit of Bacteriophage P22 in Relation to 2. Orlova, E.V., et al. (1999) Nature Struct. BioL 6, 842-6. Quatemary Assembly", will be presented at this meeting.

1004 - Pos 1005 - Pos LOW pH INDUCED RELEASE OF DNA FROM ADENOVIRUS CAPSID ON THE STABILITY OF THE STACKED DISK AGGREGATE OF TOBACCO MOSAIC Atsuko Neglshi, William G. Matthews, Douglas McCarty, Richard J Samulski, Russell Taylor, VIRUS PROTEIN. Richard Superfine, Univ. of North Carolina at Chapel Hill, Materials Science, Chapel Hill, NC Ruben Diaz-Avalos, Donald L.D. Caspar, Institute of Molecular Biophysics, Florida State 27599 University, Tallahassee, FL 32306 The stacked disk aggregate of tobacco mosaic virus protein is an exceptionally stable polymer of We are studying the structure and disruption of adenovirus to understand the infectious pathway the coat protein of TMV, which occurs naturally in plants. This agegate shows the characteristic with the potential applications to viruses as gene therapy vectors. Biological mechanisms such as green birefringence of amyloids when stained with Congo red. Structurally, it is known that the structure/function relationships and protease activity strongly depend on environmental factors aggregate consists of paired rings of protein with 17-fold synmmetry, stacked axially with a stagger (i.e. pH, ionic concentration, osmolality). We are interested in this dependence during the of 6.35° between neighboring disk pairs, making a left handed, bipolar filament. A cryoelectron multistep infection cycle of the human adenovirus. Adenovirus contains a virion-associated microscopy reconstruction of the stacked disk has shown the protein rings to be very similar to the cysteine protease known to be activated in reducing environments that is essential for the assembly A-ring pair ofthe disk crystal; however, there seem to be discrepancies between the conformations and dissassembly of the virus capsid. Using the atomic force microscope (AFM), we have of the protein in the crystal and the stacked disk, notably at the region proximal to the helical axis observed that low pH induces the release of genomic contents from the capsid. The images of the of the filament, which contains a loop that has a large temperature factor in the crystal structure. DNA which emerged from the capsid revealed loops and supercoiling. Progress in the AFM Here we present the structure of the stacked disk at low resolution using intensities extracted from imaging of the virus disruption in vitro will be reported. X-ray and electron fiber diffraction pattems collected from well-ordered arrays of stacked disk fibers, combined with phases taken from the cryoelectron microscopy reconstruction previously done, thus getting rid of some of the imaging artifacts that plague electron microscopy reconstructions.

1006- Pos 1007- Pes INHIBMON OF SENDAI VIRUS-CELL FUSION BY BLOCKING THE FOLDING OF ROLE OF RNA ON THE MEMBRANE ASSEMBLY OF HIV-1 GAG THE Fl PROTEIN TO ITS ACTIVE COFORMATION Arthur J Goff, Carol Carter, Suzanne Scarlata, SUNY at Stony Brook, Microbiology, 232 life Sergio Gerardo Peisajovleh, Jimut K Ghosh, Yechiel Shai, Department of Biological Chemistry, sciences building, Stony Brook, New York 11794-5222 Weizmann Institute of Science, Rehovot 76100, Israel The assembly of HIV-1 involves interactions with the membrane surface of host cells through Viral glycoproteins catalize the fusion between viral and cellular membranes. Initially folded into matrix and possibly nucleocapsid domains, and the binding of viral RNA to the nucleocapsid an inactive conformation, the fusion protein (Fl) of Sendai virus undergoes a conformational region. Productive assembly results in viral budding and cleavage of Gag to mature matrix, change to become active. SV-201, a peptide we identified in a conserved region of F1, is able to capsid, and nucleocapsid proteins. We are testing a model in which RNA acts as a pre-assembly inhibit viral-cell fusion, presumably by binding to its counterpart in the Fl protein. However, its factor that gives Gag the correct membrane orientation for the protein-protein interactions required detailed mechanism of inhibition remained unknown. Here, we synthesized a shorter version of for post-assembly events. To test this model in an in vitro context, we have measured the binding SV-201, namely SV-208, and compared it functionally and structurally with SV-201. of Gag to model membranes (large, unilamellar vesicles of I-pahnitoyl-2-oleoyl- Fluorescence, CD and FTIR spectroscopy studies indicate that both peptides bind to zwitterinoic phosphatidylserine) and to tRNA. We find that pre-assembly of Gag on tRNA inhibits membrane membranes, where this region adopts a a- helical structure. SV-201 promotes vesicle aggregation binding in a systematic manner. Preliminary data indicate that membrane binding induces stronger and destabilization, suggesting a role in the fusion process. Contrary to SV-201, SV-208 does not Gag-Gag interactions, that are only weakly present when bound to tRNA. inhibit virus-cell fusion. Interestingly, SV-201 is able to self-associate both in its membrane- bound state and in solution, whereas SV-208 only oligomerize in the membrane. This suggests that SV-201 inhibits virus-cell fusion by binding to its counterpart in the Fl protein in solution, before the corresponding region of Fl binds to the membrane. Based on these results we postulate a revised model of Sendai virus-induced membrane fusion.

171A 1008-1009 VIRUS STRUCTURE, ASSEMBLY, ACTIVITY MONDAY

1008 - Pos 1009- Pos HIV-1 CAPSID PROTEIN MODULATION OF CYCLOPHILIN'S MEMBRANE ELECTROSTATIC HOMOLOGY OF RETROVIRAL MATRIX PROTEINS SUGGESTS BINDING ABILITY AS MONITORED BY FLUORESCENCE SPECTROSCOPY A MECHANISM FOR PLASMA MEMBRANE TARGETING Marjorie Bon Homme, Dr. Loina Ehlrich, Dr. Carol Carter, Dr. Suzanne Scarlata, State Barry Honig, Diana Murray, Columbia University, 630 West 168th Street, New York, NY 10032 University of New York at Stony Brook, Physiology and Biophysics, Stony Brook, New York Gag polyproteins of the human immunodeficiency virus type I (HIV-I) are targeted to the host 11794-8661 cell plasma membrane where they play a crucial role in virus formation. Experimental studies Human Immunodeficiency Virus type 1 (HIV-1) requires the cellular protein, cyclophilin (CyP) A have established that a myristate and cluster of basic residues in the N-terminal matrix (MA) for infection. CyP A, a cellular chaperone with cis-trans prolyl isomerase activity, is a highly sequence are required for membrsne association of HIV-1 Gag. Calculations of the electrostatic conserved protein found in all species and cell compartments. Recently, CyP B was found to have interactions between MA and phospholipid membranes predict that, under physiological receptors on the surface of T cells. While it is known that the CA-CyP A interaction is required conditions, electrostatics enhances the membrane partitioning of HIV-1 MA by three orders of for an early event, its role is not understood. Here, we propose a model wherein CyP A has some magnitude. Three-dimensional structures of five functionally related retroviral MAs show they ability to bind membrane and this binding is altered in the presence of CA. Thus, the CA-CyP A share the same overall topology in spite of exceptionally low sequence similarity. In each interaction may provide a functional means for control of CyP interactions at the viral membrane structure, MA presents a cluster of basic residues to a surface patch that participates in membrane during viral entry into T cells. NIH GM53132 targeting during viral assembly. Examination of a structure-based sequence alignment reveals that these basic residues are not conserved in the linear sequence. Thus, these retroviral MAs share functional, structural and electrostatic homology in the absence of sequence homology. The generality of this observation is being tested by using the structure-based sequence alignment in the construction of homology models for MAs of unknown structure. As the plasma membrane is thought to be enriched in acidic lipids with respect to other intracellular membranes, electrostatics may play an important role in retroviral Gag targeting.

1010-1013 TOXIN-CHANNEL INTERACTIONS MONDAY

1010 - Pos 1011 - Pos MECHANISM OF a-SCORPION TOXIN BINDING TO SITE 3: VOLTAGE- THE DOMAIN 4 S3-S4 EXTRACELLULAR LOOP PROVIDES MOLECULAR SENSITIVITY OF AFFINITY IS DUE TO CONFORMATION CHANGES ASSOCIATED DETERMINANTS FOR BINDING OF a-SCORPION TOXINS (LqhII, LqhIII, and WITH ACTIVATION AND INACTIVATION IN THE RAT SKELETAL MUSCLE Na+ CHANNEL LqhllT) TO THE VOLTAGE-GATED RAT SKELETAL MUSCLE Na+ CHANNEL (rSkMl) (rSkMl) Zhongming Lihui Sumei Jun Dalia Mal, Tang', Lu', Kong', Gordon2, Roland G. Kallen', 'U of Zhongming Mal, Lihui Tang', Sumei Lu', Jun Kong', Dalia Gordon2, Roland G. Kallen', 'U of Penn Sch of Med, 2Tel-Aviv University Penn Sch of Med, 2Tel Aviv University The Kd values for seven mutations at six positions in the putative S3-S4 loop of Domain 4 were a-Scorpion toxins, such as LqhII from Leiurus quinquestriatus hebraeus, are responsible for a analyzed with LqhII, LqhI and Lqh aIT. These studies show that the three toxins bind to substantial slowing of the of rSkMI current decay rates (inactivation) and the appearance of a overlapping sites involving several amino acid residues in the S3-S4 loop of Domain 4, most residual current in tsA201 cells. Toxin binding equilibria and kinetics were measured from the importantly Asp'428. However, relative differences in affinity caused by mutations at the magnitudes of the fast and slow exponential current decay kinetics, which are attributed to free neighboring sites clearly indicate that the bioactive surfaces are distinguishable. and toxin-bound channels, respectively. At the holding potential of -120 mV, the LqhII association (k,,) and dissociation (kff) rate constants are -7 x 105 M' s-' and -2 x 103 s'. The voltage-dependence of LqhII k,,, and koff values are downwardly and upwardly sigmoidal, Apparent Kd of a-Scorpion Toxins with rSkMl respectively, with increasingly positive voltages with midpoint potentials at -73 and -38 mV, Mutants Lqh-II n Lqh-III n LqhaIT n respectively, (cf. G-V and h curve midpoints of -37 and -75 mV) and plateau values at > 20 mV of -6 x 104 M' s' and -1.9 x 10.2 s-, respectively. The voltage-dependence of the toxin WT 5.58±0.80 10 4.57±0.70 7 4.83±0.88 7 dissociation equilibrium constants (Kd) is upwardly sigmoidal as voltages become more positive D1428R 591±29 4 2370±173 4 4176±931 3 with a midpoint potential at -49 mV. Toxin binding appears to be affected by two components of D1428N 37.38±4.94 7 481±15 5 2955±745 3 S4 segment movements or the activation/inactivation gates or both. Q1431E 13.32±1.08 6 7.00±0.82 7 13.73±2.65 6 K1432Q 18.96±1.83 6 4.60±0.41 7 208± 38 4 S1436A 6.54±0.59 6 4.82±0.59 7 7.15±1.37 6 L1439A 8.66±1.22 6 5.87±1.09 6 6.27±1.43 6 F1441L 9.29±0.77 7 5.22±0.85 6 4.62±1.03 6

1012 - Pos 1013 - Pos EFFECT OF ,-POMPILIDOTOXIN ON Na' CHANNEL ISOFORMS EXPRESSED ON A UNIFIED NOMENCLATURE FOR SHORT CHAIN PEPTIDES ISOLATED FROM HEK 293 CELLS; CHIMERIC STUDY TO DETERMINE THE SITE OF ACTION. SCORPION VENOM: a-KTx MOLECULAR SUBFAMILIES. Issel Seysma', Eiji Kinoshita', Hiroshi Maejima2, Kaoru Yamaoka', Nobufumi Kawai3, Jan Tytgat', K. George Chandy<, Maria L. Garcia3, George A. Gutman2, Marie-France Martin- 'Department of Physiology, School of Medicine, Hiroshima University, Kasumi 1-2-3, Minami- Eauclaire4, Jurg J. van der Walt , Lourival D. Possani , 'University of Leuven, Leuven, Belgium, Ku, Hiroshima, 734-8551 Japan, 2Division of Physical Therapy, School of Medicine, Hiroshima 2University of California, Irvine, USA, 3Merck Research Lab., Rahway, USA, 4Universite de la University, 3Department of Physiology and Neurosurgery, Jichi Medical School Mediterran6e, Marseille, France, 5University of Potchefstroom, Potchefstroom, South Africa, 3-pompilidotoxin (P-PMTX) has been purified as a novel peptide neurotoxin from the venom of 6UNAM, Cuermavaca, Mexico the solitary wasp Batozonellus maculifrons and found to greatly affect the synchronized network firing of rat cortical neurons. In the present study, we investigated the effect of this toxin on Peptidyl toxins are used extensively to characterize several aspects of ion channels. Four families severd Nae channel a-subunit isoforms trangently expressed in HEK 293 cells using the whole- of peptides have been purified from scorpion venom: (I) family I contains peptides of 60-70 cell patch clamp method. ft-PMTX blocked fast inactivation without modification of the activation amino acids that modulate Nae channel activity; (2) family 2 comprises short- and long-chain process in rat brain type IIA Na+ channel a-subunit (RBIIA), but failed to affect it either in rat peptides (30-40 and 60-64 amino acids, resp.) that block K' channels; (3) family 3 contains short- heart (RHI) or skeletal muscle Nae channel a-subunit. This tissue-specific modification helped to chain insectotoxin-like peptides of -36 amino acids that supposedly inhibit Cl channels; (4) determine the binding sites of this toxin by making several RBILA/RHI chimeric Nae channels. family 4 includes peptides that modulate ryanodine-sensitive Ca2' channels. Here, the The chimeric study of domain (D) substitution between RBIIA and RHI revealed that the classification of K+-channel-blocking peptides belonging to family 2 peptides and comprising 30- sensitivity of P-PMTX was dependent on DIV of RBIIA. Furthermore, we tested whether the 40 amino acids linked by three or four disulfide bridges, is presented. Evidence is provided for the structural varieties of DIV segment (S) 3 in RBIIA and RH1 contributed to the difference in existence of 12 molecular subfamilies, named a-KTxl-12, containing 49 different peptides. sensitivity to ,-PMTX. The point mutation method demonstrated that several amino acids in Because of the pharmacological divergence of these peptides, the principle of classification was DIVS3 were critical to the P-PMTX-effect. A possible link between the site of action of P-PMTX based on a primary sequence alignment, combined with maximum parsimony and Neighbour- and fast inactivation of Na' channel will be discussed. Joining analysis (Tytgat et al. (1999) Trends in Pharmacol. Sci., in press).

172A INTERACTIONS 1014-1018 MONDAY TOXIN-CIIANNL 1014101D 1014 - Pos 1015- Pos POTASSIUM RELEASE FROM LIPOSOMES AS THE MEASURE OF THE A SYNTHETIC PEPTIDE INCREASES CHLORIDE SECRETION IN EPITHELIAL MEMBRANE DESTABILIZATION ACTIVITY OF PEPTIDES. CELLS BOTH BY FORMING A NOVEL PERMEATION PATHWAY AND Anatoll Y Silberstein', Tajib Mirzabekov2, W. French Anderson', Yanina Y Rozenberg', ACTIVATION OF AN ENDOGENOUS CHLORIDE CHANNEL 'Medical School of USC, Gene Therapy Labs, 1355 San Pablo St #205, Los Angeles, CA 90033, John M. Tomich, Kathy Mitchell, James R. Broughman, Bruce Schultz, Kansas State 2Harvard Medical School, Boston, MA University, Biochemistry, Willard Hall, Manhattan, KS 66506 A direct correlation between chloride secretion and pathophysiology of cystic fibrosis has been Potassium (K' ) selective electrode was used to evaluate the rate of K' release from large demonstrated. Based on this premise, we designed channel-forming peptides that mimic the pore- unilamellar vesicles (LUV) induced by a number of membrane-active peptides. Improvements of forming M2 transmembrane sequence of the glycine receptor, a chloride channel from brain this approach and analysis of possible pitfalls allowed us to develop highly sensitive, reproducible (Tomich et al., 1998). Here, we examined the effects of NK4-M2GlyR on whole-cell chloride and inexpensive method for the evaluation of membrane destabilizing activities of peptides and conductance in isolated T84 cells as well as its effects on chloride tansport across T84 other conmpounds. This K' release method of monitoring membrane destabilizing activities is more monolayers. NK4-M2GIyR induced a large increase in whole cell chloride conductance. The sensitive than one of the standard methods that uses ANTS/DPX as inner marker of liposomes. In resulting current showed slight outward rectification and some time-dependence at depolarized addition, this method allows us to expand the set of molecules used as inner content markers to a potentials. Treatment with DIDS reduced the NK4-M2GIyR- induced current by 8.4% at -80 mV lower size range. This method was applied to compare the membrane activities of several amphi- and 30% at +80 mV. These results suggest that there are multiple components to the NK4- pathic peptides of natural origin as well as artificially designed helices. Two different effects were M2GlyR-induced current in T84 cells. The outwardly-rectifying, DIDS-sensitive component induced by amphiphilic peptides: leakage of the inner content and vesicle aggregation. The later resembles the endogenous calcium-activated chloride current in T84 cells. When NK4-M2GlyR one is conducive with fusion. The effect of lipid composition on peptide-induced membrane was applied to the apical surface of epithelial monolayers, a large increase in short circuit current destabilization was studied. (Ise) was observed. This increase in Isc was partially blocked (<50%) by treatment with DNDS and H-8. These results suggest that the NK4-M2GlyR peptide assembles to produce a synthetic channel that introduces a novel pathway for chloride permeation in the cell in addition to activation of an endogenous chloride conductance that resembles the Ca2+ activated chloride current. The all D-amino acid peptide, D-NK4M2GlyR had a smaller effect on Isc across epithelial monolayers than the all L-isomer suggesting that the activity of the NK4-M2GIyR peptide is partially stereospecific.

1016- Pos 1017 - Pos TERTIAPIN POTENTLY AND SELECTIVELY BLOCKS MUSCARINIC K CHANNELS IDENTIFICATION OF A PEPTIDE TOXIN FROM GRAMMOSTOLA SPATULATA IN RABBIT CARDIAC MYOCYTES SPIDER VENOM THAT BLOCKS STRETCH-ACTIVATED CHANNELS. Hidetsuna Kitamura', Yoshihisa Kurachi2, MItanhiko Yamada', 'National Cardiovascular Center Thomas M Suchynsl, Janice H Johnson2, Katherine Hamer2, Henry F Clemo3, Clive M Research Institute, 2Faculty of Medicine Osaka University Baumgarten3, Joseph F Leykam4, Douglas A Gage4, Frederick Sachs', SUNY at Buffalo, 320 Tertiapin is a peptidyl toxin isolated from honey bee venoms. Tertiapin is a potent blocker of cary Hall, Buffalo, NY 14214, 2NPS Phamaceuticals, Inc., 420 Chipeta Way, Salt Lake City, UT ROMKI and GIRKI/GIRK4 channels (Jin W and Lu Z (1998) Biochemistry 37: 13291). We 84108, 3Dept. of Internal Medicine and Physiology, Virginia Commonwealth University, examined the effect of tertiapin on ion channel currents in rabbit cardiac myocytes with the patch- Richmond, VA 23298, 4Dept. of Biochemistry, Michigan State University, East Lansing, MI clamp technique. In the whole-cell configuration, tertiapin fully inhibited acetylcholine (1 tiM)- 48824 induced muscarinic K' (KAra) channel currents in atrial myocytes with the IC50 of-8 nM through We have identified a 35-amino acid peptide toxin (GsMTx-4) that specifically blocks cationic -1: 1 stoichiometry at both -40 and -100 mV. Tertiapin also inhibited the KAcI, channel stretch-activated ion channels. GsMTx-4 was isolated from the venom of the spider Grammostola preactivated by intraellular GTPyS, a nonhydrolyzable GTP analogue. The IKI channel was spatulata and has < 50%/o homology to other neuroactive peptides. However, it has 6 cysteines that >100 times less sensitive to tertiapin, and the ATP-sensitive K' channel was virtally insensitive to form an inhibitor cysteine knot consensus sequence, which is a motif commonly observed in other the toxin. Other ion channel currents in cardiac myocytes were not affected by tertiapin. At the ion channel peptide toxins. GsMTx-4 was identified by fractionating whole venom using reverse single channel level, tertiapin inhibited KACh channels from the outside of the membrane by phase HPLC, and then perfusing the fractions onto SACs in outside-out patches from adult rat reducing the NP, without changing the single-channel conductance. These results indicate that astrocytes. SAC gating properties in outside-out patches showed pronounced differences from the tertiapin potently and selectively causes a slow block of KAci, channels in cardiac myocytes in a properties of SACs in cell-attached patches. However, properties associated with the channel receptor- and voltage-independent manner. Tertiapin is a novel pharmacological tool to identify pore, such as selectivity for alkali cations, conductance (-45 pS at -100 mV) and mild the functional role of the KAC,h channel in the parasympathetic regulation of the heart beat. rectification are all unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appears to be specific since it had no effect on whole cell voltage sensitive currents. GSMTx-4 had an equilibrium affinity of -430nM for the SACs as computed from the ratio of association and dissociation rates. In hypotonically swollen astrocytes, GsMTx-4 produces an -40°/o reduction in swelling-activated whole cell current. Similarly, in a rabbit dilated cardiomyopathy model GsMTx-4 produced a near complete block of the volume sensitive cation selective current, but did not affect the anion selective current.

1018- Pos CHARACTERIZATION OF THE MAITOTOXIN-INDUCED CURRENT IN HUMAN SKIN FIBROBLASTS. Juan Ram6m Martinez-Franaols, Ver6nica Morales, Luis Vaca, Instituto de Fisiologia Celular, UNAM, Circuito Exterior S/N Ciudad Universitaria, Mexico, Distrito Federal 045 10 Mexico Maitotoxin (MTX) is a polyether produced by the dinoflagellate Gambierdiscus toxicus. This is the most potent marine toxin known to date and is responsible for ciguatera poisoning. M1TX has various effects at the celular level: it causes a rise in the intracelular calcium concentration and activates cationic channels in a wide variety of cell types. The mechanism by which MIX produces these effects is not known. Here we present the characterization of the MTX-induced current (ImTX) in human skin fibroblasts. Using the whole cell configuration of the patch clamp technique we explored the intra and extracelular divalent cations required for the activation of ImTx. Calcium and sodium influx were monitored, also, using selective fluorescent indicators for these cations. In basal conditions, the fibroblasts have a predominant potassium outward rectiflying current which can be inhibited with TEA (1mM). MTX activates a cationic current and causes a rise in the intracelular concentrations of calcium and sodium ions as monitored by the fluorescence of Fura-2 and SBFI respectively. The presence of intracelular and extracelular calcium is needed for the activation of IMT. Extracellular calcium can be partially substituted for barium, but not for magnesium or manganese. The long term objective of the present study is the understanding of the mechanisms underlying the activation of IMTx.

173A 1019-1024 PEPTIDE CHANNELS MONDAY

1019 - Pos 1020- Pos INFLUENCE OF ALCOHOLS ON VOLTAGE-SENSITIVE CHANNELS FORMED BY ION-CHANNEL FORMATION IN ARTIFICIAL LIPID MEMBRANE BY THE SYRINGOMYCIN E. BACILLUS THURINGIENSIS CRY4B TOXIN AND ITS MUTANT Yuri A Kaulin', Ludmila V Schagina2, Igor A Mezine3, Jon Y Takemoto4, John H Teeter3, Joseph Panapat Uawithyal, Umnaj Chanams', Gerd Katzenmeier', Sakol Panyim', Chartchai Krittanail, G Brand3, 'Monell Chemical Senses Center, Philadelphia, PA; Institute of Cytology of RAS, Lena Potvin2, Jean Louise Schwartz2, Chanan Angsuthanasombat', 'Institute of Molecular Biology St.Petersburg, Russia, 3500 Market Street, Philadelphia, PA 19104, 2Institute of Cytology of RAS, and Genetics, Mahidol University, Salaya Campus, Nakompathom, 73170 Thailand, St.Petersburg, Russia, 3Monell Chemical Senses Center, Philadelphia, PA, 4Utah State Univ., 2Biotechnology Research Institute, National Research Council of Canada Logan, USA We have previously shown that the cyclic lipodepsipeptide, syringomycin E (SRE), forms voltage- The Cry4B 6-endotoxin belongs to the crystal insecticidal protein family produced by Bacillus sensitive ion channels in planar lipid bilayers. Here we present data on the influence of alcohols on thuringiensis, which exhibits toxicity against mosquito larvae. We have previously reported on a the channel-forming activity of SRE in lipid bilayers. Alcohols starting from C2 increase the rate single proline substitution in the central region of a4 resulting in a nearly complete loss of of opening of SRE channels when positive voltage is applied from the side of SRE addition. larvicidal activity of the Cry4B toxin that reveals a crucial role for this amphipathic helix in toxin Alcohols also provoke a more rapid closing of these channels when the voltage polarity is function, conceivably in toxin pore-formation. In the present study, the Cry4B wild-type toxin was changed. To understand the mode of action of alcohols at the molecular level, we perfonned MD found to form ion-permeable channels by integration in artificial lipid bilayer membranes. Under simulations of SRE in three different environments: 1) in vacuo; 2) in hexane; 3) in water. A symmetrical conditions and at pH 9.0, the current-voltage relation of the single channels was minimal energy conformation of the SRE molecule in vacuo reveals a highly bent peptide ring, linear with a conductance of ca. 450 pS similar to those found for other Cry toxins. Reconstitution stabilized by electrostatic interactions between charged amino acid residues and the presence of a of the proline-substituted mutant protein in the bilayers is in progress. hydrogen bond within the SRE molecule. The results of MD simulations of SRE in water and hexane show that formation of the hydrogen bond is a more favorable process in a hydrophobic environment. Based on these data we propose a possible mechanism for the effect of alcohols on channel-fonning activity of SRE in lipid bilayers. The ability of alcohols to regulate channel activity has been shown for many native voltage sensitive channels. But these native channels are structurally complex. Therefore studying a relatively simple molecule such as SRE can provide valuable information on mechanism of toxic effects of alcohols.

1021 - Pos 1022 - Pos ALAMETHICIN AS MODEL SYSTEM FOR ION SELECTIVITY: COMPUTATIONAL GRAMICIDIN POLYMORPHISM. STUDIES B. A. Wallace, Birkbeck College, Univ. of London, London, WCIE 7HX United Kingdom D. Peter Tielemna', G. A. Woolley2, Mark S P Sansom', 'University of Oxford, Lab. of Gramicidin is a 15 amino acid polypeptide which forms ion channels in membranes. It adopts two Molecular Biophysics, Rex Richards Building, South Parks Road, Oxford, 9747 AG United major classes of structures: helical dimers and double helices. Helical dimers are the principal Kingdom, 2University of Toronto conducting fonns in phospholipid membranes; double helices have been observed in organic Alamethicin is an alpha-helical antimicrobial peptide that forms stable cation-selective channels. solvent solutions and crystals. In solution in the absence of ions, there exist at least four double In previous work we have analysed models of channels formed by parallel bundles of 4-8 helical forms, which differ in hand, stagger, and orientation of chains. The relative amounts of alamethicin molecules (1). In its native form, alamethicin contains a glutamate residue, which in each form differ with solvent type and temperature. To date, only crystals of one of these, a left- channel models combine to form a ring of charged residues. handed antiparallel form, have been produced. Different ion-containing structures are also found pKa calculations based on continuum electrostatics calculations have shown that in a model of six in solution at different temperatures, as shown by CD spectroscopy. Ion-containing crystals of two helices up to five of the glutamates are actually uncharged due to the strong local electric field distinct types have been produced when different temperatures and ion-polypeptide ratios are created by the alpha-helices and due to sidechain/sidechain interactions. Simulations have shown used: a left-handed antiparallel double helical form (PDB ID=1C4D) and a right-handed that a model in which all six glutamates are charged is unstable, while models with only one or antiparallel double helical form (PDB ID=IAV2). The two structures have similar phi, psi angles zero glutamates charged are stable (2). and molecular dimensions, but differ in the handedness of their helical twist and in their ion Here we describe an extension of the computational studies to a analogue of alamethicin that has contents and ion positions. These results indicate that the type of polymorphism previously seen in been synthetically altered to contain lysines instead of glutamates. Experimentally, this change solution for gramicidin is also found in crystals; this polymorphism is possible due to the presence leads to anion selectivity instead of cation selectivity. of alternating L- and D-amino acids in the sequence. To study the factors influencing the ionic selectivy we have performed simulations with different charge states for the lysines. In addition, we have included explicit salt concentrations of 0.1 to 1.0 M KCI.

(I) Faraday Disc. 11 1:209-223 1998 (2) Biophys J. 76:1757-1769 1999

1023 - Pos 1024 - Pos DETECTION AND PROPERTIES OF SINGLE- AND DOUBLE-STRANDED BACKBONE MODIFICATIONS IN GRAMICIDIN CHANNELS GRAMICIDIN CHANNELS Sigrid E. Schmutzer', Lyndon L. Providence2, Olaf S. Andersen2, Denise V. Greathouse', Roger Olaf S. Andersen', Roger E. Koeppe II2, 'Cornell University Medical College, New York, E. Koeppe II', 'University of Arkansas, Fayetteville, AR 72701, 2Cornell Medical College, New NY10021, 1300 York Avenue, New York, New York 10021, 2University of Arkansas York, NY 10021 Previously, we used organic synthesis to replace a peptide bond between formyl-Val' and Gly2 in The linear gramicidins assume different conformations that are environment-dependent. Two of gramicidin A by an ester bond (Jude et al. 1998. Biophys. J. 74, A387). We found that these structures could function as membrane-spanning channels: a head-to-head dimers of single- homodimeric channels with two ester-containing subunits have very short lifetimes (

174A MONDAY PEPTIDE CHANNELS 1025-1030

1025-Pos 1026- Pos GRAMICIDIN D: CORRELATIONS IN SIDE CHAIN CONFORMATIONS AND ION THE POTENCY OF DIFFERENT SULFONATED ALUMINIUM PHTHALOCYANINES COMPLEXATION TO SENSITIZE GRAMICIDIN CHANNEL PHOTOINACTIVATION AND THEIR William L. Duaxi, B. M. Burkhart', Vladimir Pletnev2, 'Hauptman-Woodward Medical Research BINDING TO MEMBRANE LIPIDS Institute, Buffalo, NY,2Shemyakin Institute, Moscow, Russia. Michael Block', Tatyana I. Rokitskaya2, Yuri N. Antonenko2, Elena A. Kotovs2, Peter Pohl', Linear gramicidin is a pentadecapeptide of alternating D- and L-amino acids that can form 'Institut fur Medizinische Physik und Biophysik, Martin-Luther-Universitit, Halle, 06097 membrane channels specific for monovalent cations. Crystal structures of ion complexes of Germany, 2A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, gramicidin D provide information on variations in side-chain conformnations on the outer surface Moscow 119899, Russia of the channel, cation positions within the channel, anion association and solvent interactions. All Binding of a photosensitizer to membrane lipids was shown to be a prerequisite for photodynamic complexes studied thus far (CsCI, RbCl, TI NO3, KCI, KI and KSCN) crystallize with two inactivation of gramicidin channels in bilayer lipid membranes. In particular, the photodynamic gramicidin dimers in the asymmetric unit, antiparallel ,-ribbons that coil into right handed P- activity of aluminium phthalocyanines (AIPcS,4) was found to depend essentially on their affinity barrels. Despite pseudo symmetry perpendicular to the P-barrel the side-chain conformations to lipid bilayer. AlPcS2 induced the highest changes in the electrophoretic mobility while differ significantly, and characteristic differences between the two dimers in the asymmetric unit adsorbing to the surface of large unilamellar vesicles (LUVs). It was also most efficient in are apparent. At least five distinctly different sites of ion coordination have been characterized in mediating photoinactivation of gramicidin channels as revealed by measurements of electric the seven complexes studied thus far. Based upon current data there appears to be one cation per current across planar lipid bilayers. Increasing the degree of phthalocyanine sulfonation channel distributed over a subset of these sites, with partial occupancies ranging from 15 to 40%. progressively reduced its affinity to the lipid bilayer as well as its potency to sensitize gramicidin The "coordination" of the ions in the channel is achieved via unusual cation-s interactions. The channel photoinactivation. With raising photosensitizer concentration, the portion of side chain conformations of the valine, leucine and tryptophan residues differ significantly and photoinactivated gramicidin channels increased up to some optimum. At higher photosensitizer consistently between the monomers related by pseudosymmetry. This asymmetry may be concentrations it was found to decrease. This was attributed to quenching of reactive oxygen correlated with ion identity and positions in the channel. Correlations observed between the tp, +, species and self-quenching of the photosensitizer triplet state by its ground state. Fluoride anions were observed to inhibit both sensitized photoinactivation of gramicidin channels and AlPcS24 and XI values of the leucines and the mp and XI values of the valines may have predictive value in fitting low resolution diffraction data and interpreting NMR solution spectra. Some evidence of binding to LUVs. Therefore, photosensitizer binding to the lipid bilayer appears to be more correlation between side chain conformational patterns and the size and location of the ions within important than its reactive oxygen quantum yield. the channel is detected. Acknowledgements: Financial support was provided by the Deutsche Forschungsgemeinschaft (Po 533/4-1).

1027- Pos 1028 -Pos ABILITY OF AROMATIC RESIDUES TO FORM HYDROGEN BONDS IS IMPORTANT ROLE OF NON-LAMELLAR PHASES IN LIPID BILAYERS BY PORE-FORMING FOR THE ORGANIZATION OF MEMBRANE SPANNING STRUCTURES. ANTIMICROBIAL PEPTIDES AS CHARACTERIZED BY SOLID-STATE NMR S Shobanal, Denise V Greathouse', Roger E Koeppe I2, Olaf S. Andersen', 'Cornell University, METHODS Weill Medical College of Cornell University, 1300 York Ave, New York, NY 10021, 2University Kevan J Hallock, Katherine A Henzler, Jose Silva Santos, Dong-Kuk Lee, John Omnaas, Henry of Arkansas Mosberg, A. Ramamoorthy, University of Michigan, Chemistry, 930 North University, Ann Arbor, MI 49109 To examine whether the ability of the amphipathic aromatic residues to form hydrogen bonds with Antimicrobial peptides have received increasing attention over the past several years as bacterial the polar groups at the membrane/solution interface could be inmportant for the organization of resistance to traditional antibiotics has become more common. Despite the work that has been membrane-spanning structures, we investigated combinations of gramicidin analogues for the accomplished, our understanding of the peptide's pore-forming mechanism is still incomplete. In formation of conducting, double-stranded (DS) heterodimeric channels (Durkin et. al., 1992 this study, we used 31p solid-state NMR of uniaxially oriented samples to characterize the lipid Biophys. J. 62 145). Gramicidin T (gT), gramicidin N (gN), gramicidin M (gM) where the phases induced in synthetic lipid bilayers by several peptides at different lipid:peptide ratios. tryptophans in gramicidin A (gA) are replaced by tyrosine, naphthylalanine and phenylalanine, Several pore-forming antimicrobial peptides were investigated, including magainin, PGLa, and respectively, were used along with the enantiomer des-D-Val'-gA- to examine the formation of LL-37, by varying the lipid and cholesterol composition of the lipid bilayer. The results show that conducting DS hybrid channels. DS hybrid channels were observed with gN/des-o-Val'-gA- and the non-lamellar phase induced by the peptide is dependent not only on the peptide identity, but also on the lipid and cholesterol concentration. The selectivity of the peptides for gM/des-D-Vall-gA-. No DS hybrid channels were observed with gT/des-D-Val'-gA- or gA/des- bacterial D-Val'-gA-. The results show that the preference for hydrogen bond formation by the tryptophan membranes is discussed in light of these results. NH or the tyrosine OH with the polar groups at the membrane/solution interface hinders the formation of conducting DS heterodimeric channels. Because naphthylalanine of gN or phenylalanine of gM cannot hydrogen bond with the membrane/solution interface, there is less penalty for burying the aromatic residues, which means the formation of conducting DS hybrid channels becomes more favorable.

1029 - Pos 1030 - Pos AVIDIN DECELERATES CHANNEL KINETICS OF BIOTINYLATED GRAMICIDIN NMR STRUCTURAL STUDIES OF RECEPTOR LINKED GRAMICIDIN CHANNELS. AS SHOWN BY SENSITIZED PHOTOINACTIVATION. Aphrodite J Anastasiadisi, C J Morton2, K Raval3, G H Talbo', F Separovic', 'University of T I Rokitskaya', Yuri N Antonenkol, E A Kotova', K RavalI2, F Separovic3, 'Moscow State Melbourne, Parkville, Melbourne, Victoria 3052 Australia, 2Monash Unversity, 3CRC for University, 2CRC for Molecular Engineering, 3University of Melbourne, Melboume, Victoria Molecular Engineering 3010 Australia Gramicidin analogues are important components of the "ion channel switch" (ICS) biosensor (I) Membrane protein functioning can be modulated by specific interactions with extemal ligands. that consists of a receptor attached to gramicidin A (gA) embedded in a lipid membrane. The We have studied the effect of a water soluble protein bearing specific binding sites on the kinetics receptor is attached to gA using streptavidin-biotin binding with the biotin group covalently joined of ion channels formed by gramicidin A (gA) in planar bilayer lipid membranes (BLM). gA to the C-terminus of gA via an amino caproyl linker, using typically five (gA5XB) caproyl groups. channels are generally assumed to represent transmembrane dimers stabilized by hydrogen bonds This biotinylated gA was studied in deuterated sodium dodecyl-d25 sulphate micelles and the between monomers. The kinetics of gA channel formation and dissociation are expected to be structure deternined using NMR spectroscopy. The confonnation of the biotinylated gA was sensitive to the peptide mobility that can be altered by the binding of extemal proteins. To study found to be almost identical to that of native gA. Hence, it appears that the addition of functional channel kinetics, we used the method of sensitized photoinactivation, in which transients of gA- groups via a caproyl linker to the C-terminus of gA does not disrupt the peptide conformation and, mediated current through BLM are induced by a flash of visible light in the presence of a therefore, preserves an active ion channel structure. The effect of avidin on the structure of photosensitizer. It has been shown (Rokitskaya et al. Biochim. Biophys. Acta 1275:221, 1996) that biotinylated gA and the role of linker length in avidin binding is being investigated in lipid the time course of the flash-induced current decrease usually follows a single exponential decay micelles. Our results suggest that avidin causes crosslinking between micelles with gA5XB but with an exponential factor (T) corresponding to the gA single-channel lifetime. Addition of avidin not when containing gA2XB. Light scattering and mass spectrometry techniques are being used to does not affect T for gA but causes a dramatic increase in? for channels formed by a biotinylated investigate these avidin-micelle complexes with preliminary results characterising the binding of analogue of gA (gA5XB). This effect is reversed by addition of excess biotin to the bathing biotinylated gA to avidin in solution. Structure determination of biotinylated gA in dodecyl solution. The deceleration of the photoinactivation kinetics is explained by reduction of lateral and phosphocholine micelles, which more closely resemble the physiological membrane than SDS rotational mobility of gA monomers and dimers upon the formation of complexes between avidin micelles, is also planned. and gA5XB and, therefore, stabilizing the channel state and increasing the single channel lifetime. (1) Cornell, B.A., Braach-Maksvytis V.L.B., King L.G., Osman, P.D.J, Raguse, B, Wieczorek L, and Pace, R. (I1997) Nature 387, 580-583.

175A 1031-1036 PEPTIDE CHANNELS MONDAY

1031 - Pos 1032 - Pos CALORIMETRIC DESCRIPTION OF STRUCTURAL CHANGES OF COLICIN-B AND CORRELATION OF ACTIVITY OF THE COLICIN El IMMUNITY PROTEIN WITH ITS CHANNEL-FORMING DOMAIN, INDUCED BY pH AND LIPID INTERACTION. ITS NEAR-UV CIRCULAR DICHROISM SPECTRUM IN ORGANIC SOLVENT. Alicia Ortega', K. Wieshuber2, M. Linnemann2, S. Lambotte2, B. Bechinger?, 'Depto. S D Zakharov, R. Taylor, W. A. Cramer, Purdue University, Dept of Bological Sciences, West Bioquimica, Fac. Medicina, Universidad Nacional Autonoma de Mexico, Mexico, 2Max-Planck Lafayette, IN 47907-1392 Institute for Biochemistry, Germany The colicin El immunity protein (ImmEl) that protects the cell against self-intoxication is a 13 Colicin-B is a protein that undergoes a transition from soluble to a transmembrane kDa integral membrane protein with state, forming and ion channel to effect its bactericidal activity. A thermolytic fragment is 20 Ttp three transmembrane helices. After predicted to form 10 a-helices possessing pore-forming properties. The protein membrane .T * extraction from membranes with n- insertion is pH dependent, suggesting that protonation is necessary for the protein to achieve a Ph. ,r \butanol, ImmEl was purified to favorable conformation to interact with its target. The effect of pH on the thermodynamic o b mt o , homogeneity in CHCI3:MeOH:H20 properties of the Colicin-B and its channel-forming fragment (CFF) was determined in solution 1 , (4:4:1), in which a near-UV CD and in. lipids by DSC. A DSC profile of Colicin-B and the CFF shown an irreversible ciasicC1W / spectrum was obtained with strong denaturation. Two superimposed endothermic transitions gave the best fit at pH 7.0 with T. of negative signals from Tyr (13 Tyr, 8 f>e* /% peaks A=54.9 and B-58.7 °C (AT,=3.9 'C) suggesting the presence of two defined structural E _ Phe, and 0 Trp in wild type) at 270- and from domains. Lower pH resulted in less stability of both transitions but with better resolution. At pH U' WIidiyw -40 y V \ F285at 255-270nm nm.typicalA signalsseries of single-Phe 3.5 the T,(A)=29.8 and the (B)=40.7 'C (AT,=10.8 'C). From the total Al, at pH 7.0, 85%, pH Cys/single-Trp mutants, where no w. 290 30 Cys 4.5, 78% and pH 3.5, 55 % corresponded to the peak A. The CFF denatures as a single peak with 2M' no,, at position 16, 50, or 94 in helices 1, a Tm=64.8°C, this grater stability as well as its AHl, and AHl are independent of pH. In 2 or 3, respectively, was replaced by liposomes, the thermal properties of Colicin-B and the CFF changed dramatically, Colicin-B Ala or Trp, revealed a relationship between the CD spectrum and Imm activity. The loss of undergoes a single transition at 47.5'C with a decrement of 15% of All, and the CFF shows more activity of C16A/C94W, C16A/C50W, and C50W/C94A ImmEl, compared to active mutants or correlated with a than 50% lost of Implications of these results will be discussed. C16W/C94A, C16W/C50A, C5OA/C94W decreased amplitude of the CD Al,,. signal(s) from Tyr. The positive CD signals centered at 290 nm from Trp at positions 16, 50, or 94, and from Phe at 260-270 nm, were the same in active and inactive ImmEl (Fig. 1). It is inferred that one or more of the 13 Tyr are determinants of ImmEl activity. Identification of that Tyr would allow determination of functionally important intramembrane sidechain interactions in ImmEl (supported by NIH GM-18457).

1033 - Pos 1034- Pos UNFOLDING PATHWAY OF THE COLICIN El CHANNEL PROTEIN ON A PROBING CONFORMATIONAL FLUCTUATIONS OF A SINGLE POLYMER CHAIN MEMBRANE SURFACE IN THE LUMEN OF A TRANSMEMBRANE PORE. Magdalen Lindeberf', S D Zakharov2, W. A. Cramer', 'Purdue, Biological Sciences, West L. Movileau, S. Howorka, X. Lu, S. Cheley, 0. Braha, H. Bayley, Medical Biochenistry & Lafayette, IN 47907, Purdue University, Dept of Bological Sciences, West Lafayette, IN 47907- Genetics, Texas A&M Health Science Center, College Station, TX 77843-1114 1392 A single polyethylene glycol (PEG) molecule of 5000 Da was attached covalently within the The channel-forming domain of lumen of the heptameric transmembrane protein pore of a-hemolysin, near the center of the large bactericidal colicin El is of a Trp j Helix f Initial Rate Distance composed intermal cavity. The properties of the modified pore were investigated by single-channel current 356 2S C e () soluble helical bundle which, upon recording. The current taces revealed a variety of low amplitude events with lifetimes of tens of 396 m 13 14 membrane binding, forms an extended seconds. The mean difference in conductance between the most common substates was 10±1 pS, 413 IV 12 4 helical net in the membrane interfacial in 300 mM KCI and at a transmembrane voltage of +100 mV. These substates are ascribed to the 424 V 6 4 layer (PNAS 95:4282, 1998). 6 mutants reorganization of the PEG molecule within the channel lumen and associated movements of the 460 VI-VII 13 1 were constructed, each containing one 495 IX 39 4 polypeptide chain. A second class of events consists of negative current spikes (120±7 pS) with a tryptophan residue at diverse locations mean lifetime of 13.7±2.2 ms. The spikes may be caused by brief excursions of the PEG polymer and a single cysteine, C509. Using into one of the constricted regions of the channel. The approach developed here should be useful stopped-flow fluorimetry to quantify changes in fluorescence resonance energy transfer firom for probing the dynamics of several polymer species at the single-molecule level and for making tryptophan to C509-AEDANS, the magnitudes of Trp-AEDANS distance change and kinetics of biosensors with responsive polymers. unfolding on a msec time scale were determined upon membrane binding (see Table). Background FRET from the nine Tyr residues was measured using a Trp- mutant and the degree of uncertainty in the orientation factor was calculated from changes in polarization for AEDANS and each Trp. The helices at the bundle surface were shown to adopt a quasi-circular conformation in the membrane interfacial layer. Comparison of the rate constants revealed the following order of unfolding events: (A) movement of the hydrophobic core helices VIHI-IX, coincident with a small shift in the outer helices; (B) major unfolding of bundle surface helices in the sequence: (i) helix I; (ii) helices III, IV, VI, VII; and (iii) helix V; (C) condensation of the surface-bound helices. Unfolding did not occur below the membrane lipid phase transition temperature (supported by NIH GM-18457).

1035 - Pos 1036- Pos CHANNEL PROPERTIES, INHIBITORS AND MODE OF ACTION OF THE TOXIN CARTOGRAPHY OF THE TOPOGRAPHY OF DIPHTHERIA TOXIN T DOMAIN. VacA OF Hdecobacserpykori. Michael Gordon', L. Senzel', K. J. Oh', R. Blaustein3, R J. Collier2, A. Finkelstein', 'Albert Mario Zoratti, I. Szabo, Francesco Tombola, S. Brutsche, M. Moschioni, C. Montecucco, E. Einstein College of Medicine, Bronx, NY, 'Harvard Medical School, Boston, MA, 3Brandeis Papini, CNR Center for Biomembranes, University of Padova University, Waltham, MA VacA, the vacuolating cytotoxin secreted by Helicobacter pylori, is believed to be a major The T domain of diphtheria toxin is known to participate in the pH-dependent translocation of the causative factor in the development of gastroduodenal ulcers. Upon activation by exposure to low catalytic domain of the toxin across the endosomal membrane. During this translocation, the T pH, the toxin causes vacuolation of cultured cells and decreases the trans-epithelial resistance of domain forms a cation-selective channel of approximately 40 pS in 1M KCI with a pH gradient of artificial epithelial cell monolayers. We have recently discovered that it forms low-conductance, 5.3 to 7.2 across the bilayer. We have previously shown that the T domain is capable of anion-selective channels upon insertion into planar bilayers as well as in the plasmamembrane of translocating the entire catalytic domain across model, planar phospholipid bilayers in the absence HeLa cells. We have characterized these channels, identified a series of inhibitors, and gathered of other proteins. Thus the T domain alone contains all the molecular machinery required to infornation on the mechanism of action of some of the blockers. These compounds have then translocate the catalytic domain across membranes. Here we present a model, containing 3 been used, along with other experimental approaches, to establish that a strict correlation exists membrane-spanning segments, of the overall topography of the T domain as seen in the open between VacA channel activity and the cellular effects mentioned above. Our data suggest that channel state (the state of the channel when the catalytic domain has been translocated). This the toxin causes cell vacuolation by inserting into - and increasing the anion permeability of - the model was derived from experiments in which either a histidine tag (H6 tag) or biotin was membrane of late endosomal compartments after endocytosis. attached at residues that were mutated to cysteines. From the sign of the voltage gating induced (supp. by CNR Target Project Biotechnology, grant 97.01168.PF49) by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain were on the cis or trans side of the membrane, and by inference, which segments spanned the membrane.

176A MONDAY PEPTIDE CHANNELS 1037-1042

1037 - Pos 1038- Pos heLICAL CRYSTALS OF ANTHRAX TOXIN PROTECTIVE ANTIGEN PA63 FORMED EFFECT OF LIPID COMPOSITION AND IONIC STENGTH ON THE ASSOCIATION ON LIPID TUBULES. OF MASTOPARAN X WITH BICELLES: A1NEXTENSIVE NMR ANALYSIS Elizabeth M. Wilson-Kubalek', C. J. Miller2, R. A. Milligan', R. J. Collier, Alok KC Mltra', 'The Jennifer A. Whles', Robert Brasseur2, Regitze R. Vold', Elizabeth A. Komives', 'UC San Diego, Scripps Research Institute, 2Harvard Medical School 9500 Gilman Dr., La Jolla, CA 92093-0359, 2Facultd Universitaire des Sciences Agronomiques de Gembloux The active 63kD component (PA63) of anthrax protective antigen oligomerizes, generating ring- Mastoparan X (MPX) is a lytic peptide found in wasp venom. Many ionophoric peptides are like heptameric structures that form pores in the endosomal membrane which serve to deliver the thought to exert their activity through either a membrane-thinning mechanism or formation of an toxic lethal and/or edema factor to the cytosol. We have used the method of crystallization oligomeric pore. In our work, 2H NMR studies revealed that MPX associated with both neutral described in Kubalek et al. (1998) [Proc. Natl. Acad. Sci, USA. 95:8040-45] to form helically and acidic bicelles with its helical axis parallel to the bilayer surface, and that MPX was more ordered arrays of PA63 oligomer on preformed lipid tubules. The helical crystals diffract to -28A deeply buried in the acidic bicelles. Complementary high resolution NMR studies showed that resolution when embedded in negative stain or in vitrified buffer. In order to study the structure of although MPX appeared to maintain an amphipathic a-helical conformation in all systems studied, the PA63 and PA63-ligand complex in 3-dimensions we are applying helical-image processing the association of MPX with acidic bicelles in the absence of KCI appeared to be different. In algorithms to analyze minimal-dose images of the crystals. The investigation should elucidate the acidic bicelles, 1D peptide spectra showed significant line broadening and there was no observable mode of interaction in the protein-oligomer-ligand complex in situ and the mode of interaction of TOCSY transfer for residues 1-10. These results were found to be consistent with a model in which the oligomer with the bilayer. MPX rests parallel to the acidic bicelle normal and is buried deeply within the bicelle interior resulting in a faster T2 relaxation rate due to the slow tumbling of the bicelles. Overall, we have concluded that the biological activity of MPX can be explained using a membrane-thinning mechanism. Results from '5N relaxation studies will also be presented.

1039 - Pos 1040- Pos DETERMINATION OF THE CONFORMATION OF A MELITTIN-INHIBITOR EFFECT OF A PORE-FORMING PEPTIDE ISOLATED FROM THE VENOM OF COMPLEX. PARABUTHUS SCORPIONS ON GRANULOCYTES. Yuen-Han Lam', E Fakaris', C J Morton2, F Separovic', S R Wassall3, 'University of Melboume, Suzanne Maria Bosteels', Leentje Moerman', Jean Willems', Jan Tytgae, Jurg van der Wale, Parkville, Melboume, Victoria 3052 Australia, 2Monash University, 31UPUI Fons Verdonck', 'K.U.Leuven Campus Kortrijk, E. Sabbelaan 53, Kortrijk, B-8500 Belgium, Melittin is a 26-residue, amphipathic, a-helical polypeptide that forms ion channels in 2K.U.Leuven, 3University of Potchefstroom, South-Africa biological membranes and induces cell lysis. Its precise characterization in a lipid bilayer is An new pore-forming toxin has been isolated from the venom of Parabuthus schlechieri, a difficult but can be attempted by rotational resonance (RR), a solid state NMR method which scorpion living in South-Africa. This new lysin-rich, cystein-free peptide showed no sequence pennits measurement of homonuclear distances by reintroducing the dipolar coupling that is homology with other reported primary structures of pore-forming peptides. It shares, however, the removed by magic angle spinning techniques. cationic amphiphilic properties with other pore-formers like mastoparan and mellitin. The Several '3C labelled sites in melittin bound to the phospholipid compound caused degranulation of human granulocytes, similar to mastoparan. Upon addition of ditetradecylphosphatidylcholine were studied. The sites are near the N-terminus and, particularly, the peptide to granulocytes a transient increase in calcium in Fura-2 loaded cells was observed, in the region around Prol4 where a kink in the helix occurs to an extent heavily dependent on the combined with a decrease of the fluorescent signal. The action of the new substance on calcium molecular environment. The measurements were first made on peptide powders dried from water signalling was highly similar to that of fMLP, a chemoattractant and degranulating substance and and methanol, and gave results in agreement with X-ray crystallography and solution NMR to the pore-former mastoparan, both substances known to elicit Ca release by activation of timeric measurements, respectively. In contrast, molecular modelling with the interatomic distances G-proteins. Ca release was induced at concentrations below the tresbhold for installing pores determined in dry and hydrated lipid indicate a change in peptide conformation corresponding to a permeable towards calcium. In conclusion, a new pore-forming peptide from scorpion venom has sharper bend between terminal helices. been characterised that induced Ca release by a mastoparan-like mechanism. Melittin's cytolytic activity is inhibited by a 6-residue peptide; Ac-IVIFDC(Acm)-NH2. The interaction with melittin and the nature of the melittin-inhibitor complex were studied in solvent. Chemical shift changes in the amide region, primarily the trytophan indole amide, were observed and suggest that binding of the inhibitor may affect the melittin structure. The structure of melittin bound to inhibitor in a lipid environment will be studied using RR to measure intermolecular distances.

1041 - Pos 1042 - Pos DISTRIBUTION OF MOLECULAR ELECTROSTATIC POTENTIAL OF STRUCTURES AMYLOID BETA PROTEIN (1-42) (APP) FORMS TETRAMERIC PORES IN LIPID FORMED BY AMPHOTERICIN B IN MEMBRANE. BILAYERS: AN ATOMIC FORCE MICROSCOPC (AFM) STUDY Maciei Baglaskl', A. Tereszczyn', H. Resae, E. Borowski', 'Technical University of Gdansk, Hai Lin', Timothy Alig2, Joseph A Zsadzinski 2, Ratneshwar Lal', 'Neuroscience Research Inst, Narutowicza St 11/12, Gdansk, 80-952 Poland, 2Battelle-Pacific Northwest National Laboratory University of Califomia, Santa Barbara, CA 93106, 2Dept of Chemical Engineering Amphotericin B (AmB) is a polyene macrolide antifungal antibiotic used to treat systemic fimgal APP forms senile plaques in the brain and cerebrovasculature of the patients with Alzheimees infections. The antibiotic forms channels in cell membrane but the molecular mechanism of disease (AD). However, the role and mechanism of APP toxicity is poorly understood. Recent antifungal action of AmB is still not clear. It is accepted that the chemotherapeutic application of biochemical, electrophysiological, and cell biology studies have suggested that small globular AmB is based on higher affinity of AmB to ergosterol (fungal sterol) than to cholesterol (non-fibril) ApPs form cation-selective channels in lipid bilayer and cell membrane and induce (mammalian sterol). AmB molecules are amphipathic, can associate with each other, and can form cellular degeneration by calcium uptake. In this study, APiP(142) was reconstituted in unilamellar complexes with sterols. Present work is aimed at further studies of molecular properties of phospholipid vesicles, which were then fused to form a planar lipid bilayer. Imaging with an supramolecular structures formed by the antibiotic. Therefore, the molecular electrostatic potential AFM under aqueous buffer showed that APP(I-42) forns multimeric ion-channel like structure (MEP) was calculated for different complexes formed by AmB in model membrane and water: (i) with a central pore surrounded by predominantly 4 symmetrically distributed subunits. The outer different structural AmB dimers, (ii) AmB-cholesterol and AmB-ergosterol primary complexes diameter of these channels is approximately 9-12 nm and the top of the pore extrudes (iii) and AmB-sterol channels. The calculations were performed within continuum treatment of the approximately I em from the top of the lipid bilayer. This is the first direct structural environment (water and phospholipids). It was found that AmB-AmB head-to-tail dimer is more than demonstration of APP channel formation consistent with existing model, which may provide a hydrophilic head-to-head one. It appears that screening of the AmB negative potential by direct the mechanism for in AD. sterols can be responsible for stability of the antibiotic channel and its proper hydrophobic contact pathway underlining APP toxicity with phospholipids. It was also found that the distribution of MEP at the entrance of AmB- cholesterol and AmB-ergosterol channels is qualitatively different which can be due to the size of Supported by a grant from the Alzheimers Disease Program, Department of Health, Califomia. the channels.

177A 1043-1048 MEMBRANE DYNAMICS & BILAYER PROBES MONDAY

1043 - Pos 1044 - Pos NANOMETER MOVEMENT OF CELL MEMBRANES INDUCED BY VOLTAGE: AFM LIGHT SCATTERING EXPERIMENTS TO PROBE MEMBRANE FLUCTUATIONS OF DETECTION OF FLEXOELECTRIC EFFECTS. MONODISPERSE UNILAMELLAR LIPOSOMES. Ping C Zhang, Kenneth Snyder, Steven Besch, Fred Sachs, University at Buffalo Sophie Pautot', David A Weitz2, 'University of Pennsylvania, 209 south 33rd street, Philadelphia, HEK293 cells were voltage clamped and touched with the cantilever of a modified Quesant AFM. PA 19104, Harvard University Depolarization produced an outward movement and movements only occurred when the tip was in We have developed a novel emulsion technique which allows one to produce controlled contact with cells. At a fixed indentation force, the steady state displacement was linear with the monodisperse liposomes. By using an inverted emulsion we define the size distribution, the lipid test potential and was independent of the holding potential. The sensitivity was a 0.05nm/mV at composition and the medium encapsulated inside the liposomes. We characterize the size and the InN and saturated at an indentation force of - 3.5 nN. Some cells had sensitivity > 1 nm/tOO mV. polydispersity by static and dynamic scattering. In the case of 1pm diameter Egg Phosphatidyl The rise time was independent of potential and greater than the cantilever response time, perhaps Choline (EggPC) liposomes, we measure an autocorrelation function using dynamic light reflecting cytoskeletal viscosity. The displacements seemed isotonic and flexoelectric (an scattering whose initial decay exhibits a dependence varies with wave vector as qv, where 2

1045- Pos 1046- Pos CHARACTERIZING RAFTS IN LIVING CELLS BY LOCAL VISCOUS DRAG TIME EVOLUTION OF LIPID BILAYER DOMAINS ADSORBED ON MICA STUDIED MEASUREMENTS USING ATOMIC FORCE MICROSCOPY Amid Pralle, P. Keller, E.-L. Florin, K. Simons, J.K.H. Holrber, European Molecular Biology Adrian S Muresan, Shaohua Xu, Ka Yee C. Lee, The University of Chicago, 5735 S. Ellis Ave, Laboratory, Meyerhofstr. 1, 691 17 Heidelberg, Germany Chicago, IL 60637

Lipid rafts have been showvn to play an essential role in protein sorting and cellular signaling. The lateral mobility of bilayers and their interactions with the substrate are of crucial importance Their proposed function as reaction platform depends on their size and stability. These parameters in obtaining a uniform coverage of the surface when making planar supported lipid bilayers (PSB) can be determined from diffusion meauements as long as the immediate membrane environment via vesicle rupture. Using atomic force microscopy (AFM) we monitored the shape change for of the protein can be approximated as homogenous twvo-dimensional lipid bilayer. To fulfill this patches of egg-phosphatidylcholine (PC) bilayer adsorbed on mica as a function of time. The prerequisit in living cells, a laser trap was used to confine the motion of a bead bound to a raft shape of the egg-PC bilayer domains adsorbed on mica was found to change as a function of time. protein to a small area (d5100nm) and measured its local viscous drag by high reolution single An initial asymmetrical bilayer domain tends to become circular with time which suggests that particle tracking (Ax

1047 -Pos 1048- Pos ASYMMETRIC DISTRIBUTION OF ELECTRIC-FIELD INDUCED MEMBRANE A NEW MICROPROBE FOR MEASURING ELECTRICAL PROPERTIES OF PORES IN DOPC VESICLES. MEMBRANE SURFACES. Ephrein Tekle, Boon P Chock, National Institutes of Health, Bldg 3, Rm 203, 9000 Rockville Beate M. Kllsgen, Wolfgang Becker, Klaus D. Kramer, Free University Berlin, Arnimallee 14, pike, Bethesda, MD 20892 Berlin, D 14195 Germany The method of parelectric spectroscopy bases on the response of permanent dipoles to an The induction and distribution of field induced membrane pores in 1,2-Di-oleoyl-sn-glycero-3- oscillating electrical field E(X) in the radio frequency range of (1-1000)MHz. The dispersion phosphocholine (DOPC) vesicles was monitored using standard video microscope. The (£'(a))) and absorption (e"(w)) curves are related to the dipole mobility and to the dipole density pernmeabilization pattern upon field application shows asymmetric pore formation at the vesicle within the volume. In hydrated lipid membranes, the total response of the composite system of hemispheres facing the electrodes. Single membrane pores as large as -6 micrometer in diameter water and lipid can be separated into the respective contributions. The dipole moments of the are visible as the hemisphere facing the cathode. Further images revealed these pores are created membrane headgroups may serve as internal sensors to detect changes of molecular packing (for by partial loss of the phospholipid bilayer from the vesicle membrane. Measurement of the vesicle example during the main phase transifion). Adsorption processes onto the membrane surface (due size after the field showed up to -14% of the membrane surface can be lost due to pure formation. to molecules or even other membranes that bind to the surface) should become discernible As the anode facing hemisphere, large visible pores are seldom found within the time resolution of whenever they change the number of accessible dipoles or reduce their mobility. The response of the detection system. However, estimates based on the extent of lipid loss indicate that pores of the water dipoles reflects the hydration state of the surface. equivalent area as that formed at the cathode faring hemisphere must have formed and resealed. In The new probe consists of a metallic conductor (R-2pm) that can be coupled to standard coax contrast to what is already known in the electroporation of cell membranes, results of systems. It has a preliminary surface contact area of -10pm2. Spatial averaging of the signal permeabilization patterns from these vesicle systema shows that (i) Asymmetric pore formation origin will be restricted to smaller regions than with the inital probe, at the expense of temporal ensues despite the absence of a resting tranamembrane potential. (ii) The effective pore area resolution. This in principle allows the scanning of the surface for spatially separated induced as both hemispheres facing the electrodes is approximately equal, however, (iii) many (dipole) domains. Results obtained during first experiments with the new probe will be presented and small pores are formed at anode faring membrane and small number but larger pores at the discussed in the context of further and cathode faring hemisphere. biophysical applications developments.

178A MONDAY MEMBRANE DYNAMICS & BILAYER PROBES 1049-1054

1049 - Pos 1050- Pos CYCLIC SHRINKAGE OF LIPOSOMES AND DIRECT EXPULSION OF INNER Leishmanta asazoaensis ENTRY AND EXIT FROM MACROPHAGES VESICLES. Alexandra Corrales', Marcela Camacho', Maria Elisa Forerol, Isabel Llano2, Alison Agnew3, F. Nomura, K. Takiguchi, H. HotanL, Dept. of Mol. Biol., School of Sci., Nagoya Univ., Nagoya 'Centro Internacional de Fisica, Laboratorio de Bioflsica, Ed. manuel Ancizar. Ciudad 464-8602, Japan Universitaria, Bogota, A.A. 4948 Colombia, 2AG Zellulare Neurobiologie, Max-Planck Institut fur To study the dynamic behavior of biomembrane in aqueous solution, the transformation of biophysikalische Chemie, Gottingen, Germany, 3School of Biology, University of Leeds, Leeds, liposomes caused by surfactants were monitored by direct, real-time observation using dark-field Great Britain optical microscopy. Liposomes are closed vesicles of lipid bilayer membranes, and have been well studied as a simplified model of biological membrane. When Triton X-l00 was added to a Leishmania invade macrophages by phagocytosis, but whether the parasite contributes membrane liposome solution, spherical liposomes became smaller in size with maintaining their spherical to the so-formed parasitophorous vacuole (PV) or how the parasites subsequently exit shapes. During this process, the shrinking liposome exhibited two phases altematively, i.e., its macrophages after replication is not known. Using whole cell patch clamp recordings we membrane fluctuated vigorously or kept still. Some liposomes encapsulated a few smaller measured capacitance (Cm) to monitor changes in macrophage surface area during Leishmania liposomes inside. When Triton X-100 was added, the outer liposomes shrank smaller, and infection. Within 6 hours of infection (pi) there is a 31 % significant decrease in macrophage Cm surprisingly, the encapsulated liposomes escaped when the outer liposomes were vigorously suggesting that the PV membrane is contributed by the macrophage membrane. During the first 48 fluctuating in shape. Since the outer liposomes prepared by our method are unilamellar, indicating pi, before parasite replication starts, there is a recovery of cell surface area and as parasite that Triton X-100 transiently generates holes on a liposome and thereby excludes inner vesicles replication progresses there is a continued increase in macrophage Cm by as much as 56% at 96 directly. hours. At 120 pi we detected a decrease in macrophage Cm and parasite number to similar values measured at 24 pi. These, suggest that these cells contain recently invaded parasites and the culture has started a new infection cycle. Parasite exit may be the result of membrane lysis or PV membrane fusion to the macrophage membrane. Using leupeptin, a membrane fusion inhibitor, we observe no changes in parasite number at 120 pi. This result suggest that L. amazonensis enters by phagocytosis and exits the macrophages by a membrane fusion event.

1051 - Pos 1052 - Pos DIRECT VISUALIZATION OF LIPID DOMAINS IN FREE BILAYERS. A TWO TWO-PHOTON FLUORESCENCE MICROSCOPY STUDIES OF BIPOLAR PHOTON FLUORESCENCE MICROSCOPY STUDY TETRAETHER ARCHAEBACTERIAL LIPOSOMES. Luta A Bagatofll, Enrico Gratton, University of Illinois at Urbana-Champaign, Physics/LFD, Parkson Lee-Gau Chong', L. Bagatolli2, E. Gratton2, T. Khan', 'Temple University School of 1110 W. Green St., Urbana, IL 61801 Medicine, 3420 N. Broad St., Philadelphia, PA 19140, 2University of Illinois Combining the sectioning capability of the two-photon microscope and the partition and spectral properties of LAURDAN, PRODAN and N-Rhodamine-DPPE on the solid and fluid lipid phases, The effects of temperature (10-65 °C) and pH (7.2 and 2.7) on Laurdan fluorescence intensity temperature-induced changes on single giant unilamellar vesicles (GUVs) were studied. We images of giant unilamellar vesicles (GUVs) composed of the polar lipid fraction E (PLFE) from obtained images of GUVs composed of phospholipid binary mixtures labeled with the different thermoacidophilic archaebacteria S. acidocaldanius have been studied using two-photon fluorescent probes using polarized laser excitation. To characterize the lipid phase-state we used excitation. The excitation generalized polarization (GP) of Laurdan fluorescence in PLFE GUVs the LAURDAN Generalized Polarization function (GP). At temperatures corresponding to lipid always showed a broad distribution (> 0.3), and the GP values were low (< 0). When excited with domain coexistence we observed concurrent fluid and solid domains in GUVs composed of the y-direction polarized light, Laurdan fluorescence in the center cross-section of PLFE GUVs DPPE/DPPC, DLPC/DPPC, DLPC/DSPC, DLPC/DAPC and DMPC/DSPC. In all cases the exhibited a photoselection effect in favor of the x-direction. These results suggest that the domains grew and moved on the vesicle surface as we decreased the temperature. The size of the emission dipole of Laurdan is aligned parallel to the membrane surface and the Laurdan solid domains was up to several microns (-30 ism), displaying different shapes dependent on the chromophore resides in the polar head-group region of PLFE. The GP exhibited a small but lipid mixture. In the case of DPPE/DPPC (7:3 mol/mol) we observed mainly dendritic shape. abrupt change near 50 TC, which may correspond to a conformational change in the polar head- Instead, in the PC mixtures (1:1 mol/mol) the solid domains showed line, circular and dendritic groups of PLFE. Most interestingly, the two-photon Laurdan fluorescence images showed shape depending on the hydrophobic chain length difference of the lipid components. As judged remarkable snowflake-like domains in PLFE GUVs at pH 7.2 and low temperatures. These by the LAURDAN GP values, DPPE/DPPC showed pure gel and pure fluid domain coexistence domains, attributable to lipid lateral separation, were stable and laterally immobile. The while the PC mixtures showed an increasing influence of the fluid phase on the solid phase as the relationship of these results with membrane packing shall be discussed. [Supported by grants hydrophobic chain length difference between the components decrease. In all cases the solid from the NIH (RR03155) and NSF (MCB-9513669)] domains span the lipid bilayer showing monolayer coupling.

Supported by grants from the National Institutes of Health (RR03155).

1053 - Pos 1054 - Pos ASSOCIATION OF A FLUORESCENT AMPHIPHILE WITH LIPID BILAYER MECHANISM OF THE LIPID BILAYER MAIN PHASE TRANSITION STUDIED BY VESICLES IN THE SOLID-LIQUID-DISORDERED PHASE COEXISTENCE REGION. FLUORESCENCE SPECTROSCPOPY Antje Pokorny, Paulo F.F. Almeida, Winchil L.C. Vaz, University of Coimbra, Coimbra, 3000 Arimatti R Jutila, Paavo K.J. Kinnunen, University of Helsinki, P.O. Box 8 (Siltavuorenpenger Portugal 10A), Helsinki, FIN-00014 Finland We have previously reported on the kinetics and thermodynamics of association of a fluorescent amphiphile with cholesterol containing lipid vesicles exhibiting either solid-liquid-ordered (s-1 o) Lateral organization of lipid bilayers in the course of the main phase transition was studied by or -1 liquid-ordered-liquid-disordered (I d) phase coexistence (Pokomy et al., 1999, Biophys. J., in fluorescence spectroscopy. Large unilamellar vesicles of DMPC and DPPC were labeled with press). In the present study we extend the investigation to systems exhibiting s-l d phase trace amounts of pyrene and diphenylhexatriene (DPH) lipid analogs. separation, such as mixtures of dimyristoylphosphatidylcholine (DMPC) and distearoyl- phosphatidylcholine (DSPC). In analogy to the previous study, we first determined binding and Increase in the efficiency of excimer pyrene can kinetics of insertion and desorption of the amphiphile from single-phase vesicles as a function of formation of be due to enhanced lateral diffusion or enrichment of the probe to lateral domains. On temperature. The kinetic data obtained from mixed-phase vesicles was then analyzed using a the other hand, fluorescence anisotropy of DPH embedded in bilayer depends mainly on membrane fluidity. combination of the results from single-phase vesicles. We find that in regions of s-l d phase separation, the kinetics of association are well described by a combination of the rates obtained from the single-phase systems. This finding is in contrast to the observation that in regions of In brief, the data describes the transition as a three phase process, as follows. (i) Well below the solid-fluid coexistence of the type s-I , found in mixtures of cholesterol with temperature of maximum heat capacity (T.) decrease in anisotropy is paralleled with increase in phosphatidylcholines, the kinetics of association are significantly slower as compared to single- excimer formation as the bilayer becomes more fluid, lateral diffusion increases, and pyrene-lipids phase vesicles. We conclude that a solid-fluid coexistence of the s-I . type behaves very different enrich into the boundaries between gel and fluid domains. (ii) The middle phase (22.0-23.0 'C and from a s-l d phase separation with respect to the insertion of amphiphiles. Furthermore, we can 37.0-41.6 'C for DMPC and DPPC, respectively) includes T. and is characterized by decrease in conclude that the s-l d phase coexistence that exists in a DMPC/DSPC mixture differs from the both anisotropy and excimer formation indicating further fluidization and decomposition of lipid one that exists in pure DMPC vesicles around the phase transition temperature. domains, respectively. (iii) Further increase in temperature induces changes similar to those below [Supported in part by TMR ERBTMRXCT96 and PRAXIS/PCNA/C/BIO173/96 grants.] T.. These model systems were further studied by time resolved fluorescence spectroscopy. The results are in accordance with the model describing membrane at T,, as a 'pseudocrystalline' superlattice of fluid and gel state lipids.

179A 1055-1060 MEMBRANE DYNAMICS & BILAYER PROBES MONDAY

1055- Pos 1056- Pos POLYPHOSPHOINOSITIDE DISTRIBUTION AND DYNAMICS IN LIVING CELLS: PERTURBATION OF MEMBRANE ORGANIZATION BY THE ANTIMICROBIAL EVIDENCE FOR LATERAL SEGREGATION OF PHOSPHATIDYLINOSITOL 4,5- PEPTIDE PGLA BISPHOSPHATE Susanne Gangl', Erich Staudegger2, Karl Lohner2, Sylvie Blondelle3, Gottfried Grabner', Gottfried Edward Tall', Srinivas Pentyala2, Suzanne Scarlata', Mario Rebecchi', 'Departments of Kohler', 'University of Vienna, Althanstrasse 14, UZAII, Vienna, Austria A-1090, Physiology & Biophysics, Stony Brook, NY 11794, 2Department of Anesthesiology, SUNY at 2Osterreichische Akademie der Wissenwchaften, 'Torrey Pines Institute for Molecular Studies Stony Brook, Stony Brook, NY I1794 Lipid-peptide interactions are analyzed by fluorescence spectroscopic techniques for a 21-residue We investigated the distribution and dynamics of phosphatidylinositol 4,5-bisphosphate antimicrobial peptide from frog skin and model membranes. In particular specific docking of the (PtdIns(4,5)P2) in the plasma membrane of living fibroblasts. Relative levels of this lipid were peptide to the membrane surface and incorporation into the lipid bilayer is followed and the measured indirectly using the pleckstrin homology (PH) domain of PI-PLC-6,, a protein that binds resulting perturbations of the acyl chain order are analyzed. Ptdlns(4,5)P2 with high affinity and specificity. The PH domain, fused to Enhanced Green The location of this cationic peptide within model membranes composed of DMPC and DMPG Fluorescent Protein (EGFP), was viewed by epifluorescence microscopy. The PH domain-EGFP was investigated by fluorescence emission using a derivative of PGLa carrying a single tryptophan fusion protein was highly concentrated at the cell borders, indicating predominant plasma residue. Different fluorescence probes reporting specific physico-chemical properties of the lipid membrane localization, as previously described. Plasma membrane fluorescence, however, was bilayer are applied. The change of the shape and position of the emission spectrum of 12-(9- far from uniform and was most concentrated in highly dynamic ruffles that were identified anthroyloxy) stearic acid in the presence of PGLa indicate restricted mobility, decreased concurrently by phase contrast microscopy. The relative fluorescence intensities of these microviscosity and a lower degree of water accessibility within the acyl chain region, both in the structures were much greater than predicted for a uniformly distributed plasma membrane protein. gel and in the liquid-crystalline phase. A model for the interactions between PGLa and membranes Elimination of PtdIns(4,5)P2 binding by a point mutation in the fusion protein prevented its is proposed. association with the entire plasma membrane. These results imply a lateral segregation of PtdIns(4,5)P2 which may have important implications for its function.

1057 - Pos 1058- Pos REGULATION OF BACTERIAL MEMBRANE FLUIDITY PYRENE-LABELLED CHOLESTEROL: AN EFFECTIVE REPORTER OF THE Marta S Fernandez', R Mejia', M. C. G6mez-Eichelmann2, 'CINVESTAV-IPN, P.O. Box 14- BEHAVIOUR OF CHOLESTEROL IN MEMBRANES 740, Mexico, DF 07000 Mexico, 2Instituto Investigaciones Biomedicas, UNAM Jea,-Franqois Tocanme, Bernard Lagane, Laurence Cezanne, Andr6 Lopez, IPBS/CNRS, 205 Bacteria, as the rest of living organisms, react to temperature changes through several adaptive route de Narbonne, Toulouse, 31077 France mechanisms. Abrupt temperature upshifts, in particular, give rise to the cellular heat-shock Cholesterol is an essential component of eukaryotic cell plasma membranes known to modulate response, which consists in the overexpression of the heat-shock proteins (HSPs). Physical the activity of transmembrane proteins, through specific interactions or modifying the physical membrane changes associated to thermal stress can be studied with dipyrenylpropane, an properties of the lipid bilayer. This key role is now reinforced with the recent introduction of the intramolecular excimer-forming fluorescent probe. A fluidity parameter obtained with the probe is concept of cholesterol-rich lipid rafts in membranes. Although of prime interest, approaches quantitatively sensitive to membrane order alterations induced both, by temperature and by enabling an in situ investigation of the lateral distribution, dynamics and more generally of the variation in the unsaturated acyl chain proportion in lipids. Studies in Escherichia coli, show that behaviour of cholesterol in cell plasma membranes are still lacking. In this respect, we turned our following the sudden temperature upshift that elicits the heat-shock response. it is possible to attention toward I-pyrenemethyl-30-hydroxy-22,23 bisnor-Scholenate (PMC) as a reporter of the detect a marked increase in membrane fluidity. Thereafter, such "hyperfluidity" decreases behaviour of cholesterol in membranes. As a first result, absorbance measurements showed that in gradually towards the steady-state level at the high temperature. This regulation occurs while the egg-yolk phosphatidylcholine vesicles, PMC undergoes a concentration-dependent self- heat-shock response -ascertained by overexpression of HSPs-, is still turned-on. It has been association process, characterized by a self-associafion constant of 82.6 Ml. This 100-fold suggested that membrane fluidity decrease is too rapid to be accounted for by changes in the increase in K as compared to that measured for pyrene-labelled phospholipids (K < IM ')(1), bacterial lipids and that it could be due, instead, to the effect of some HSPs. We demonstrate, strongly suggests that self-association of PMC molecules results mainly from self-recognition of however, that the fluidity control is attributable to membrane remodeling through a profound the cholesterol moieties. Accordingly, addition of cholesterol to DPPC vesicles at fixed PMC/lipid change in fatty acid composition during the heat-shock response. Dipyrenylpropane also allows ratio (2 mol%) resulted in an increase in the excimer/monomer ratio indicating recruitment of the the evaluation of E. coli homeoviscous response, which, for a 30 to 45°C temperature upshift, probe by the liquid ordered cholesterol-rich phase in proportion as it formed. Having established shows an efficacy of 59%. that PMC is a good substitute of cholesterol, the phase behaviour of this probe in various lipids (including sphingolipids) and in natural membranes is currently under investigation. (1) S. Maz6res, J.F. Tocanne and A. Lopez. Eur Biophys J& Biophys Letters (1997) 26: 85

1059 -Pos 1060- Pos THE EFFECT OF ACYL CHAIN UNSATURATION ON FLIP-FLOP RATES AND A FREE VOLUME MODEL FOR BODIPY FLUORESCENCE IN MONOLAYERS MEMBRANE PERMEABILITY AS FOLLOWED BY DITIDIONITE QUENCHING.OF N- Mohammed Dahim, Nancy K Mizuno, Howard L. Brockman, Hormel Institute, University of NBD-PEs Minnesota, Austin, MN 55912 William Stillwell, Victiria Armstrong, Laura J. Jenski, IUPUI, 723 W. Michigan, Indianapolis, Lipids containing the 5,7-dimethyl BODIPY fluorophore exhibit concentration-dependent Indiana 46202 emission properties in membranes. We earlier reported that lipids containing the BODIPY group We are interested in determining the effect phospholipid-associated docosahexsenoic acid (DHA, mixed ideally with more naural lipids in monolayers, but exhibit exceptionally large partial 22:6 4.7.10.13.16.19) has on a variety of basic membrane properties. Here we employ dithionite molecular areas. Improvements in purification of the BODIPY-containing lipids and quenching of N-NBD-PE fluorescence to compare the effect of DHA to a variety of other common instrumentation for measuring fluorescence in monolayers have allowed the properties of the membrane acyl chains on flop-flop rates and membrane permeability. Two types of experiments molecules to be studied over a wider range of lipid compositions and lipid packing densities. This were conducted. In one the basic membrane was kept constant (egg PC) and membrane properties has revealed that the BODIPY-containing phosphatidylocholine is smaller than reported, being were followed by dithionite quenching of N-NBD-PEs having different acyl chains (N-NBD-16:0, comparable in size to l-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine. Momomer emission 16:0 PE; N-NBD-18:1, 18:1 PE; N-NBD-egg PE; N-NBD-18:0, 22:6 PE; and N-NBD-22:6, 22:6 decreases continuously with increasing fluorophore concentration, independent of lipid PE). In the second set of experiments the fluorescent probe was kept constant (N-NBD-egg PE) concentration. Excimer emission increases with both BODIPY and total lipid concentrations. and the bulk membrane phospholipids varied (14:0, 14:0 PC; 16:0, 16:0 PC; 18:0, 18:1 PC, 18:1, Analysis reveals a continuous relationship between excimer emission vs. BODIPY concentration 18:1 PC; 18;0, 18:2 PC; 18:2, 18:2 PC; 18:0, 22:6 PC and 22:6, 22:6 PC). Preliminary results and BODIPY free volume over a wide range of surface pressures and lipid compositions. Thus, indicate that for the egg PC membranes the flip-flop rates of the various N-NBD-PEs were the spectral properties of this BODIPY fluorophore may be useful to quantitatively assess changes dictated by NBD and were almost acyl chain independent. However, the flip-flop rates for N- in its lateral concentration in membranes, independent of lipid composition. Supported by NBD-egg PE were highly dependent on the degree of acyl chain unsaturation in the bulk HL17371, HL 49180 and the Hormel Foundation. membrane PCs. The lowest flip-flop rates were measured for bilayers made from the less saturated PCs while the highest rates were associated with the DHA-containing PCs. Acyl chain- dependence on membrane permeability to dithionite paralleled the flip-flop rates.

180A MONDAY MEMBRANE DYNAMICS & BILAYER PROBES 1061-1066

1061 - Pos 1062 - Pos TRANSMEMBRANE DIFFUSION OF SPIN-LABELLED AND FLUORESCENT INTERFACIAL MEMBRANE DYNAMICS AS STUDIED BY 'HNMR USING DIETHER PHOSPHOLIPIDS IN THE PLASMA MEMBRANE OF MAMMALIAN EXCHANGE LABELED HYDROXYL RESIDUES CELLS. Klaus Beyer, Volker Kurze, Bernhard Steinbauer, Thomas Huber, University of Munich, P. Fellman, P. Herv6, T. Pomorski, P. Miller, D. Geldwerth, A. Herrmann, Philippe F. Devaus, Schillerstr.44, Munich, 80336 Germany IBPC, 75005 Paris, F and Humboldt Universitat zu Berlin, 10115 Berlin, G. The polar interface ofmixed phospholipid membranes was studied by 2H NMR spectroscopy as a We have synthesized diacyl, alkylacyl- and di-alkyl glycerophospholipids with a short sn-2 chain fimction of the interfacial hydration. Covalent labeling of the membrane interface with deuterium bearing either a nitroxide group or a 7 nitro-2,1,3 benzoxadiazol4-yl (NBD). The translocation was achieved by chemical exchange of the labile hydroxyl hydrogens of phosphatidylglycerol or towards the inner leaflet could be determined by back exchange or in the case of NBD labelling cholesterol. A novel hydration procedure permitted continuous water uptake, membrane annealing with dithionite. In human erythrocytes, the spin-labelled phosphatidylcholine analogues moved and dehydration under NMR control. Planar orientation gave well resolved 2H NMR signals of very slowly, while the phosphatidylserine (PS) analogues accumulated on the inner leaflet with interlamellar D20 and of the phospholipid headgroup OD residues. This technique afforded an tl/2 at 37°C for the diacyl, alkyl-acyl and dialkyl of 2.3 min, 3.5 min and 41 min, respectively. accurate deennination of the number of water molecules associated with the membrane interface. ATP depletion or incubation with N-ethylmaleimide inhibited the rapid PS translocation. The Spontaneous evaporation of interlamellar water from the oriented multibilayers also allowed the dialkyl PS bearing a NBD group translocated very slowly in human erythrocytes. On the other observation of the conformational and dynamical response of the lipid headgroups and of the hand, in human fibroblasts the dialkyl NBD-PS and spin-labelled PS were both transported rapidly cholesterol molecule with respect to decreasing membrane hydration. The rate of deuteron by a process which requires ATP hydrolysis. These results prove that the ether bonds do not exchange among headgroup and water residues was determined as a function of interfacial prevent completely PS binding and translocation by the aminophospholipid-translocase in spite of hydration using the inversion transfer technique. It will be shown that the PG headgroup a probable hinderance due to the ether linkage on the sn-2 chain. Because of the high stability of undergoes changes of both conformation and average orientation with respect to the membrane the ether linkage the spin-labelled and the NBD di-ether analogues should be useful to investigate surface and that the off axis motion of the cholesterol molecule decreases upon removal of lipid traffic in cultured cells. interbilayer water. The rate in EYPG membranes decreases by roughly an order of magnitude whereas the exchange in the POPC/cholesterol mixture remains almost unaffected.

1063 -Pos 1064 - Pos SIMULATION STUDIES OF HIGH-FIELD EPR SPECTRA OF SPIN-LABELLED INVESTIGATION OF EXTRINSIC PROBE AND DRUG CANDIDATE COMPOUND LIPIDS IN MEMBRANES. LOCATIONS IN DMPC VESICLE PREPARATIONS BY NMR AND DIFFERENTIAL V. A. Livshits', Derek Marsh2, 'Centre of Photochemistry, Russian Academy of Sciences, SCANNING CALORIMETRY, 117427, Moscow, Russian Federation., 2Max-Planck Institut fir biophysikalische Chemie, D- J. C. Smith, D.J. Barrow, Jr., S. Chandrasekaran, L. Strekowski, Dept. Chemistry Georgia State 37070 Gottingen, Germany University, Atlanta, GA The high-field (i.e., 94 GHz) membrane EPR spectra of lipids spin-labelled in their fatty acid NOESY spectroscopy and DSC are being used to investigate the locations assumed by the chains have been simulated by using two limiting motional models. The aim was to identify the potential-sensitive molecular probe Oxonol V (OX-V) and the drug candidate compound LS8 (N- dynamic origin of the residual (gr-g,.) anisotropy observed in the non-axial EPR spectra of [2-(dimethylamino)ethyl]-2-(2'-naphthyl)quinoline-4-amine) in small unilamellar DMPC vesicles cholesterol-containing membranes. It is concluded that the residual spectral anisotropy arises from and in multilamellar DMPC preparations (MLV). When added as a solid at 15 mole percent to in-plane ordering of the lipid chains by cholesterol. The partial averaging of the (g&-g,y) preformed MLV, LS-8 causes, at 0.4 and 23 hours after addition, a nominal 0.2 and 3.5 °C l respective increase in the width of the to transition excess heat was best described restricted axial rotation with a in the of gel liquid crystalline phase capacity anisotropy by frequency region ?ri profile; the pretransition profile is broadened into the base line within 40 minutes. Tm is reduced 0.5-lx109 s-'. Simulations for slower axial rotation of unrestricted amplitude produced less by <0.5 'C by LS8. These perturbations are similar to those caused by OX-V (Biochim. Biophys. satisfactory fits. In phospholipid membranes not containing cholesterol, the non-axial anisotropy is Acta, 994, 164-176 (1988)), a type A perturbant, and indicates significant bilayer penetration by almost averaged, even in the gel phase. The unrestricted axial rotation in the gel phase completely LS-8 within the given time intervals. Quiet band noesy techniques (J. Mag. Res. 132, 34 is of comparable frequency to that of the limited axial rotation in the liquid-ordered phase of (1998)) have been used to demonstrate that a cross peak is observed between the DMPC terminal methyl membranes cholesterol. These results on in-plane ordering by cholesterol in the liquid- containing group protons and those of the OX-V phenyl group under conditions where the DMPC fatty acid ordered could be for current proposals regarding domain formation in cellular phase significant methylene protons are removed as a possible spin diffusion pathway. OX-V thus appears to membranes. penetrate the bilayer to the level of the terminal methyl groups. The interacting spin-pair model of Macura and Ernst (Mol. Phys. 41, 95-1 17(1980)) is being used to obtain the cross relaxation rates between the OX-V phenyl protons and those of DMPC moieties. Preliminary rate values (normalized to the number of DMPC moiety protons) of 10.2 sa' are observed to the DMPC fatty acid methylenes, the terminal methyl group, and to the choline N(CH3)3 protons.

1065 - Pos 1066 - Pos INTERACTION OF PHENOTHIAZINE DRUGS WITH SODIUM DODECYLSULFATE: LIPID REARRANGEMENTS ASSOCIATED WITH POLYETHYLENE GLYCOL (PEG)- MAGNETIC RESONANCE AND SPECTROPHOTOMETRIC DATA MEDIATED FUSION OF HIGHLY CURVED VESICLES Marcel Tabak, Wilker Caetano, Victor Evguenievich Yushmanov, Instituto de Quimica de Sao kervin o evans', B R Lentz2, 'university of north carolina chapel hill, 418 mejb, cb#7260, chapel Carlos/Universidade de Sao Paulo, Av.Dr.Carlos Botelho,1465, Sao Carlos, Sao Paulo 13560-970 hill, nc 27599-7260, 2University of North Carolina Brazil Location and dynamics of two phenothiazine drugs, chlorpromazine (CPZ) and trifluoroperazine We have used several fluorescent probes to follow changes in DOPC:DL15.3PC (85:15) small, NMR and spectrophotometric studies (TFP), in membrane systems have been addressed by ESR, unilamellar vesicles (SUVs) during fusion. SUV diameters were estimated as 19 32 nm based of CPZ and TFP in sodium dodecylsulfate (SDS) ionic micelles. Changes in absorption spectra of on quasi-elastic light scattering, and - 20 um based on outer/inner leaflet lipid ratio. Thb3/DPA the M) in the presence of increasing concentrations of SDS M) drugs (3.10-5 (3.l100-2.10.2 movement between vesicles followed a two-step path, with fast initial movement (416 x 103 s-' ) the formation of mixed aggregates at low concentrations with further solubilization in suggested followed by a much slower event (6.36 x NOf3 s- ). We have used the head-group labeled lipid SDS stearic acids, and 'H micelles. ESR spectra of two lipophilic spin probes, 5- and 16-doxyl NBD-PS (through dynamic quenching and chemical reduction) to follow the lipid movement from SDS M) protons, NMR (200 MHz) chemical shifts and spin-lattice T, relaxation times of (2.10.2 outer to inner leaflet and between vesicle inner leaflets during fusion. Rapid lipid rearrangement enabled the estimation Both CPZ and TFP (1-10 mM) of dynamics of hydrocarbon chains. occurs during the first phase of the process. This initial lipid rearrangement is followed by a and inside the hydrophobic restricted chain mobility both near the polar micelle-water interface slower rearrangement. TMA-DPH lifetime measurements suggest that outer leaflet packing core relaxation rates T-' increased 1.5-3 micelle between pH 1.0 and 9.0: correlation times TC and changes during the second, slow event such that water and probe are excluded from the upper times at 5mM of drug. In the polar headgroup region, the effect of TFP increased twice when TFP regions of the leaflet. Lipid mixing (following using the chain-labeled probe DPH-pPC) occurred displacement in mixed was double protonated (pH<4). Different patterns of SDS NMR peak mainly during the initial rapid event. Implications of these results and their relation to previous micelles with CPZ and TFP show that interaction changes with relatively small differences in drug results with less highly curved vesicles (diameter 45 nm) for the mechanism of the fusion CAPES, FAPESP and FINEP structure, being modulated by electrostatic forces. Support: CNPq, process will be presented. Supported by USPHS grant HL45916 to BRL. Brzilian agencies.

181A 1067-1072 MEMBRANE DYNAMICS & BILAYER PROBES MONDAY

1067- Pos 1068 - Pos EFFECT OF CURVATURE ON THE LOCAL STRUCTURE OF MODEL MEMBRANES PHASE SEPARATION AND SHAPE DEFORMATION ON MEMBRANES Anat Hatzor, T. G. D'Onofrio, C. D. Keating, M. J. Natan, P. S. Weiss, The Pennsylvania State Yi Jiang, Turab Lookman, Avadh Saxena, Los Alamos National Laboratory, MS B258, Los University Alamos, NM 87545 The physical properties and the biological functions of membranes are dependent on the local structure of the lipid bilayer. We describe methods to modulate the local structure of model Within a coupled-field Ginzburg-Landau model we study the dynamics of phase separation and membranes through control of membrane curvature. Our model membranes of choice are giant accompanying shape deformation on a two-phase elastic membrane. Using an exact periodic unilamellar vesicles prepared from multi-component lipid mixtures. We use lipid components domain wall solution we solve for the equilibrium shape and phase(concentration) field. We known to phase segregate into domains large enough to resolve with optical microscopy. observe preferential phase separation in regions of differing curvature on a variety of vesicles and Fluorescence microscopy techniques indicate properties of the local structure and enable us to estimate the degree of deformation of the membrane. We also investigate the effects of observe domain rearrangement as a function of curvature. Methods to manipulate the curvature deformation on the phase separation kinetics. include the polymerization of tubulin encapsulated within the vesicle and "stretching" the membrane in the grasp of an optical trap, either trapping the membrane directiy or using a bead as a "handle." The combination of the optical trap with a flowcell enables us to control the forces used to modulate the curvature. We measure the local composition dependence of these mechanical properties, seeking to correlate local structure with models of biological function.

1069- Pos 1070- Pos MULTISCALE PROPERTIES OF THE AQUEOUS BOUNDARY OF BIOLOGICAL COMPUTATIONAL STUDIES OF INSERTION OF IONS INTO THE INTERFACE OF MEMBRANES FROM SIMULATION LIPID BILAYER MEMBRANES Thomas Huber', Klaus Beyer2, 'Adolf-Butenandt-Institut der Universitat Milnchen, Hougjle Yang', See-wing Chiu2, Shankar Subramaniam3, H. L. Scott4, Eric Jakobsson5, 'Dept. of Schillerstrasse 44, Munich, 80336 Germany, 2University of Munich, Schillerstr.44, Munich, 80336 Biochemistry, UIUC, 2Beckman Institute, 3National Center for Supercomputing Applications, Germany 4Dept. of Physics, Oklahoma State Univ., 5Dept. of Molecular and Integrative Physiology Biomolecular hydration represents an important contribution to structure defining forces. Partitioning of ions into headgroup regions of membranes is difficult to predict by continuum Interfacial water molecules are part of the structure of the aqueous boundary of biomolecules such theory owing to the dynamical nature of the system and the complex nature of the dielectric as biological membranes. In contrast to the macromolecular surface residues this water is boundary at the interface. In order to gauge the variation in dielectric at the membrane-electrolyte characterised by fast positional exchange. The individuality of such water molecules is lost over interface, we have utilized a long-time molecular dynamics trajectory with enhanced sampling of short times. the configuration space spanned by the ion in conjunction with molecular mechanics. To get A different aspect of the hydrating water will be presented here. The standard picture of the appropriate sampling, a curve of energy vs. depth for each ionic species requires approximately microscopic behaviour of water molecules as obtained by MD simulation will be extended to a 1000 CPU-hours on a fast workstation or a processor of a supercomputer such as the SGI Origin. mesoscopic scale. Short length- and time-scale dynamics is smeared out to get a better picture of We compare the results obtained with this atomistic method with the Born energy approach to the hydration layer in total. assess the dielectric constant variation at the interface. The application of Boltzmann statistics to The validity of the model will be shown by comparison with NMR data. the results can predict the distribution of ions within the interfacial region, and hence the contribution of different electrolytes to the dipole potential (Clarke and Lupfert, 1999, Biophys J). A larger goal is to be able in general to predict and characterize in detail ionic and molecular Huber, T. (1999) Untersuchungen zur mikroskopischen Struktur einer biologischen Membran, Dissertation Universitlt Milnchen penetration into surfaces. To date computations have been done for sodium, potassium, and chloride. Support provided by fnancial grants from NIH and NSF and computer time from the National Center for Supercomputing Applications.

1071 - Pos 1072 - Pos MODEL STUDY OF TRACER DIFFUSION IN LIPID-STEROL BILAYERS. SOFTLY SUPPORTED PLANAR BILAYERS FOR RECONSTITUTION OF INTEGRAL Ilpo Vattulainen', Hong Zhu2, James M Polson2, Martin J Zuckermann2, 'Dep. of Chemistry, MEMBRANE PROTEINS: A UNIQUE PEG-LIPID AS A CUSHION AND TETHER. Technical University of Denmark, Building 207, Lyngby, 2800 Denmark, 2Dep. of Physics Michael L Wagner', Lukas K Tamm2, 'University of Virginia, Health Sciences Center, Box McGill University, Canada 10011, Charlottesville, VA 22906-001 1, 2Department of Mol. Physiol. & Biol. Phys. University of Virginia Health Sciences Center We study tracer diffusion of lipid and sterol-like molecules using off-lattice Monte Carlo There is increasing interest in solid-supported planar lipid-protein bilayers as models of biological simulations in conjuction with a minimal model for lipid bilayers which yields a phase diagram membranes and as a physiological environment for immobilizing membrane receptors. The consistent with experimental observations for several lipid/cholesterol systems. We study the structure and behavior of membrane proteins and lipids are studied in supported bilayers by a tracer diffusion process in various phases both above and below the main transition temperature variety of surface-sensitive techniques and functionally reconstituted membrane receptors are over a wide range of cholesterol concentrations and characterize the role played by the energetics, promising for the construction of receptor-based biosensors and molecular libraries for mass- the lipid chain conformational ordering and the area per molecule in the tracer diffusion process. screening of ligand-receptor interactions. A common problem of bilayer membranes that are The effect of phase boundaries on the tracer diffusion coefficients is examined in considerable directly supported on hydrophilic substrates are non-physiological interactions of integral detail. In spite of minimal nature of the model, the calculated results are in full qualitative membrane proteins with the substrate resulting in an immobilization of these proteins. To alleviate agreement with experiments for tracer diffusion in a DMPC/cholesterol system. Further studies in these problems we are developing polymer supported planar bilayers (PSPB). We have supported a model of lipid/lanosterol bilayers, for which experimental diffusion results are not available, lipid bilayers on a unique polyethylene glycol (PEG)-lipid tether, on quartz surfaces, and predict that the behavior of tracer diffusion in this case is quite similar. investigated bilayer morphology, and lateral diffusion behavior with total intemal reflection fluorescence microscopy (TlRFM) and fluorescence recovery after photobleaching (FRAP). We have produced both cushioned and tethered PSPB with the same polymer system. We report conditions for producing stable, uniformly fluorescent PSPBs where the lipids have high lateral diffusion coefficients (0.8-1.0 l10-' cm2/sec) and mobile fractions (75-80%). Characterization of the PEG-lipid as well as initial results on the functional incorporation of membrane proteins will be reported.

182A MONDAY MEMBRANE DYNAMICS & BILAYER PROBES 1073-1078

1073 -Pos 1074 - Pos OF ELECTROSTATICS AND SURFACE CHEMISTRY ON BIOANALYTES WETTING OF PHOSPHOLIPID MEMBRANES ON SOLID SURFACES INTERACTIONS WITH SURFACE SUPPORTED LIPID BILAYERS. Julia Nissen, Joachim Radler, Lehrstuhl fur Biophysik, Physik Departsnent, TUMJames-Franck- N. Asanov, Philip B. Oldham, Mississippi State University, Box 9573, Mississippi StraBe, 85748 Garching State, MS 39762 There is a substantial body of evidence which suggests that phospholipid liposomes self-assemble Solid supported membranes are of great interest as model systems for cell surfaces, as well as into planar bilayers on hydrophilic solid supports. Since a thin layer of water (15-20 Angstrom) is natural host matrices for membrane spanning proteins in practical applications. We present a study rapped between the support and the polar headgroups of the lower leaflet of the bilayer, the of the wetting behaviour of phospholipid membranes on a broad variety of surfaces. Using supported planar membrane mimics many of the properties of natural cell membranes. In fluorescence microscopy the spreading of a single bilayer-flow field and its one-dimensional present work, a new spectroelectrochemical system is described which allows simultaneous interface is analysed by image processing. investigation and control of interactions between planar bilayers and solution phase analytes such The kinetics of the bilayer yields information about the interaction between membrane and as proteins, DNA oligonucleoutides, surfactants, and peptides. substrate, which are crucial in the formation of defect-free, self-healing bilayers. Beside bare SiO2 The kinetics of adsorption and spreading of unilamellar liposomes on the surface of hydrophilic substrates surfaces covered with biopolymers were used. quartz oxide (ITO) films were studied as a function of electrochemical polarization. To investigate the influence of defects on the motion of the membrane, artifical pinning centers ITO and quartz surfaces were chemically modified to obtain hydrophobic, hydrophilic, cationic or were created by standard photolithography. The evolution of a distorted membrane rim is well surfaces. The results indicate that the interactions between planar bilayers and different described by computer simulated stochastical growth process (KPZ-equation)p. analytes are driven by van der Waals, electrostatic, hydrophobic and steric forces. Electrochemical polarization substrate allows modifying kinetics of interactions between solution phase analytes receptor molecules immobilized in the bilayer.

1075 Pos 1076- Pos EFFECT OF ETHYLENE OXIDE AND PROPYLENE OXIDE BLOCK COPOLYMERS WHY DOESN'T SKIN LEAK (MUCH)? WATER FLUX ACROSS MODEL STRATUM PERMEABILITY OF BILAYER LIPID MEMBRANES TO SMALL SOLUTES CORNEUM MEMBRANES. Yu, Yuri N. Antonenko, O.O. Pomaz, N.S. Melik-Nubarov; A.N. Belozersky Institute, Nell Kitson', Myma Monck2, Sherry Wang2, Jenifer L. Thewale, 'University of British Columbia, University, Moscow. Dermatology, 835 West 10th Ave, Vancouver, BC V5Z 4E18Canada, 2Simon Fraser University ethylene oxide and propylene oxide block copolymers (pluronics) on the transport of a We are investigating the relationship between phase behaviour and water flux across model number of weak acids and bases across model lipid membranes has been studied. It has been membranes based on those that provide the permeability barrier in mammalian epidermis that pluronics facilitate the permeation of small solutes across lipid bilayers, the effect ("stratum comeum intercellular membranes"). Models were prepared by layering multilamellar being dependent nature of the compound. 2.5- 4-fold increase of the rate of permeation has vesicles of varying lipid composition onto Millipore filters and drying. They were then mounted observed for 2-n-undecylmalonic acid, decane-1,10-dioic acid, hydroxyoctanoic acid, and in diffusion chambers over an aqueous salt solution, and water flux to dry nitrogen was measured chloroquine, the increase of the permeation for caproic acid, acetic acid and decylamine does not as a function of temperature by Fourier transform infrared spectroscopy, using the H20 absorbance exceed 1.2 times. No acceleration is detected for ammnonium chloride, caprylic acid, and 1,10- band at 3744 cm '. Model stratum corneum (SC) membranes were prepared from an equimolar diaminodecane. The effect of pluronics correlates satisfactory with the compound molecular mixture of ceramide, cholesterol (CHOL) and palmitic acid (PA). The control membrane was volume and its ability to form hydrogen bonds. Pluronic does not induce the increase in the equimolar sphingomyelin:CHOL:PA. The absolute value of the water flux across the control electrogenic conductivity of the planar bilayers under our experimental conditions, thus the effect membrane was 8 times that of the model SC membrane, at room temperature. From about 40°C accounted for by the formation of channels or water pores. It has been proposed that up, water flux across the two membranes was identical. We are presently correlating these pluronics accelerates the processes of the solute permeation via an increase of the innermembrane observations with the results of solid state deuterium NMR studies on analogous membranes. It rather than by the enhancement of the rate of the solute adsorption/desorption. appears that filter-deposited model SC membrane phase behaviour is consistent with previous studies of fully hydrated multilamellar dispersions. Thus we have shown that the solid phase formed by the model SC membrane restricts water flux compared with the liquid crystalline phase formed by the control membrane.

Pos 1078- Pos PHOSPHOLIPASE C INDUCED FORMATION OF DIACYLGLYCERIDES IN INTERACTION OF WATER SOLUBLE IRON PORPHYRINS WITH IONIC ZWITTERIONIC GIANT UNILAMELLAR VESICLES. MICELLES. Juha Holopainen', Paavo K.J. Kinnunen2, 'Institute of Biomedicine, Siltavuorenpenger ItOA, Shirley M.C. Gandini, Victor E. Yushmanov, Marcel Tabak, Instituto de Quimica de Sao Helsinki, FIN-00014 Finland, 2University of Helsinki Carlos/Universidade de Sao Paulo Porphyrins (Pph) and their analogues are useful in biochemical and medical applications. In this have extended our previous studies on enzymatic hydrolysis of zwitterionic work we have studied the interaction of two water soluble, anionic and cationic, iron porphyrins, phospholipids presented in the form of giant unilamellar vesicle (GUVs, diameter 10-100 tm) meso-tetrakis (4-sulfonato-phenyl)porphyrin (FeTPPS4) and meso-tetrakis (4-N- (Holopainen et al., Biophys. J., in press). The enzymatic hydrolysis of phosphatidylcholine by methylpyridiniyumyl)porphyrin (FeTMPyP) with micellar membrane model systems. We use phospholipase C (PLC) presented in GUVswas studied. Three different lipid compositions were VIS-UV optical absorption, resonance light scattering (RLS) and NMR spectroscopies. In the used, follows (1) l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), (2) 1,2- presence of cationic CTAC a complex equilibrium (three species) is observed for FeTPPS4, similar dimyristoyl-sn-glycero-3-phosphocholine (DMPC), or (3) a mixture of SOPC and N-palmitoyl- to what occurs for the free base (S.C.M.Gandini et al., Langmuir v.15, p.6233-6243, 1999), sphingomyelin (C16:0-SM) (molar ratio 75:25). We show that PLC causes a stepwise shrinkage of involving aggregation of Pph which is dependent upon [CTAC] / [PPh]. Aggregates dissociated phase SOPC GUVs continuing beyond vesicle size is smaller than the optical resolution of into monomers atpH < 4. At alkaline pH and excess concentration of surfactant aggregates bound the microscope. Likewise, incorporation of 25 mol%of SM into SOPC GUVsdoes not affect the to the micelles are observed. The formation of mixed aggregates of porphyrin-surfactantat low the enzymatic reaction. Importantly, giant multilamellar vesicles are not degraded by concentrations is supported by RLS data. For zwitterionic HPS the aggregates are not observed of PLC. similar to what occurs for the free base. Binding contants of FeTPPS4 of the order of 104M-'are activity of PLC is strongly dependent on the phase state of the bilayer. In brief, whereas fluid also similar to those observed for the free base. Our data suggest that both anionic and cationic GUVs are readily hydrolyzed by PLC, gel state (T=13-16°C) DMPC GUVs remain iron porphyrins are able to bind to the hydrophobic corein membrane mimetic systems, and that unaffected even after prolonged incubation time (2 brs) in the presence of PLC. the equilibrium monomer-aggregates remains effective upon the binding. Support: CNPq, FAPESP, FINEP Brazilian agencies.

183A 1079-1084 CHOLESTEROL-LIPID & PROTEIN-LIPID INTERACTIONS MONDAY

1079- Pos 1080- Pos 7-KETOCHOLESTEROL, BUT NOT 25-HYDROXYCHOLESTEROL, FORMS EXCHANGE OF MONOOLEOYLPHOPHATIDYLCHOLINE WITH MEMBRANES IMMISCIBLE DOMAINS IN MODEL MEMBRANES AND MURINE AORTIC SMOOTH CONTAINING CHOLESTEROL MUSCLE CELLS. Natalia G. Stoicheva, Doncho V. Zhelev, David Needham, Duke University, Science Dr., Durham, R. Preston Mason, Yong-Jian Geng, Jane Ellen PhilUps, Membrane Biophysics Lab, NC 27708 Cardiovascular & Pulmonary Research Inst,, Department of Medicine, MCP Hahnemann We have studied the exchange of monooleoylphosphatidylcholine (MOPC) with vesicle University, Allegheny Campus, 320 East North Avenue, Pittsburgh, PA 15212-4772 membranes containing cholesterol from 0 to 60 mol %. Micropipet manipulation was used to 7-ketocholesterol (7-keto) is one of the major oxygenated products present in oxidized low-density expose a single lipid vesicle to a flow of MOPC solution (0. 025 lM to 500 FM). The presence of lipoproteins and in atherosclerotic plaque where it is believed to play a role in arterial pathology. only 3 mol % cholesterol in the vesicle membrane was sufficient to inhibit the partitioning of 25-hydroxycholesterol (25-OHC) is also found in plaque and is thought to be obtained through the MOPC micelles ( at a concentration of 100 FM ) into the membrane, while it did not affect the diet. When applied to cells, 7-keto contributes to the formation of extracellular crystals whereas exchange of MOPC monomers and microaggregates both into and out of the membrane. The 25-OHC does not. Direct membrane effects of these oxysterols independent of receptor binding experimental data show that cholesterol prvents micelle-membrane fusion and a possible may mediate this biological activity. In this study, small-angle x-ray diffraction approaches were mechanism is discussed. Cholesterol thereby stabilizes the membrane in solutions of up to I mM used to examine the membrane interactions of oxysterols in well-defined lipid vesicles and murine MOPC that would otherwise dissolve it. aortic smooth muscle cell membranes. Diffraction analyses demonstrated that 7-ketocholesterol was located in the bilayer hydrocarbon core/phospholipid head group, whereas 25-OHC appeared in the phospholipid head group region only. Cells grown in the presence of the 7-keto developed extracellular crystals concomitant with the formation of discrete membrane 7-keto domains with a unit cell periodicity of 35.4 A. Membrane domains were formed within 4 hours and persisted up to 72 hours, after which cells showed signs of declining viability. 25-OHC did not form domains in the membrane or extracellular crystals. We conclude that 7-keto is able self-associate into immiscible membrane domains, providing direct evidence that these domains may contribute to the formation of atherosclerotic sterol crystals.

1081 - Pos 1082- Pos SOLID STATE NMR AND X-RAY DIFFRACTION STUDIES OF CHOLESTEROL SPONTANEOUS CURVATURE, BENDING MODULUS AND SHAPE OF DMPC GIANT MOLECULAR ORGANIZATION IN POLYUNSATURATED PHOSPHOLIPID UNILAMELLAR VESICLES CONTAINING CHOLESTEROL AND LANOSTEROL MEMBRANES. Mads C Sabra', Jonas R Henriksen', Hans-GUnther D6bereiner9, 'Technical University of Michael R Brzustowicz', Mustapha Zerouga2, Vadim Cherezov3, Martin Caffrey3, William Denmark, Building 207, Lyngby, 2800 Denmark, 2Max-Planck-Institut fir Kolloid und Stillwell2, Stephen R. Wassall', 'Department of Physics, Indiana University Purdue University Grenzflachenforschung Indianapolis, Indianapolis, IN 46202, 2Department of Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN 46202, 3Departnent of Chemistry, The Ohio State We study the effects of the sterols cholesterol and lanosterol on the morphology and the elastic University, Columbus, OH 43210 properties of DMPC bilayer membranes. Phase contrast video microscopy is used to record Our current solid state NMR studies of [2,2,3,4,4,6-2H6]cholesterol mixed with 22:6-22:6PC (1,2- thermal shape fluctuations of single giant unilamellar vesicles (H.-G. Dobereiner, Fluctuating didocosahexaenoylphosphatidylcholine) membranes in 1:1 mol ratio reveal unique molecular Vesicle Shapes, in Giant Vesicles, pp 150-167, Eds. P.L. Luisi and P. Walde, John Wiley & Sons, organization. Membrane intercalation of only 10 mol% and a tilt angle ao = 22° are identified for 2000) as a function of sterol content and temperature. Analysis of the fluctuation spectrum within cholesterol in the dipolyunsaturated membrane by 2H NMR, which compares with typically >50 the framework of the Area-Difference-Elasticity Model (L. Miao et al. (1994) Phys. Rev. E 49, mol% incorporation and a tilt angle oo = 16° in PCs (phosphatidylcholines) containing a saturated 5389) allows us to deduce the spontaneous curvature (H.-G. DWbereiner et al. (1999) Eur. acyl chain. Low- and wide-angle X-ray diffraction studies indicate that membrane insoluble Biophys. J. 28, 174) as well as the bending modulus of the vesicle membrane. Variations in these cholesterol exists outside the membrane in the form of monohydrate crystals. Also in contrast to elastic parameters with temperature and sterol content are discussed in relation to the phase PCs with a saturated chain, a transition from lamellar (L.,) phase to isotropic (I) and inverted diagram of DMPC-cholesterol (J.H. Ipsen et al. (1987) Biochim. Biophys. Acta 905, 162). hexagonal (Hi,) phases was observed via 31P NMR when the temperature of the dipolyunsaturated membrane was raised from 20°C to 50°C. Intercalation of [2,2,3,4,4,6-2H6]cholesterol was detected in all three phases. Experiments underway will determine the role of cholesterol in the formation of non-bilayer phases and the structure of the isotropic phase. Cholesterol's low solubility in 22:6-22:6PC membranes and the formation of non-bilayer phases at physiological temperatures denote a major distinction in cholesterol / phospholipid interactions. These results correlate with the compositional distribution observed in the disk membranes of the retinal rod outer segment where cholesterol and large amounts of 22:6-22:PC are present.

1083 - Pos 1084- Pos COMBINED MOLECULAR DYNAMICS AND MONTE CARLO SIMULATION OF COMBINED MOLECULAR DYNAMICS AND MONTE CARLO SIMULATION OF DPPC- CHOLESTEROL LIPID BILAYERS AT 33% AND 50% CHOLESTEROL POPC AND DPPC - CHOLESTEROL LIPID BILAYERS AT LOW CHOLESTEROL CONCENTRATIONS CONCENTRATIONS Eric H. L. See-Wing ChIul, Jakobsson2, Scott3, 'University of Illinois at Urbana-Champaign, 405 Hugh L Scott', Eric Jakobsson2, See-Wing Chit3, 'Okl homa State University, Stillwater, OK N. mathews, Urbana, IL 61801, 2Ctr for Biophys. & Comp. Biology, NCSA, Beckman Inst., U. 74078, 2Ctr for Biophys. & Comp. Biology, NCSA, Beckman Inst., U. Illinois, Mol. & Integrative Illinois, Mol. & Integrative Physiology, 524 Burrill Hall, 407 S. Goodwin Ave,, Urbana, Illinois Physiology, 524 Burrill Hall, 407 S. Goodwin Ave,, Urbana, Illinois 61801, 3University of Illinois 61801, 3Oklahoma State Univ. at Urbana-Champaign, 405 N. mathews, Urbana, IL 61801 We have applied a hybrid equilibration and sampling procedure for the atomic level simulation of We have applied a hybrid equilibration and sampling procedure for the atomic level simulation of a hydrated lipid bilayer to bilayers consisting of dipalmitoyl phosphatidylcholine (DPPC) and a hydrated lipid bilayer to bilayers consisting of dipalmitoyl and palmitoyl-oleyl cholesterol plus > 32 waters per molecule. The procedure is applied to bilayers of 64 molecules of phosphatidylcholine (DPPC,POPC, resp.), cholesterol, and water. The procedure is applied to DPPC and 64 molecules of cholesterol, and bilayers of 48 molecules of DPPC and 24 molecules bilayers consisting of 94 DPPC molecules with 6 cholesterol molecules, and 120 POPC molecules of cholesterol. After equilibration three continuous fixed pressure and temperature molecular with 8 cholesterol molecules. All bilayers contain in excess of 32 waters per membrane molecule. dynamics runs, separated by 10,000 Configurational Bias Monte Carlo steps, were carried out for After equilibration three continuous, fixed pressure and temperature molecular dynamics runs, each system. Properties of the systems were calculated and averaged over the three separate runs. separated by 10,000 Configurational Bias Monte Carlo steps, were carried out for each system. Analysis of the hydration environment in the head groups reveals bonding between water Properties of the systems were calculated and averaged over the three separate runs. We find that molecules and and in cases cholesterol, some bridging between neighboring cholesterols by water. cholesterol whole-molecule tilting is greater than that found in bilayers containing higher The evolution of the radial distribution function between cholesterol oxygen clearly shows the cholesterol concentrations, and this tilting has a pronounced effect on order parameters of aggregation of cholesterol molecules over the course of the simulation in the 33% system. neighboring lipid chains. In the POPC simulation we find that cholesterol affects the conformation Diffusion is anisotropic in the both systems, favoring motion perpendicular to the membrane. of the saturated chain mode strongly than the unsaturated chain. We have calculated pair of Tilting cholesterol molecules is greater in the 33% system than in the 50% system. Based on the correlation functions between water oxygens and lipids, between water oxygens and and simulations we suggest possible mechanisms for attractive interactions between cholesterol cholesterol, between PC and cholesterol, and between cholesterol and cholesterol. Based on the molecules that promote aggregation in bilayers. simulations we examine possible mechanisms for direct and water-mediated interactions between cholesterol molecules and between cholesterol and lipid molecules in bilayers.

184A MONDAY CHOLESTEROL-LIPID & PROTEIN-LIPID INTERACTIONS 1085-1090

1085 - Pos 1086- Pos MOLECULAR DYNAMICS STUDIES OF THE EFFECTS OF DMSO, CHOLESTEROL STRUCTURES OF DPPC AND DMPC MEMBRANES WITH CHOLESTEROL, AND CHOLESTEROL SULFATE ON THE ELECTROSTATIC PROPERTIES OF LIPID ERGOSTEROL AND LANOSTEROL AT LOW AND HIGH CHOLESTEROL BILAYER. CONCENTRATIONS AS REVEALED BY MOLECULAR DYNAMICS SIMULATION. Alexander M Smondyrev, Max L Berkowitz, University of North Carolina at Chapel Hill, Max L Berkowitx', Alexander M Smondyrev2, 'UNC-Chapel Hill, CB#3290, Chapel Hill, NC CB#3290, Chapel Hill, NC 27599-3290 27599-3290, 2University of North Carolina at Chapel Hill, CB#3290, Chapel Hill, NC 27599-3290

We performed molecular dynamics simulations of DPPC, DPPC:Cholesterol and Using the molecular dynamics simulation technique we studied the properties of several DPPC:Cholesterol Sulfate bilayers in water and simulation of the DPPC bilayer in DMSO. The membranes composed of a mixture of phospholipid and sterol molecules. Our simulations were value of the dipole potential for the DPPC membrane in water (+600 mV) is in a good agreement performed at constant temperature and pressure on a nanosecond time scale. In one series of with experiments. In membranes containing mixtures of DPPC and cholesterol the dipole potential simulations we studied the properties of DPPC:Cholesterol and DPPC:Cholesterol Sulfate is positive and is larger (+900 mV at 50 mol% sterol). Our simulations of the DPPC bilayer in membranes at 1:1 ratios. Compared to membranes with cholesterol, inclusion of cholesterol DMSO predict that the dipole potential becomes negative (-350 mV). Similar behavior was sulfate in lipid bilayers increases the area per DPPC:sterol heterodimer, decreases the order in observed in the DPPC:Cholesterol Sulfate bilayer, where the dipole potential also became negative DPPC acyl chains and increases the number of hydrogen bonds between water and membrane. (-200 mV). We calculated separate contributions to the dipole potential due to lipid headgroup, The dipole potential for the DPPC:Cholesterol Sulfate membrane changes its sign compared to two ester groups, sterols, DMSO and water. We also discussed the changes in membrane pure DPPC and DPPC:Cholesterol membranes that may contribute to the reduction of the structures and their possible effects on the solvation forces. We propose that the dipole potential hydration forces. We also investigated changes occurring in mixed lipid:cholesterol membranes at would become zero for the DPPC membrane surrounded by the mixture of DMSO and water at low sterol content (8:1 ratio) when DPPC is replaced with DMPC and cholesterol is substituted some DMSO:water ratio. We predict that similar effect can be observed in lipid membranes which with ergosterol or lanosterol. The modulations of membrane structures were characterized by contain both cholesterol and cholesterol sulfate at some ratio. Our results may at least partially order parameters in lipid hydrocarbon tails, membrane areas, electron density profiles and dipole explain certain well known effects of DMSO and cholesterol sulfate such as, for example, potentials. We also determined whether changes in the structure of sterol molecules had any effect promotion of the cell adhesion by cholesterol sulfate and inducing of the cell fusion by DMSO. on their mobility. Our results are in a good agreement with the data available from experiments.

1087 - Pos 1088 - Pos EFFECTS OF MODIFICATIONS TO STEROLS ON THE INTERFACIAL BEHAVIOR RELAXATION FROM LASER-INDUCED NONEQUILIBRIUM IN LIPID- OF SPHINGOMYELIN AND PHOSPHATIDYLCHOLINE. CHOLESTEROL MIXTURES Xln-MIn Li, Maureen M. Momsen, Howard L. Brockman, Rhoderick E. Brown, University of Frank Richter', Gert Rapp2, Leonard Finegold3, 'European Molecular Biology Laboratory, TU Minnesota Munich, 2MpI for Colloid and Interface Science, c/o HASYLAB, DESY, Notkester. 85, Hamburg, The structural features of cholesterol that affect its interfacial elastic packing interactions with 22603 Germany, 3Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104 16:0 sphingomyelin (SM) and dipalmitoylphosphatidylcholine (DPPC) have been investigated by In a cholesterol range typical for mammalian membranes, cholesterol and the host lipid are not using a Langmuir-type film balance. Structural changes included alteration of the polar perfectly miscible. Following a detailed investigation of this fluid-fluid miscibility under near- 'headgroup' region (e.g., 6-ketocholesterol or cholesterol sulfate) as well as deletion of the equilibrium conditions (Biophys. J. 76(1999) A63), we here continue to far from equilibrium nonpolar 'tail' (e.g. 5-androsten-33-ol). Analysis of the elastic area compressibility moduli (Cs-') conditions. Using time-resolved x-ray diffiaction, we study how the near- equilibrium miscibility as a function of sterol mole fraction permitted insights into the in-plane packing interactions at gap affects the relaxation from controlled far from equilibrium conditions, induced by a membrane-like surface pressures (is . 30 mN/m). The following hierarchy in the reduction of millisecond laser temperature jump. T-jump experiments were carried out on a suspension of in-plane elasticity was observed (cholesterol > cholesterol sulfate > 6-ketocholesterol _ multilamellar DMPC vesicles containing 15 mol% cholesterol, which concentration lies in the 5-androsten-33-ol). Sterol mole fractions producing liquid-disordered phase (X < 0.25) led to heart of the gap. A temperature amplitude of 20 C within 2 ms was achieved, and jumps were pressure-dependent 'softening' of the DPPC condensed phase (higher in-plane elasticities; lower performed in steps of 10 C from 10 C upwards. We apply two independent strategies to analyse Cs.') relative to that of pure DPPC condensed phase. Yet, sterol mole fractions producing liquid- the mixture's structural response to the T-jump: (1) We evaluate the flatness of the relaxation ordered phase (X 2 0.3) exhibited an abrupt decrease in-plane elasticity (higher Cs-') that curve, which can be linked to metastable states, and (2) we perform a thermal gradient analysis, surpassed the pure condensed phases of DPPC or 16:0 SM. When mixed with equivalent sterol and compare the results with near-equilibrium data. Both methods disclose that the relaxation mole fractions, the in-plane elasticity of 16:0 SM was lower than that of DPPC. The results depends on the initial state, and not on the state which the system acquires immediately after the provide insights into the structural features of sterols and sphingolipids likely to be important in temperature jump. Moreover, the relaxation for base temperatures in the gap (i.e. below 50 C) 'raft' or microdomain formation. [Support: NIGMS RO1-45928 & Hormel Foundation] follows passive cooling. For starting temperatures above the gap, the relaxation path appears to display metastable intermediates. Acknowledgements: NATO CRG970255, BMBF 03SATU25.

1089- Pos 1090 - Pos EFFECTS CAUSED BY A CATIONIC PEPTIDE ON THE POLARITY PROFILES OF CHOLESTEROL SULFATE AND CALCIUM AFFECT STRATUM CORNEUM LIPID FROZEN AND FLUID PHASE ANIONIC LIPID BILAYERS: A COMPARATIVE STUDY ORGANIZATION OVER A WIDE TEMPERATURE RANGE. WITH SODIUM IONS AND CHOLESTEROL Joke A. Bouwstra', G.S. Gooris', F.E.R. Dubbelaar', M. Ponec', 'LACDR, Leiden University, Roberto M Femandez, M. Teresa Lamy-Freund, Universidade de Sao Paulo PO Box 9502, Leiden, 2300 RA Netherlands, 'Department of Dermatology, LUMC The cationic tridecapeptide a-MSH is known to interact with anionic vesicles of DMPG, partially The skin barrier is mainly located in the intercellular lipid matrix in the upper layer of the skin, the penetrating the lipid membrane. Phospholipid spin label derivatives show that the peptide stratum comeum (SC). The main SC lipids are ceramides (CER), free fatty acids (FFA) and increases the bilayer packing at all depths, for the liquid-crystal phase membrane. However, in the cholesterol (CHOL) that are organized into two lamellar phases with periodicities of 13 and 6 nm. gel phase, the peptide increases the bilayer packing at the 5" C-atom position and at the membrane A comparable organization has been observed by SAXD in equimolar CHOL:CER:FFA mixtures core, but decreases it close to the 12 C-atom. Those different results can be correlated with at room temperature. Upon raising the temperature from 20° to 95°C, a new 4.3 nm phase changes in the bilayer polarity, measured by the isotropic hyperfine splitting at high temperatures, appeared. This phase is absent in intact human SC. Since pH, cholesterol sulfate (CS04) and Ca2+ and by the hyperfine splitting z component at -150 °C, associated to the lipids in the gel phase: levels vary with SC depth, their effect on the lipid phase behavior was investigated. CS04 and above the phase transition a-MSH increases the polarity at all the bilayer positions, while it increased pH promote the formation of the 13 nm lamellar phase. Furthermore, CS04 reduces the clearly only increases the frozen membrane polarity at the 12"' carbon position. Those results, amount of crystalline CHOL and causes a shift in the formation of the 4.3 nm phase to higher apart from showing that a bilayer polarity profile can be different when measured with frozen and temperatures. This phase also becomes weaker. These effects of CS04 can be counteracted by fluid-phase membranes, they suggest that the increase in packing yielded by a-MSH could be Ca2+. When extrapolating these results to the in vivo situation, it seems that CS04 is required to related to an increase in water concentration at a particular micro-environment. That discussion is dissolve CHOL in the lamellar phases and to stabilize SC lipid organization. Therefore, a drop in extended to the effects of monovalent ions and cholesterol on the membrane packing and polarity. CS04 content in superficial SC layers is expected to destabilize the lipid lamellar phases and may (Supported by FAPESP and CNPq) facilitate the desquanmation process.

185A 1091-1096 CHOLESTEROL-LIPID & PROTEIN-LIPID INTERACTIONS MONDAY

1091 - Pos 1092 - Pos X-RAY DIFFRACTION ANALYSIS OF HUMAN OCULAR LENS FIBER CELL PROBING MEMBRANE ACTIVE PEPTIDES WITH NEUTRON DIFFRACTION. PLASMA MEMBRANES Thad A. Harroun, Malcolm J. M. Darkes, Jeremy P. Bradshaw, University of Edinburgh, R. F. Jacob, R.P. Mason, Membrane Biophysics Laboratory, MCP Hahnemann University School Summerhall, Edinburgh, EH9 lQH United Kingdom of Medicine, Pittsburgh, PA 15212 Neutron diffraction is a common technique for measuring the structure of the lipid bilayer normal The human ocular lens is a transparent tissue consisting almost entirely of a large number of rigid, to the membrane plane, and has been used effectively to study peptide-lipid interactions. However, elongated, prismatic cells known asfiber cells. The plasma membranes of these cells are unique an expanded variant of the technique can be used to measure the entire three-dimensional structure in that they contain: (1) the highest cholesterol/phospholipid (C/P) mole ratios known for of the membrane, providing even more direct information on the orientation and organisation of mammalian cell membranes (with C/P mole ratios ranging from 1 to 4), and (2) a phospholipid membrane active peptides within the bilayer. Our goal is to apply this technique to current composition of gicater than 50% sphingomyelin (SPM) and SPM derivatives. In our laboratory, problems in membrane biophysics. We will show preliminary data on the structure and orientation small angle x-ray diffraction (XRD) approaches are being used to explore the structural of two types of membrane active peptides; fusion peptides and voltage gated ion channels in characteristics of isolated human fiber cell plasma membranes, especially the contributions of model bilayers. Fusion peptides occur in many viruses and promote the fusion of two membranes cholesterol and SPM. The results of XRD analyses demonstrate the presence of immiscible for the purpose of viral DNA delivery; a mechanism for which there are many models but little cholesterol domains in the oriented membrane samples with a constant unit cell periodicity of 34.0 direct evidence to distinguish between them. One effective fusion peptide comes from the simian A, observed over a broad range of tempratures (5-401C) and relative humidities (31-97%). By immunodeficiency virus, and is of the class of viral proteins that inserts obliquely into bilayers. contrast, the unit cell periodicity of the sterol-poor phospholipid bilayer region is highly Voltage gated ion channel formation is a characteristic of several naturally occurring anti- modulated by these experimental conditions. These findings provide direct evidence for distinct microbial peptides as well as the isolated trans-membrane segsnents of larger proteins, the variety cholesterol domains in the plasma membranes of fiber cells under physiologic-like conditions. of which is only recently being explored. Even the synthetic amphipathic peptides, such as We hypothesize that SPM at such high levels contributes specifically to human lens membrane (LSLLLSL)3, exhibit such characteristics. The data we will present involves these two peptides. structure by stabilizing cholesterol domains and attenuating structural damage due to oxidative insult, a consequence of lifelong exposure to visible and ultraviolet light. To test these hypotheses, model membrane systems composed of SPM and cholesterol were examined using XRD and spectroscopy approaches. The formation of separate and stable cholesterol domains in SPM-enriched bilayers was observed at C/P mole ratios that reproduce levels found in fiber cell membranes. In addition, SPM demonstrated potent (>70%) and dose-dependent inhibition of lipid peroxidation in liposomes enriched with polyunsaturated fatty acids at physiologic concentrations (0.5-1.5:1). These data indicate that cholesterol is organized into discrete domains in human fiber cell plasma membranes and that SPM modulates domain formation while increasing resistance to oxidative damage.

1093 - Pos 1094 - Pos INTERACTION OF ANTIBACTERIAL PEPTIDE MAGAININ 2 WITH THE ABILITY OF PHOSPHATIDYLETHANOL TO PROMOTE MEMBRANE MULTILAMELLAR LIPID BILAYER SYSTEMS PROBED BY NEUTRON- AND X-RAY CURVATURE IS STRONGLY ENHANCED BY CALCIUM SCATTERING Yangehlh Lee, Nathan Janes, Emanuel Rubin, Theodore F. Taraschi, Thomas Jefferson Tim Salditt', Christian Muenster', Burkhard Bechinger2, 'Universitaet Muenchen, Geschwister- University, 1020 Locust Street, Philadelphia, PA 19107 Scholl-Platz 1, Muenchen, 80539 Germany, 2MPI Biochemie Martinsried In tissue exposed to ethanol, phospholipase D (PLD) catalyzes a transphosphatidylation reaction of phosphatidylcholine (PtdCho) to form an unusual anionic phospholipid, Phosphatidylethanol The membrane-active 23-residue helical peptide magainin 2 belongs to a class of antibacterial (PtdEt) (1,2). We have found that PtdEt is unique among phospholipids for its extraordinary (and fungicidal) peptides, which are part of the vertebrate immune system and interact directly ability to promote membrane curvature and destabilize the bilayer structure. To investigate with the lipid bilayer of the bacterial cell wall. Despite recent advances, many aspects of lipid- whether cations effect membrane curvature, the ability of a series homologous cations peptide interaction are to date not well understood. We have studied the bilayer structure, the (Ca2+,Mg5',Zn2+,Ba2+ and Al3+) to promote the formation of nonbilayer structures in a elasticity and fluctuation behavior, as well as the lipid chain packing in model systems of PtdEt/PtdEtn (phosphatidylethanolamine) matrix have been investigated. We have chosen to multilamellar membranes (POPC, POPS, DMPC, DLPC, DOPC). focus on the cation/lipid molar ratio between 0 and 1. Among these selected ions, calcium promotes curvature structure far in excess of the other cations with the order of For this have been on silicon and substrates and Ca2+>>AI3+>Mg2e>Ba2+. After thermodynamic analysis, the imparted membrane curvature stress purpose, highly aligned samples prepared glass ranged from -32 J per mole for Ba2+ to +90 J per mole for The enhanced membrane were investigated by x-ray and neutron scattering in a temperature and humidity controlled Ca2+. Ca2+ chamber. While the was destabilizing properties of PtdEt in tissues may play a role in alcohol-induced tissue injury. scattering length density profile determined by reflectivity measurements, US PHS diffuse and neutron was used to characterize the fluctuations and Supported by Grants AA07215, AA07463, AA07186. 1. Lee et al. Biophys.J. 65, 1429- x-ray scattering bilayer elasticity 1432 (1993). 2. Lee et al. Biochem. 35, 3677-3684 (1996). properties. The lateral membrane structure on molecular length scales was probed by synchrotron based grazing incidence x-ray diffraction. The results obtained for different lipids are discussed as a function of peptide concentration.

1095- Pos 1096- Pos MOBILE NEUTRAL LIPIDS AND OXIDATIVE BURST IN NEUTROPHILS. LOW CONCENTRATION OF A NON-ANESTHETIC MODIFY PROPERTIES OF A Ana Camps, Eva Burger, Lucia H.M. Bruneri, Universidade de Sao Paulo, Av. Prof. Lineu MODEL BILAYER Prestes, 580, Sao Paulo, SP 05508-900 Daphea Scharf, L A Davies, QF Zhong, J S Johansson, M L Klein, University of Pennsylvania, Neutrophils exposed to pro-inflammatory stimuli give rise to a prominent signal in 'H-NMR 3400 Spruce Street, Philadelphia, PA 19104-4283 spectra probably due to mobile neutral lipids (triglycerides) in the plasma membrane. The A number of chemically similar compounds to known potent inhaled anesthetics (IAs) have been correlation between the appearing of these NMR signals and the organization of the NADPH observed to be non-anesthetics / non-immobilizers (Eger et al., 1994). To explore this observation oxidase system in the neutrophil membrane is here studied. NMR spectra (Bruker DPX 300) and we study the non-anesthetic hexafluoroethane (C2F), an analogue of the IA halothane NADPH oxidase system activity (luminol-enhanced chemiluminescence triggered by soluble or (C2F3BrCIH), in a DPPC bilayer. Our goal is to demonstrate differences in the lipid response to a opsonized-stimulus) of neutrophils recovered from air pouch infected mice were determined after non-anesthetic when compared with halothane [Biophys. J. 75:2123 (1998)]. A concentration of one or 15 days of infection. While the NMR spectra from cells recovered after one day of infection C2F6 comparable to the halothane in clinical usage (6.5 mole%) was incorporated in a model of were poor, a high-resolved spectrum showing prominent signals in the triglyceride region was hydrated liquid-crystal phase (La) of a DPPC bilayer (64 lipids and 28 water/lipid head-group) observed after 15 days. The intensity of luminol-enhanced chemiluninescence of neutrophils [Biophys. J. 69:2558 (1995)]. A I ns constant pressure and temperature MD trajectory was exposed to soluble stimulus was not modified by the time of infection indicating that generated. Compared with DPPC La phase, the area per lipid head-group dropped from 61.8Ag to modifications in the plasma membrane, observable by NMR, do not alter the response. However, a 56.5A2, the lamellar d-spacing increased from 67.3A to 73.OA, while the volume remained four times higher chemiluminescence for cells recovered after 15 days of infection were observed for unchanged, showing a tendency to drop. Calculated 15-30% drop in the gauche/trans rotamer neutrophils exposed to the opsonized stimulus. These results indicate the simultaneity of the defect ratios along the lipid acyl chains resulted in significant changes in the segment order appearing of NMR-visible lipids with the expression of opsonin-receptors in the phagocyte parameters. Increased hydration (- 25%) was found for the choline groups, which exhibit larger membrane. distances from the bilayer center. These represent dramatic changes, which are experimentally Supported by FAPESP, CNPq and CAPES measurable. Halothane lacked such measurable effects [Biophys. J. 75:2123 (1998)].

Supported by National Institutes of Health under grant P-01 GM 558776.

186A MONDAY CHOLESTEROL-LIPID & PROTEIN-LIPID INTERACTIONS 1097-1098

1097 -'Pos 1098- Pos EFFECT OF SURFACE AREA CHANGE ON CORTICAL TENSION IN PASSIVE GALACTOSYLCERAMIDE MEMBRANE DISTRIBUTION - A GLYCOSPHINGOLIPID NEUTROPHILS. TRANSFER PROTEIN STUDY Brian Albarran, Hie Ping Ting-Beall, Robert M Hochmuth, Duke University, Dept of Peter Mattjus', Julian G Molotkovsky2, Helen M Pike', Rhoderick E Brown', 'University of Biomedical Engineering, Durham, NC 27708 Minnesota, 801 16th Avenue NE, Austin, Minnesota 55912, 2Russian Academy of Sciences, The simplest parameterized model of the "resting" neutrophil views the cell as being composed of Moscow, Russia. a cortex surrounding a liquid-like highly viscous cytoplasm. In this "liquid drop" model (Evans and Yeung, Biophys. J., 56: 151, 1989), a resting cell behaves under a steady suction pressure as a We have used the glycolipid transfer protein (GLTP) to study the partitioning of Newtonian liquid drop with a constant cortical tension. Using micropipette techniques, we galactosylceramide in bilayer membranes. We previously showed that the GLTP mediated transfer measure this tension to be -23.5 ± 3.5 pN/pm, in good agreement with our earlier work (Needham process is significantly retarded by the presence of negatively charged lipids (5-10 mol%) in the and Hochmuth, Biophys. J., 61:1664, 1992). In order to gain better insight into the origin of the donor vesicle surface. In this study we examined donor vesicles composed of both sphingomyelin cortical tension, we measure the effect of surface area change on cortical tension using newly (SPM, natural and synthetic) and phosphatidylcholine (PC) at different molar ratios. We found developed micropipette methods. In one set of experiments, neutrophils were suspended in either that the galactosylceramide transferred from PC donor vesicles was faster than from SPM vesicles. hypertonic, isotonic (control), or hypotonic solutions and their cortical tension measured. Area Gradually increasing the content of PC of mixed PC/SPM vesicles did not however increase the dilations of -20% resulted in increased cortical tension, represented in an apparent elastic area transfer rate in a linear fashion, but rather showed a considerably slower transfer rate for vesicles expansion modulus of -45 pN/tLm. In an alternate set of experiments, neutrophils were entirely containing 5-50 mol% PC, compared to vesicles containing only SPM or PC. It is possible that aspirated into one pipette and their cortical tension measured with a second pipette. Area dilations galactosylceramide partitioning among the two phospholipids changes at different mixing ratios of -20% resulted in increased cortical tension and an expansion modulus of -60 pN/tm. and diminishes accessibility to GLTP, or that the transbilayer distribution of galactosylceramide Therefore, the cortical tension in passive neutrophils increases when their surface area is expanded between the inner and outer leaflets in the mixed vesicles is affected. To determine the possible as originally observed by Needham and Hochmuth. Supported by NIH HL23728. lateral or transbilayer distribution of galactosylceramide in SPM/PC mixed bilayer vesicles, fluorescence investigations are being initiated. This will provide insights into the glycosphingolipid membrane distribution/localization, which have recently received much attention in the literature from a wide variety of fields. Support: NIGMS 45928, Academy of Finland.

MONDAY INTRACELLULAR CALCIUM SIGNALING II 1099-1102

1099 - Pos 1100 - Pos CHARACTERIZATION OF RYANODINE RECEPTOR AND SERCA ISOFORMS FROM CHARACTERIZATION OF RYANODINE RECEPTORS FROM ZEBRAFISH THE THERMOGENIC HEATER ORGAN OF MARLIN AND SWORDFISH. SKELETAL MUSCLE. Jeffery M Morrissette', Jens Franck2, Barbara A Block', 'Stanford University, Hopkins Marine Tobias Janowitz, Peter Koulen, Barbara E. Ehrlich, Yale University, New Haven, CT 06520 Station, Oceanview Blvd., Pacific Grove, CA 93950, 2Occidental College Calcium (Ca) signaling is mediated by two sources for Ca ions. Ca can enter through Ca channels A thermogenic organ, derived from extraocular muscles, is found beneath the brain in located in the plasma membrane and can also be released from intracellular stores through Ca- open ocean fish. The heater organ warms the brain and eyes up to 20°C above ambient water channels of the sarcoplasmic/endoplasmic reticulum (SR/ER), the IP3 and ryanodine receptors temperature. Heater organ cells are packed with mitochondria and express a modified muscle (RyR). We isolated SR vesicles from zebrafish skeletal muscle and incorporated these vesicles in phenotype with an extensive transverse-tubule network and sarcoplasmic reticulum (SR) enriched planar lipid bilayers. Single channel currents measured using barium as the current carrier had a in Ca2+ pumps (SERCA) and ryanodine receptors (RyR). Thermogenesis has been hypothesized to conductance similar to RyR type 1 from mammalian skeletal muscle (RyRI). However, RyR from be associated with release and reuptake of Ca2+, but the mechanism has yet to be examined. Ca2+ zebrafish skeletal muscle showed significant differences to RyRI with regard to Ca activation and fluxes in heater SR vesicles were measured using fura-2 fluorescence. Heater tissue expresses the inactivation. Channels activation occurred at lower Ca concentrations and maximal activation was neonatal SERCA IB and upon the addition of MgATP, heater SR vesicles rapidly sequestered maintained at higher Ca concentrations than RyRI. Also, inactivation was incomplete even at Ca2 . Uptake was thapsigargin sensitive, and maximum loading ranged between 0.8-1.0 Psmol high Ca concentrations with RyR from zebrafish skeletal muscle. This Ca dependence of the RyR Ca2+/mg protein. Upon the addition of 10 mM caffeine or 350 FM ryanodine, heater SR vesicles of skeletal muscle from zebrafish is similar to the Ca dependence of mammalian type 2 and 3 RyR released only a small fraction of the loaded Ca2+, but a much larger RyR release event could be and to other teleost and amphibian RyR isoforms. Our results have important implications for the unmasked when the activity of SERCA pumps was limited by reducing the MgATP. RNAse interpretation of the role of the RyR in Ca signaling when comparing zebrafish and mammalian protection assays revealed that heater tissue expresses a unique RyR isoform that is specific to muscle physiology, especially when analyzing mutations underlying physiological changes in slow-twitch muscle fibers in fish. [3H]ryanodine binding assays indicated that this RyR displays a zebrafish. Supported by a BASF scholarship (PK), a German National Merit Scholarship left-shifted Ca2 activation curve and in the presence of adenine nucleotides this isoform lacks Foundation scholarship (TJ), and grants from the American Heart Association and the NIH (BEE). Ca2"-dependent inhibition. These RyR properties are similar to the characteristics found in the RyRs of malignant hyperthermia muscle. The fast Ca2+ sequestration of the heater SERCA IB, coupled with a unique RyR, may be responsible for the cycling of Ca2+ that results in ATP turnover and heat generation.

1101 - Pos 1102 - Pos DEPLETION OF INTRACELLULAR Ca2+ BY CAFFEINE AND RYANODINE INDUCES RYANODINE RECEPTORS ARE CLUSTERED IN NEURITES OF CULTURED APOPTOSIS OF CHINESE HAMSTER OVARY CELLS TRANSFECTED WITH EMBRYONIC RAT spinal AND CORTICAL NEURONS. RYANODINE RECEPTOR. Manana Sukhareva, S. Smith, J. Barker, NIHININDS, 36 Convent Dr, Bethesda, MD 20817 Zui Pan', Derek Damron2, Anna-Liisa Nieminen3, Manjunatha B Bhat', Jianjie Ma', 'Dept. of Recent studies have shown a wide distribution of ryanodine receptors (RyRs) throughout the CNS Physiology & Biophysics, Case Western Reserve Univ., Cleveland, OH 44106, 2Center for suggesting that intraneuronal Ca2" stores may play an important role in neuronal calcium Anesthesiology Research, Cleveland Clinic Foundation, Cleveland, 3Dept. of Anatomy, Case dynamics. Using BODIPY-ryanodine and confocal microscopy we found the presence of RyRs in Western Reserve University, Cleveland cultured embryonic rat spinal and cortical neurons. After treatment of neurons with I pM Recent studies have suggested a central role for Ca2" in the signaling pathway for cells undergoing fluorescent ryanodine in the presence of millimolar Ca2" and Mg2" we observed fluorescence in apoptosis, and the anti-apoptotic effect of Bcl-2 has been implicated with changes in intracellular the cell bodies of 90% of the cells and randomly distributed fluorescent spots in selected neurites Ca2+ homeostasis. Here we report that depletion of Ca2+ from the endoplasmic reticulum (ER) possibly revealing clusters of RYR. Removal of Ca2" abolished these spots. Addition of 2 mM leads to apoptosis in Chinese hamster ovary (CHO) cells. Stable expression of ryanodine receptor caffeine increased the number of spots and noticeably changed the pattern of fluorescence towards (RyR) in the ER membrane enables rapid and reversible changes of both cytosolic Ca2+ and ER a more uniform spot distribution. I mM tetracaine significantly reduced ryanodine binding in the Ca2+ content via activation of the RyR/Ca2' release channel by caffeine and ryanodine. The presence of caffeine in neurites, but not in cell bodies. Since ryanodine binds tightly only to the changes in morphology and chromatin structure of the cells undergoing apoptosis or necrosis as a open conformation of Ca2" release channels, our observations suggest that the fluorescent spots result of ER Ca2" depletion were characterized with confocal microscopic imaging and DNA are clusters of active RYRs. Patch-clamp studies of reconstituted synaptosomes revealed caffeine laddering assays. We found that sustained depletion of the ER Ca2" store led to apoptosis in CHO and ryanodine sensitive channels. Thus, RyRs clustering in growing processes of embryonic cells, whereas co-expression of RyR and Bcl-xL in these cells prevented apoptotic cell death but neurons may play critical roles in the process of synapse formation and neurotransmitter release. not necrotic cell death. The anti-apoptotic effect of Bcl-xL did not correlate with changes in either the Ca2+ release process from the ER or the capacitative Ca2+ entry pathway across the plasma membrane. The data suggest that Bcl-xL likely prevents apoptosis of cells at a latter stage after ER Ca2+ release. Supported by NIH and AHA.

187A 1103-1108 INTRACELLULAR CALCIUM SIGNALING II MONDAY

1103 - Pos 1104 - Pos SPATIAL RELATIONSHIPS OF Ca'2-ACTIVATED CHANNELS, Ca2" CHANNELS, PEROXIDES OF VANADATE STIMULATE CALCIUM UPTAKE INTO AN 1P3r AND AND RYANODINE-RECEPTORS (RyRs) THAPSIGARGIN-INSENSITIVE, ACDIFIED STORE IN CHO CELLS. James L. Kenyon, University of Nevada, Reno, MS 352, Reno, NV 89557 Madallna Condrescu, John P. Reeves, UMDNJ Grad. Sch. Biomed. Sci., Dept. Pharmacology & Physiology, 185 South Orange Avenue, Newark, NJ 07103 Ca"-activated currents in chick dorsal root ganglion neurons are activated by Ca2s influx or Ca2" CHO cells were treated for 5 min with an equimolar mixture of 100 IsM H202 and sodium ortho- release by RyRs. At 20 ° C, the rates of deactivation of Ca?'-activated currents activated by Ca" vanadate. This procedure, which generates peroxovanadate, a powerful inhibitor of tyrosine influx were similar in cells dialyzed with no exogenous Ca2" buffer and in perforated patch phosphatases, induced a sustained increase in Ca or Ba influx, as measured in fura-2 loaded cells. recordings (half-times of deactivation, t5, were 629 + 47 and 509 * 56 ms, n=7). t, was shorter in The Ca or Ba influx was blocked by SK&F 96365 (20 pM), an inhibitor of store-operated cells dialyzed with 2 mM EGTA (20 'C, free Ca2+=l00 nM, 112 25 ms, n=7) and in perforated channels, and by the tyrosine kinase inhibitors genistein (50 pg/ml) and herbimycin A (2 pM). patch recordings at 35 OC (96 * 18 ms, n=6). These data imply that the neurons have little Peroxovanadate treatsnent had no effect on the IP3-sensitive Ca pool, measured by the addition of endogenous mobile buffer and that warming and 2 mM EGTA have similar effects on the recovery the purinergic agonist ATP. However, the amount of calcium in an acidified, IP3- and of intracellular Ca2+. The amplitudes of Ca +-activated currents, activated at 20 °C by influx or by thapsigargin (Tg)-insensitive pool increased more than 2-fold following peroxovanadate treatment. release (10 mM caffeine), were similar in cells dialyzed with no exogenous buffer, with 2 mM This pool was released into the cytosol by treatment with ionomycin (2 pM) plus either NH4CI (20 EGTA, and in perforated patch recordings. In contrast, 2 mM BAPTA (free Ca2+ = 100 nM) mM) or nigericin (10 pM) after discharge of the IP3- and/or Tg-sensitive pool. Dissipating the prevented the activation of these currents. Substantial niflumic acid-sensitive currents were pool's proton gradient with either balfilomycin Al or nigericin did not prevent the accumulation of activated by dialysis with 5 pM free Ca2+ (2 mM HEEDTA). These data are compatible with Ca induced by peroxovanadate. The effects of peroxovanadate were blocked by 30 min separations between Ca2+-activated channels, Ca2+ channels, and RyRs of up to 200 nm. Supported pretreatment with brefeldin A (10 pg/ml) or nocodazole (2 pg/ml), agents which disrupt the Golgi by Western States Affiliate of AHA. complex. The results suggest that tyrosine hyperphosphorylation activates a Ca influx pathway that is coupled to Ca sequestration within an acidified, Tg- and IP3-resistant Ca pool that may be associated with the Golgi complex.

1105- Pos 1106 - Pos CHARACTERIZATION OF A PLASMA MEMBRANE CALCIUM OSCILLATOR IN THE ROLE OF ELECTROSTATIC INTERACTIONS IN THE MEMBRANE RAT PITUITARY SOMATOTROPHS LOCALIZATION OF C2 DOMAINS Melanlja Tomic, Taka-aki Koshimizu, Davy Yuan, Silvana A. Andric, Dragoslava Zivadinovic, Diana Murray', Stuart McLaughlin , Barry Honig', 'Columbia University, 630 West 168th Street, Stanko S. Stojilkovic, NICHD-NIH, 49 Convent Dr, Room 6A36, Bethesda, MD 20892 New York, NY 10032, 'SUNY Stony Brook Rat pituitary somatotrophs generate spontaneous [Ca2+]i oscillations, which rise from fluctuations Subcellular targeting is often accomplished through protein domains that interact with specific in the influx of external Ca2+ and propagate within the cytoplasm and nucleus. Caffeine and membranes. C2 domains are implicated in the subcellular localization of many proteins that ryanodine, modulators of ryanodine-receptor channels, and the depletion of intracellular Ca2+ function in interfacial signal transduction. Although they share the same structural fold, C2 stores did not affect the global nature of spontaneous [Ca2]1 signals. Bay K 8644, an L-type Ca" domains perform multiple functions. Among the C2 domains that are targeted to membranes in a channel agonist, initiated [Ca2], signaling in quiescent cells, increased the amplitude of [Ca+]i calcium dependent manner, there is more than one mode of protein-lipid interaction. For example, spikes in spontaneously active cells, and stimulated growth hormone secretion, while nifedipine, a upon binding calcium, the C2 domain from protein kinase Cf3 preferentially associates with blocker of L-type Ca2+ channels, had the opposite effect. Ni2+, a blocker of T-type Cae channels, membranes containing the acidic lipid phosphatidylserine (PS), while the C2 domain from abolished spontaneous [Ca2+], oscillations. Spiking was also abolished by the removal of cytosolic phospholipase A2 penetrates into the hydrocarbon core of zwitterionic extracellular Nae and by the addition of 10 mM Ca2+, Mg2+, or Sr2+, the blockers of cyclic phosphatidylcholine membranes. Two mechanisms have been widely discussed: when calcium nuclootide-gated channels. RT-PCR and Southern blot analyses indicated the expression of ions bind to C2 domains they may increase the long-range electrostatic attraction to membranes mRNAs for these channels in somatotrophs. Growth hormone-releasing hormone, an agonist that containing PS and/or form specific chelates with phosphate groups on the lipids. Calculations stimulated cAMP and cGMP production, initiated spiking in quiescent cells and increased the presented here suggest a new mechanism for membrane targeting: calcium ions neutralize clusters frequency of spiking in spontaneously active cells. These results indicate that in somatotrephs a of acidic residues in the calcium binding loops, decreasing the desolvation penalty associated with cyclic nucleotide-controlled plasma membrane Ca+ oscillator is capable of generating global Ca2+ bringing the C2 domains close enough to the membrane surface to allow for favorable short-range signals spontaneously and in response to agonist stimulation. The Ca2+-signaling activity of this interactions. The electrostatic potentials on C2 domain surfaces are correlated with the type of oscillator is dependent on voltage-gated Ca"+ influx, but not on Ca2+ mobilization. membrane targeted. This correlation is not evident from comparisons of the relevant sequences nor from the structures themselves.

1107 - Pos 1108 - Pos NUCLEOPLASMIC Ca+ LOADING REGULATED BY PERINUCLEAR Ca2+ ROLE FOR SERCA3 IN THE REGULATION OF CALCIUM TRANSIENTS IN MOBILIZATION LYMPHOCYTES Bernard Abrenica, James S. C. Gilchrist, University of Manitoba, 351 Tache Avenue, Winnipeg, Carla Selarretta', Gary Shull', Jonathan Lytton', 'University of Calgary, Biochem & Mol Biol, Manitoba R2H 2A6 Canada 3330 Hospital Drive NW, Calgary, AB T2N 4N1 Canada, 'University of Cincinnati Regulation of nucleoplasmic Ca" concentration may occur by the mobilization of perinuclear Antigen activation of lymphocytes is mediated by IP3-induced Ca2+ release from intracellular luminal Ca2+ pools involving specific Ca" pumps and channels of both inner and outer perinuclear stores, consequently stimulating Ca2+ influx, which contributes to a sustained and essential membranes. To determnine the role of perinuclear lumenal Ca", we examined freshly cultured 10 elevation of [Ca2+],. Two Ca+ pump isofonns, SERCA2b and SERCA3, are responsible for day-old embryonic chick ventricular cardiomyocytes. We obtained evidence suggesting the loading calcium stores in lymphocytes. We tested the hypothesis that SERCA3 has a unique role existence of the molecular machinery required for regulating bi-directional Ca2+ fluxes using in regulating lymphocyte Cae+ signaling using cells isolated from mice homozygous for an confocal imaging techniques. Embryonic cardiomyocytes were probed with antibodies specific inactivated SERCA3 gene. Changes in intracellular Ca2+ following stimulation were deternined for ryanodine-sensitive Ca2+ channels, SERCA2-pumps, and fluorescent derivatives of ryanodine by monitoring Fluo-3 fluorescence using flow cytometry. In splenic B cells and thymocytes, the and thapsigargin. Using immunocytochemistry, confocal imaging showed the presence of RyR2 rate of decline of [Ca2+]i following stimulation was significantly reduced in the serca3f'-) cells Ca2+ channels and SERCA2-pumps highly localized to regions surrounding the nucleus, referable compared to wild-type controls. In addition, the peak [Ca"+]j in stimulated serca3(') thymocytes to the nuclear envelope. Results obtained from Fluo-3/AM loaded ionomycin-perforated was reduced compared to control cells. Experiments conducted in the absence of extracellular Ca2+ embryonic cardiomyocytes demonstrated that gradual increases of Ca2+ from 100 nM to 1600 nM revealed that compared to wild type, serca3(') splenic T cells had a smaller antigen-responsive Ca2+ was localized to the nucleus. SERCA2-pump inhibitors Tg, cyclopiazonic acid as well as Ca2+ store, which correlated with a smaller increase in [Ca2], when extracellular Ca2+ was re- low micromolar concentrations of ryanodine inhibit Ca2+ loading of the nucleus. Our results show introduced. We were unable, however, to observe a difference in the Ca2+ transient between that the perinuclear lumen in embryonic cardiomyocytes is capable of independently regulating serca3() and wild type splenic T cells upon stimulation in the presence of extracellular Ca2+. nucleoplasmic Ca2" fluxes. Further, we observed that serca3() splenocytes stimulated in culture secrete a reduced level of (Supported by the Manitoba Health Research Council) interleukin-2, a cytokine whose expression in T cells is calcium-dependent. These results indicate that SERCA3 makes a critical contribution to Ca2+ signaling in lymphocytes.

188A MONDAY INTRACELLULAR CALCIUM SIGNALING II 1109-1114

1109 - Pos 1110 - Pos THAPSIGARGIN-INDUCED DECREASE OF CALCIUM WAVE VELOCITY IN THE PI 3-KINASE INHIBITORS, WORTMANNIN AND LY294002, INHIBIT SPERM- CARDIAC MYOCYTES. INDUCED EGG ACTIVATION IN XENOPUS LAEVI& Grit Landgrafe, Helmut Podhaisky2, Manfred Wussliagi, 'Dept. of Physiology, 2Insitute of Richard Nuccitelli, Nguyet M Nguyen, Hyun Kim, University of California, Davis, 1 Shields Numerical Mathematics Martin Luther University Halle-Wittenberg Ave., Davis, CA 95616 Thapsigargin, a potent and highly specific inhibitor of the sarcoplasmic reticulum Ca2'-ATPase, Fertilization is accompanied by a wave of increased free [Ca2+]i in all eggs studied to date. was reported to enhance the propagation velocity of caffeine-stimulated calcium waves in rat However, it is not fully understood how the sperm activates this release of Ca+ upon fertilization. cardiac myocytes at a concentration of 10 tM (1). Here we demonstrate (i) a concentration- There is evidence that PIP2 hydrolysis is required for the Ca2' increase, but it is not known how dependent reduction of the propagation velocity of spontaneous calcium waves in ret cardiac PLC is stimulated to initiate this hydrolysis. One possibility is that PLC is recruited to the myocytes by thapsigargin within a range of 0.1 nM to 20 nM and (ii) that spontaneous calcium membrane via the binding of its plekstrin homology domain(s) to PIP3. PI 3-kinase is the enzyme waves are lacking at higher thapsigargin concentration than 20 nM. The wave speed of the responsible for the formation of PIP3 so we are investigating the possible involvement of PI 3- controls principally depends on the interwave period (dispersion relation), and on the extracellular kinase in the Xenopus egg activation cascade. Two inhibitors of P13-kinase are Wortmannin and [Ca2'] (2). The effect of thapsigargin, however, was maximum at 20 nM. Data of propagation LY204002. We injected mature, jelly-intact Xenopus eggs with either of these compounds and velocity v versus concentration c ofthapsigargin were fitted by a decaying one phase exponential: studied both activation and Ca2 release upon sperm addition. LY294002 was found to reduce sperm-induced egg activation and to completely block cleavage. At 20 uM final concentration, v = a*exp(b/[c+d]) with a 53 Ism/s, b 0.6 nM, d 1.2 nM. 81% of the LY294002-injected eggs did not exhibit fertilization envelope (FE) liftoff, cortical The results are interpreted in terms of a diffusion-reaction system and simulated by the Tang- contraction or rotation compared to only 19%/o inhibition in the 16 solvent control eggs injected Othmer model (3) describing the propagation of calcium waves in cardiac myocytes. (Supported with the same amount of DMSO. For Wortmannin, we found that at 100 nM final concentration, by the BMBF Wu 1999). 80% of the injected eggs were activated but none cleaved, while 100% of the solvent control- injected eggs were activated and cleaved. At 200 nM final concentration, 0% of Wortmannin- (I) V. Lukyanenko et al., J. Physiol., in press (1999) injected egg could be activated by sperm. Confocal imaging of eggs co-injected with (2) M.H.P. Wussling and Th. Mair, Lecture Notes in Physics, in press (1999) Wortnannin or LY294002 and Oregon Green-dextran indicated that the increased [Ca2+]i was (3) Y. Tang and H. G. Othmer, Biophys. J. 67, 2223 (1994) inhibited under normal insemination conditions but could be observed with very high sperm densities. However, the Ca2+ waves observed under these conditions were abnormal. In those eggs exhibiting waves, the wave propagation velocity was much slower than normal. In some eggs no waves occurred, but brief cortical "hot spots" were observed in which Ca2+ increased for 10-20 seconds. Both slow waves and hot spots were completely inhibited by Wortmannin when higher concentrations of 200 nM were used.

1111 - Pos 1112 -Pos CALCIUM RELEASE FROM INTERNAL STORES IN LATERODORSAL THALAMIC SUPPRESSION OF Ca2+ EFFLUX FROM INTRACELLULAR Ca2+ STORES BY TPEN, A NUCLEUS NEURONS. MEMBRANE PERMEANT, LOW AFFINITY CALCIUM BUFFER, IN FROG Ilya Krugllkov', A Eremin', A Tarasenko', A Ivanov', V Snitsarev2, N Voitenko', 'Bogomoletz SYMPATHETIC NEURONS Inst. of Physiology, 2University of Iowa Zoltin Cseresanys, S.I. McDonough, M.F. Schneider, Department of Biochemistry and Thalamic associative nuclei including the laterodorsal (LD) one play a major role in the Molecular Biology, School of Medicine, UM Baltimore, Baltimore MD 212 organization of rhythmic thalamocortical activity. However, data about the participation of The membrane permeant Ca2+ buffer TPEN was used to alter Ca2+ concentrations within internal calcium stores in calcium signaling in these neurons are still missing. The cytoplasmic intracellular Ca2+ stores in individual neurons. We used whole-cell measurements of fura-2 free Ca2+ concentration ([Ca2']i) was measured in acutely isolated LD neurons of P12 rats, using fluorescence to calculate the cytosolic free [Ca2+] during and after periods of elevated Ca2+ influx Fura2-AM based microfluorimetry technique. An initial caffeine application did not influence and of Ca2+ release, and a computer model to simulate the experimental data. The slow [Ca2+]i. However, after cell stimulation with 50mM KCI caffeine induced [Ca2'], elevation of component of decay of [Ca21 after 50 mM K+-induced Ca2+ entry was abolished reversibly by 184±28nM both in normal and calcium-free solutions. The second application of caffeine in 400 RiM TPEN, but not by 100 FiM. The transient delayed rise of cytosolic [Ca2'] after rapid Ca2+ calcium-free solution did not produce [Ca2+]i elevation indicating the depletion of ryanodine- uptake by release activated calcium transport (RACT; Cseresnyes et al., Neuron 19, 403-419, dependent calcium stores. Administrations of potential InSP3-induced Ca2+ release agonists such as 1997) was also reversibly eliminated by 400 'M TPEN. Caffeine-induced [Ca2+] oscillations were glutamate (10M), acetylcholine (100pM) and ATP (100IpM) produced Ca2+ transient elevation of also abolished by 400 jiM TPEN. All the effects of TPEN are attributable to Ca2+ binding to 129±25nM, 259±44nM and 243±36nM respectively. The ssme applications of glutamate and TPEN within intracellular stores. 10 jM TPEN, which should completely buffer heavy metals acetylcholine, but not ATP, in Ca2+-free solution and after conditioning depolarization with KCI (Hofer et al., Journal of Cell Biology 140, 325-334, 1998), had no effect on any Ca2+ signals, resulted in transient [Ca2']J elevation of 20±6nM and 19±4nM respectively. Caffeine application indicating that heavy metal buffering was not involved in the observed effects of TPEN. All after challenging cells with glutamate or acetylcholine did not evoke rise in [Cal']i. Thus, we may experimental observations could be simulated assumning (Hofer et al., 1998) free TPEN to be assume that both InsP3 and ryanodine receptors in these neurons may be localized within the same highly membrane permeant, Ca2+-TPEN to be impermeant and TPEN to have low Ca2+ affinity intracellular Ca2+ pools, though this hypothesis requires further investigations. Supported by (KD=135 ILM). Our results indicate that elevated store Ca2+ concentrations may be present during CRDF grant UBI-318 to N.V. the slow decay of [Ca2J] after high-K+ induced Ca2+ entry and after Ca2+ uptake by RACT. Supported by NIH (RO1-NS33578).

1113 - Pos 1114 - Pos INITIATION SITES OF CALCIUM OSCILLATIONS IN FROG SYMPATHETIC DIFFERENTIAL CALCIUM BUFFERING IN MOTONEURON POPULATIONS THAT NEURONS. ARE SELECTIVELY VULNERABLE AND RESISTANT DURING MOTONEURON Stefan . McDonough, Z. Cseresnyes, M. F. Schneider, Dept Biochem, Univ. MD Med Sch, 108 DISEASE N. Green St., Baltimore MD 21201 Bernhard U. Keller, Bodo K Vanselow, Mario B Lips, Jiri Palecek, Center of Physiology, Fluorescence from frog neurons loaded with the Ca2'-sensitive dye fluo-4-AM was recorded with Humboldtallee 23, Gottingen, 37073 Germany the Nikon RCM-8000 video-rate confocal microscope. In most cells, the fluorescence increase Motoneurones are particularly vulnerable both in human forms of amyotrophic lateral sclerosis evoked by 10 mM caffeine, corresponding to Ca2+ release from intracellular stores, arose not (ALS) and corresponding mouse models of the disease. While some populations like hypoglossal uniformly throughout the cell, but spread from 14 distinct initiation sites at the edges of the cell. or spinal motoneurones are selectively impaired, oculomotor neurones are largely resistant to These initiation sites often occurred distal to the direction of microperfused caffeine application motoneuron damage. Selective vulnerability has been closely linked to neuron-specific disruptions and were seen -10 min after bath application of caffeine, implying that they were not due to of calcium signaling. To investigate underlying mechanisms, we performed a quantitative analysis nonuniform local caffeine concentrations. The same pattems of release and spread were seen in of calcium homeostasis in oculomotor, hypoglossal and spinal motoneurones from mice by Ca2+-free external solution. Fluorescence spread from the initiation sites around the edges and simultaneous patch clamp recordings in sliced tissue and microfluorometric calcium then into the center of the cell; the patterns of spread suggested propagation occurred via measurements. Endogenous calcium homeostasis was quantified by using the "added buffer" regenerative Ca2e-induced Ca2+ release and not via passive diffusion from a point source. approach. In oculomotor neurones, endogenous calcium binding ratios were found to be K, = 264, Repeated caffeine applications followed by washout evoked Ca2+ release from the same initiation indicating that 99-8 % of cytosol calcium ions were taken up by endogenous buffers. This ratio sites each time. When steady caffeine application evoked Ca2+ oscillations, the calcium wave for was 5 6 fold larger compared to those of more vulnerable motoneurones in the nucleus each individual calcium peak during up to 28 oscillations originated from the same single site or hypoglossus (Ks = 41) and spinal cord (Ks = 50). Recovery of calcium transients was few sites. Oscillation frequency (n = 14) was not correlated with the number of Ca2+ release sites. characterized by an "effective" extrusion rate of 156, 145 and 60s-' in oculomotor, spinal and These observations suggest that these neurons contain sites primed for Ca2+ release, possibly due hypoglossal motoneurones, respectively. With respect to pathophysiological conditions, our to increased ryanodine receptor (RyR) or SERCA pump levels, or, as the patterns of Ca2+ release measurements suggest that the selective vulnerability of hypoglossal and spinal motoneurones suggested, due to higher Ca2+ sensitivity of RyRs at the initiation sites. Such sites might enable partially results from exceptionally low calcium buffering capacities, and that addition of local intracellular Ca + regulation to govern [Ca2+] over the entire cell. ,,exogenous" buffers could represent a valuable strategy for motoneuron protection.

189A 1115-1120 INTRACELLULAR CALCIUM SIGNALING II MONDAY

1115 - Pos 1116 - Pos CYTOPLASMIC Ca'+ HANDLING AND DYNAMICS OF Ca2+ GRADIENTS IN IMPAIRED CALCIUM RELEASE CEREBELLAR PURKINJE NEURONS ADRENAL CHROMAFFIN CELLS. MAINTAINED IN CULTURE Fernando D Marengo, Jonathan R Monck, UCLA School of Medicine Mary D. Womack, Kamran Khodakhah, UCHSC, 4200 East Ninth Avenue, Denver, CO 80262 The temporal and spatial pattern of cellular Ca2+ redistributions is influenced by endogenous Ca2+ Inositol trisphosphate (InsP3)-evoked calcium release has been shown to be important for long buffers. We used pulsed laser imaging to measure the development and dissipation of Ca2+ term depression (LTD) in Purkinje neurons in cerebellar slices. Depression of responses to gradients induced by activation of voltage-sensitive Ca+ channels in patch-clamped bovine iontophoretically-applied glutamate in cultured Purkinje neurons is assumed to share the same adrenal chromaffin cells loaded with the Cae indicators (rhod-2, OGB-SN). Cai+ gradients underlying mechanism as LTD in slices. However, it has been suggested that LTD in cultured appeared rapidly (5 ms) upon membrane depolarization and dissipated over several hundred Purkinje neurons does not require InsP3-evoked calcium release. We have directly examined the milliseconds following repolarization. The Ca2+ gradient dissipation was described by two properties of InsP3-sensitive calcium stores in cultured neurons and compared them to those of exponentials (r'S: 7.6 and 156 ms). Neither inhibition of Ca2+ uptake by the endoplasmic reticulum acutely dissociated Purkinje neurons. Whole cell patch clamp pipets were used to introduce caged (thapsigargin) or mitochondria (FCCP/oligomycin) affected the dissipation times, suggesting that InsP3 and the calcium indicator fluo3. In cultured neurons InsP3-evoked calcium responses were passive Ca+ buffers slow Ca2+ redistribution. small and rose slowly (mean DF/F=0.08+/-0.2, n=9). InsP3 produced no change in calcium levels We used a radial diffusion model considering Ca2+ diffusion and binding to intracellular Ca2+ in 80% of cultured Purkinje neurons tested. At similar concentrations InsP3 produced large, buffers to simulate Ca2+ gradients. A 3-D optical sectioning model using the microscope PSF was rapidly rising calcium responses in all acutely dissociated Purkinje cells tested (mean DF/F = included to simulate the effects of out-of-focus light on the modeled Ca2+ gradients and to allow 3.3+/-1.8, n=7). Caffeine had little effect on calcium levels in cultured Purkinje cells, yet evoked comparison with the measured gradients. The size and rate of dissipation of the measured Ca+ large calcium transients in acutely dissociated Purkinje cells. These results demonstrate that gradients matched the simulations when we included a high capacity immobile endogenous buffer release of calcium from intracellular stores is severely inpaired in cultured Purkinje neurons. capable of capturing -99.9%/o of the Ca2+ influx (ic-1000). Finally, simulations without exogenous Cultured Purkinje neurons appear to be an inadequate model for elucidating the role of calcium buffer suggest that Ca2+ signal due to Ca?+ channels extends less than l&m from the cell stores in LTD. membrane by endogenous Ca + buffer.

1117- Pos 1118 - Pos EFFECTS OF EGTA ON SINGLE ACTION POTENTIAL INDUCED SOMATIC Ca2 CHANGES IN CALCIUM CHANNEL ACTIVITY IN DORSAL HORN NEURONS OF TRANSIENTS IN MOUSE HIPPOCAMPAL NEURONS DIABETIC RATS Valentlm Urs Nigerl, Istvan Mody, UCLA School of Medicine, 710 Westwood Plaza, RNRC, Nasa Voltenkol, Ilya Krsglikov', Elena Kostyuk2, 'Bogomoletz Inst. of Physiology, Kiev, Los Angeles, CA 90095 Ukraine, 2Inst. of Endocrinology We have previously found that spinal dorsal horn neurons from strptozotocin-diabetic rats, an We have recorded action potential (AP) induced Ca2+ transients from the soma of granule cells in animal model for diabetes mellitus, show the prominent changes in the mechanisms responsible mouse hippocampal slices. Signal averaging and low-noise fluorescence detection (Axopatch- for [Ca2'] regulation (Voitenko et al, Neuroscience, 1999). The present study aimed to further amplified photodiode system) pernmitted the use of a low affinity Ca2e indicator dye (Oregon chracteize the effects of steptozotocine-induced diabetes on neuronal calcium homeostasis. The Green 488 BAPTA-5N). This minimized signal filtering and buffering associated with high- cytoplasmic C?' concentration ([Cal'] ) was measurd in Fura-2/AM loaded dorsal horn neurons affinity Ca2+ probes and therefore enabled us to faithfully resolve changes in somatic [Ca2+] on a from acutely isolated spinal cord slices using fluorescence technique. We studied Ca2' entry millisecond timescale. through plasmalemmal Ca2' channels during potassium (50 mM KCI)-induced depolarisation. The We experimentally varied Ca2+ entry and intracellular Ca?+ buffering by altering the number of K+-induced [Ca2'], elevation was inhibited to a different extent by nickel ions, nifedipine and t- APs and the concentration of EGTA (20-200 pM) in the patch pipette, and analyzed the effects on conotoxin suggesting the coexpression of different subtypes of plasmalemmal voltage-gated Ca2' amplitude and timecourse of the fluorescence transients. The l0-900/o rise time of Ca2+ transients channels. The suppression of [Ca2+]i transients by Ni2+ (50 pM) was the same in control and associated with single APs was 2.8 ms. The AP-induced transients decayed bi-phasically with cf.a diabetic neurons. On the other hand, inhibition of [Ca2+]i transients by nifedipine (50 pM) and Do- ranging between tens to hundreds of milli-seconds depending on the number of evoked APs and conotoxin (1 pM) was much greater in diabetic neurons compared with normal animals. These t;.,. being constant at around 2 s. Surprisingly, the addition of as little as 100 pM EGTA data suggest that under diabetic conditions the activity of N-and L- but not T-type voltage-gated significantly accelerated f,,,, retarded r,,,, and reduced the maximal AF/F. These salient features Cal' channels substantially increased in dorsal horn neurons. Supported by CRDF grant UBI-318 were consistent with a numerical simulation for intraneuronal Ca2+ buffering. Hence, this to N.V. technique allows us to dissect the properties of endogenous Ca2e buffering following brief Ca2' entry resulting from physiological stimuli.

1119 - Pos 1120 - Pos CHANGES IN CALCIUM HOMEOSTASIS OF MURINE PRIMARY SENSORY THE Ca2' CHANNEL BLOCKER, NI2, INIHIBITS DIRECTED MIGRATION OF NEURONS UNDER INFLAMMATION KERATINOCYTES IN A PHYSIOLOGICAL ELECTRIC FIELD. Vyacheslav Shishkln', P Kostyuk', A Klishin2, N Voitenko', 'Bogomoletz Inst. of Physiology, DoBns R TroliNager, R Rivkah Isseroff, Richard Nuccitelli, University of California, Davis, 2Loyola Unversity Chicago Molecular and Cell Biology, 1 Shields Ave., Davis, CA 95616 Changes in neuronal calcium homeostasis were studied on acutely isolated dorsal root ganglion Directed nigration of keratinocytes is essential for wound healing. Previously we showed that (DRG) neurons of carrageenan-injected mice. [Ca2+]i was measured using indo-I based human keratinocytes in vitro migrate to the cathode in direct current electric fields, a process microfluorimetry. In control cells 10pM CCCP applied before KCI-depolarization induced an termed galvanotaxis. Here we investigated the role of calcium influx on the directionality and increase of the amplitude of depolarization-induced [Ca2l+, indicating the participation of migration speed of keratinocytes during galvanotaxis. Cultured human keratinocytes were plated mitochondria in fast uptake of Ca2+ from the cytosol during the peak of transient. In carrageenan- on collagen-coated glass bottoms of plexiglass chambers and allowed to adhere for 2 h at 37'C in injected animals this increase became diminished. CCCP, applied after the transient has reached keratinocyte growth medium (1.8 mM Ca2+). Cells were exposed to a constant physiological its peak, induced an additional rise of [Ca2l, that reflected massive release of Ca?+ previously electric field strength of 100 mV/mm for 1-2 h. Calcium channel inhibitors, verapamil (10 mM), accumulated by mitochondria. In small (nociceptive) neurons from mice with inflammation such amiloride (10 mM) and nickel (5mM) were used to block Ca2+ entry. Migration paths, speeds and an elevation was dramatically smaller. As it was previously shown endoplasmic reticulum take directionality of travel were determine by computer assisted image analysis. After 1 hr of part in [Ca2']i homeostasis of large but not small DRG neurons by releasing Ca2' through galvanotaxis, nickel, but not verapamil and amiloride inhibited keratinocyte migration speed (0.7 ryanodine receptors and InsP3-receptors. [Ca2+], transients evoked by 20mM caffeine and 100pM ± 0.1 pm/min vs. 0.2 ± 0.03 pm/min) and directionality, indicated as cosine (0.6 ± 0.7 vs. 0.2 ± ATP were significantly smaller in animals with inflammation with respect to control mice. Such a 0. 1), as compared to controls. Nickel's inhibition of directed migration but not speed of movement difference became more considerable when caffeine or ATP were applied after KCI- was reversible. These results suggest that calcium influx is required for directed migration of depolarization. We conclude that inflammation is associated with substantial decrease of keratinocytes during galvanotaxis. However, directionality of migration and migratory speed are mitochondrial Ca2+ uptake and subsequent release into the cytosol in all types of DRG neurons, controlled by separate mechanisms. and diminishing of Ca2' accumulation ability of endoplasmic reticulum in large (proprioceptive) neurons. Supported by CRDF grant UBI-318 to N.V.

190A MONDAY MODYITAELLRCLIMSGAIGIINTRACELLULAR CALCIUM SIGNALING II 1121-1126

1121 - Pos 1122 - Pos INTERFERING WITH INTRACELLULAR CA'+ RELEASE AFFECTS CAPACITATIVE POLYAMINE BLOCK OF RYANODINE RECEPTOR CHANNELS CA'+ ENTRY IN CHINESE HAMSTER OVARY CELLS TRANSFECTED WITH Fiona C Mead, Alan J Williams, NHLI, Imperial College, London, UK, Dovehouse St.,, RYANODINE RECEPTOR LONDON, SW3 6LY United Kingdom Zul Pan, Jianjie Ma, Dept. of Physiology & Biophysics, Case Western Reserve Univ., Cleveland, Polycationic shaker B N-type inactivation peptides interact with the cytosolic vestibule of the OH 44106 ryanodine-modified SR Ca"-release channel causing discrete blocking events, probably as the Capacitative Ca2+ entry (CCE) is a general feature of many non-excitable cell types triggered by result of interactions with negatively charged sites in this region of the channel (Mead et al. depletion of intracellular Ca2+ pools. The mechanism of signal transduction from the intracellular (1998), J.Memb.Biol. 163:225-234). Here we describe block of this channel by neomycin, another organelle to the plasma membrane remains largely unknown. To study CCE, thapsigargin (TG) polycation that inhibits ryanodine receptor-mediated Ca2" release and [3H]-ryanodine binding. and ionomycin are commonly used to deplete the intracellular Ca2+ stores. We report here a new Block was investigated using purified, ryanodine-modified, cardiac channels with K' as the method to interfere with endoplasmic reticulum (ER) Ca2+ release and thus affect CCE via caffeine permeant ion. Neomycin induces well defined blocking events to a level clearly distinguishable and ryanodine in Chinese hamster ovary cells (CHO) stably expressing ryanodine receptor. Using from the closed state, permitting quantification of block uncontaminated by normal closings. In fluorescence of Fura-2, cytoplasmic Ca + of single cell was measured. Caffeine (10 mM) induced contrast to most other blockers of this channel neomycin was effective from both the cytosolic and rapid Ca2' release with partial depletion of ER Cs+ stores. Addition of 2 mM Ca+ in extracellular lurninal sides of the membrane. In both cases block was dependent upon concentration bath solution restored the ER Ca2+ stores through CCE. Ryanodine binds to activated RyR and (KD(cytosolic) = 42±7.3 nM (n=5); KD(luminal) = 250±30.5 nM (n=6)) and voltage. Cytosolic locks the Ca+ release channel in a permanent open state. Thus, the combined application of block was seen only at positive potentials, luminal block was seen only at negative potentials with ryanodine and caffeine resulted in a sustained depletion of ER Ca" stores, as TG failed to elicit the following parameters; cytosolic : Kbi(0)=534.8±35nM, zS=1.09±0.035; luminal: any Ca2+ release even after the Ca2+ reloading. Following the caffeine/ryanodine-induced depletion Kb(0)=1.03±0.01pIM, z&=-0.578±0.005. These data indicate that neomycin has access to binding of ER Ca2+ stores, a significantly larger CCE was observed, which could be blocked by SKF sites from both the cytosolic and luminal faces of the ryanodine receptor channel and are 96365 but not by verapamil. This method provides an alternative and convenient model for future consistent with the proposal that these regions of the channel contain negatively charged residues. studies of CCE.

1123 - Pos 1124 - Pos LUMINAL TRYPSIN ALTERS THE RESPONSE OF THE SHEEP CARDIAC RYR TO RYANOID-INDUCED RE-LOCATION OF A BLOCKING SITE IN THE RYR2 LUMINAL Ca+ CHANNEL. Lilien Ching, Alan J. Williams, Rebecca Sitsapesan, NHLI, Imperial College of Science, Biavna Tannai, Luc Rueste, John L. Sutko3, William Welch4, Alan J. Williams', 'NHLI Imperial Technology and Medicine, Dovehouse Street, London, SW3 6LY United Kingdom College London, UK, 'Dept. Chem. Univ. Sherbrooke, Quebec, 3Dept. Pharmacol., Univ. Nevada, Luminal Ca2+ can modulate RyR channel gating but there is controversy over whether Ca2+ acts Reno, 4Dept. Biochem. Univ. Nevada, Reno, Mail Stop 330, Reno, Nevada 89559 via luminal binding sites (Sitsapesan et al., 1994) or flows through the channel and modifies The interaction of ryanodine with RyR channels produces a reduction in the rates of translocation gating by binding to cytosolic sites (Tripathy et al., 1996). The luminal face of the channel was of permeant cations reflecting altered permeability, affinity and a possible ryanodine-induced therefore exposed to trypsin to establish if putative luminal Ca+-binding sites could be conformational change in the channel. Alteration of ryanodine structure yields ryanoids that bind proteolytically degraded. Native RyR2 were incorporated into bilayers and voltage clamped at at the ryanodine site and reduce conductance, however the amplitude of the reduced or fractional +40mV in symmetrical 250 mM Cs+. Increasing luminal Ca"+ from 10 piM to I mM increased the conductance state (FC) is influenced by ryanoid structure. RyR modification by ryanoids also open probability (Po) of ATP-activated channels from 0.192 ± 0.127 to 0.362 ± 0.165 (SEM, n = alters the interaction of impermeant (blocking) cations with RyR. Here we describe the influence 4). After incubation of the channel with 400 pglml luminal trypsin, increasing luminal Ca2+ from of a group of ryanoids on block of RyR by tetraethylamnnonium (TEA). Single channel K' 10 pM to 1 mM caused a reduction in Po from 0.122 ± 0.103 to 0.053 ± 0.038 (SEM, n = 4). currents in RyR2 were monitored with and without 20 mM TEA between -80 and +80 mV in both Subsequently, lowering luminal [Ca2+] to approximately 90 nM led to an increase in Po to 0.110i± control and ryanoid-modified states. Woodhull (J.G.P.:61; 687-708, 1973) blocking parameters 0.096 (SEM, n = 4). These effects were not observed with heat-inactivated trypsin. Our results (effective valence: za, dissociation constant Kb,ts) were as follows; no ryanoid z-0.8 1, indicate that luminal Ca2+ may exert both activating and inactivating effects on RyR by binding to Kb4o)=142mM; ryanodol (FC=67%) z&=0.77, Kb,o)=107mM; 9-hydroxy-21-azidoryanodine luminal sites on the channel but that only activating sites are affected by the action of trypsin. (FC=55%) zo=0.52, Km(ofs79mM; 21-amino-9a-hydroxyryanodine (FC=45%) z&d0.57, Supported by the BHF. Kbso)=230mM; 21-p-nitrobenzoyl-amino-9-hydroxyryanodine produces two modified states (FC=28%) z&=0.56, Kb,os=70lmM and (FC=17%) a=0.1, Kb(o)=1506mM. Ryanoid-induced channel modification results in alteration of both parameters with an apparent re-location of the TEA site within the voltage drop across the channel that varies with FC. These observations add support to the proposal that ryanoid binding produces structural re-arrangements in RyR.

1125- Pos 1126 - Pos EXCESS NOISE IN THE RYANOID-MODIFIED CONDUCTANCE STATE OF THE DEIS INCREASES OPEN PROBABILITY AND CONDUCTANCE AND INDUCES A RYR2 CHANNEL SUBCONDUCTANCE STATE IN RYR2 Alan J. Williams', Naeemah Haji', Bhavna Tanna', William Welch2, Luc Ruest3, John Sutko4, Adam Parker Hill, Rebecca Sitsapesan, NHLI, Imperial College of Science, Technology and 'NHLI, Imperial College, London, U.K., 2Dept. Biochemistry, Univ. of Nevada, Reno, 3Dept. Medicine, London, UK. Chimie, Univ. Sherbrooke, Canada, 4Dept. Pharmacology, Univ. of Nevada, Reno With Ca2" as the permeant ion, diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) irreversibly Ryanoids bind to RyR channels and reduce conductance to levels determined by structural features increases the open probability (Po) and conductance of RyR2 (Sitsapesan, 1999. J. Memb.Biol., of the ryanoid (Welch et al., Biochemistry: 36; 2939-2950, 1997). The subconductance states 168,159-168). To investigate if the permeant ion can influence the action of DIDS we have studied induced by different ryanoids also vary in their substructure. Some, such as those induced by DIDS-induced modification of RyR2 using K' as the permeant ion. Purified channels were ryanodine, display little excess noise when compared to the normal open state. In contrast, the incorporated into bilayers under voltage-clamp conditions in symmetrical 210 mM KW. Cytosolic noise of the modified state induced by 21-amino-9a-hydroxyryanodine is approximately twice that DIDS (1 mM) irreversibly increased conductance from 694.2±2 pS to 728.9±5 pS (SEM; n=4) and of the normal open state and that induced by 805-amino-9a-hydroxyryanodine is approximately Po to almost I (n=20). At increasingly positive and negative holding potentials the duration and five times that of the normal open state with clear transitions between two levels (a and J). The frequency of channel closings increased. Voltage-dependent inactivation was also observed, but at probability of the a and P levels of the 8[-amino-9a-hydroxyryanodine-induced modified state negative potentials only (79% of channels inactivated at -80 mV; n=14). In addition, DIDS occurring is influenced by trans-membrane voltage. For a typical RyR2 channel in symmetrical induced the channel to dwell in a subconductance state at a level approximately 20% of the control 610 mM K+ with 500 nM cytosolic 81-amino-9a-hydroxyryanodine Pa varies with holding conductance (145±6 pS (SEM; n=4)). Unlike the other effects of DIDS, the induction of the potential as follows (holding potential(mV)/Pa): -80/0.80; -60/0.78; 40/0.67; -20/0.35; 20/0.31; subconductance state was fully reversible after washout of DIDS. Our results demonstrate that the 40/0.24; 60/0.20; 80/0.19, reflecting variations in the mean dwell times in the two levels. At - effects of DIDS depend on the permeant ion and indicate that DIDS binds to multiple sites on 6OmV Pa is 0.76±0.01; mean, is 4.30±0.16 ms; mean is 1.19±0.20 ms, at +6OmV Pa is RyR2; at least one site to irreversibly increase conductance and Po, and at least one other site to 0.21±0.02; mean., is 1.72±0.03 ms; meanp is 6.01±0.26 ms (n=5). These data indicate that in reversibly induce gating in the subconductance level. addition to determining the amplitude of the reduced conductance state, ryanoid structure Supported by the BHF. influences the nature of the ryanoid-induced subconductance state.

191A 1127-1132 STORE-OPERATED CA2.CA2+ MONDAY

1127- Pos 1128 - Pos ACTIVATION OF STORE-OPERATED CALCIUM CHANNELS BY EXTRACELLULAR CRAC CHANNELS ARE THE ONLY CALCIUM INFLUX PATHWAY STIMULATED IN ALKALINIZATION IN JURKAT T-CELLS. CYTOTOXIC T LYMPHOCYTES BY TARGET-CELL CONTACT. David Sepulveda', P. Harikunuar', Madalina Condrescu2, John P. Reeves2, 'Univ. Puerto Rico- Adam Zwelfach, UCHSC, 4200 E. 9th Ave., Denver, CO 80262 Cayey, A. R. Barcelo Ave., Cayey, PR 00736, 2UMDNJ-Grad. Sch. Biomed. Sci., Dept. Pharmacology & Physiology, 185 S. Orange Ave., Newark, NJ 07103 T cell receptor-stimulated Ca2+ influx in helper T lymphocytes occurs through depletion-activated Activation of store-operated calcium channels (SOCs) is a critical feature of Ca signaling in Ca2+ release-activated Ca2+ (CRAC) channels, a mechanism known as capacitative calcium entry lymphocytes. Previous work from this laboratory showed that cytosolic alkalinization accelerated (CCE). The mechanism of Ca2+ influx in cytotoxic T cells (CTLs), which kill relevant targets by thapsigargin (Tg)-induced activation of SOCs. Here we report that extracellular alkalinization releasing vesicles containing pore-forming peptides such as perforin, is unkmown. We used activates SOCs in the absence of agents that induce Ca release. Fura-2 loaded Jurkat T-cells were imaging techniques to investigate whether target-cell stimulated calcium influx in a CTL line incubated in a Mg-free physiological salt solution (PSS) buffered to pH 7.3, 8.3 or 9.3 with 20 occurs through CRAC channels. [Ca2+]i responses in AJYs consist of both release from mM Bis-Tris SOC was s Propane. activity assayed after 90 by the addition of 5 mM BaCI2. The intracellular stores and influx. Release precedes influx. CTLs possess CCE, because thapsigargin rate of Ba influx increased in the order pH 7.3<8.3<<9.3; Ba influx was inhibited by SK&F 96365 and oligomycin, consistent with SOC activity. Increased Ba influx was observed after 10 (TG) elevates [Ca24li. Target cell contact depletes the TG-sensitive Ca2+ store. Two tests for sec at pH 9.3, and became maximal by 60 sec. A small, gradual release of Ca from internal stores non-CCE pathways were negative. There was more influx in cells stimulated with TG than in was observed at pH 9.3, but ionomycin-sensitive Ca stores remained >600/o full after 120 s at this cells stimulated with TG and targets, and there was no difference in the divalent cation pH. At pH 7.3, Tg induced much greater levels of Ca release, but Ba influx was much less than permeability of influx stimulated by target cells, TG or TG+target-cells. With all three that seen at pH 9.3 without Tg. Activation of SOCs at pH 9.3 was inhibited 500/o by I mM Mg stimulations, the sequence was Ca2+(l)>Sr2+(0.09)>Ba2+(0.03). ICRAC in Jurkat cells showed did not inhibit SOCs activated We conclude that although Mg by Tg. extracellular alkalinization a similar sequence: These results that CRAC channels activates SOCs independently of Ca release from the bulk of intracellular stores. Possible Ca2+(l)>Sr2+(0.06)>Ba2+(0.02). suggest mechanisms include an antagonism of Mg-dependent inactivation of SOCs and/or activation of Ca are the sole Ca2+ influx pathway stimulated in CTLs by contact with target cells. release from a small subset of internal stores that is tightly coupled to SOCs.

1129- Pos 1130 - Pos A CYTOSOLIC CALCIUM CLAMP FOR CONTINUOUS MEASUREMENT OF OXIDATIVE STRESS ACTIVATES TRP3 CATION CHANNELS CALCIUM ATPase ACTIVITY AND MODULATION IN SINGLE T CELLS. Klaus Groschner', B. Lintschinger2, C Romanin3, W. Schreibmayer4, Michael Xi Zhu5, M. Diana M Bautista, Richard S Lewis, Department of Molecular & Cellular Physiology, Stanford Balzer-Geldsetzer2, 'Dept. of Pharmacol. and Toxicol., Karl-Franzens University Graz, University Medical Center, 300 Pasteur, Stanford, California 94305 Universitltsplatz 2, Graz, Styria A-8010 Austria, 2Dept. of Pharmacol. and Toxicol., University of Following a rise in [Ca2]j, activation of extrusion via the plasma-membrane Ca2+-ATPase Graz, 3Institute for Biophysics, University of Linz, Austria, Altenbergerstr. 69, Linz, Austria, (PMCA) is biphasic, consisting of a rapid increase followed by a slow enhancement or 4Dept. of medical Physics, University of Graz, 5Neurobiotechnology Center, The Ohio State modulation. To quantitate the Ca2+sensitivity and kinetics of modulation in intact single cells, we University developed a Ca2 -clamp technique. Indo-l-loaded Jurkat T cells, pretreated with thapsigargin to Changes in the cellular redox state are typically associated with altered ion channel functions. In open CRAC channels, are voltage clamped in the perforated-patch mode while [Ca ]i is particular oxidative controlled in the stress-induced activation of nonselective cation currents has been through changes the holding potential. The Ca> clamp generates [Cas+] steps implicated in oxidant-induced cell depolarization and injury. The present study was designed to ranging from 300-1500 nM with risetimes of several seconds and duration >5 min. In T cells in test for a possible regulation of Trp3 cation channels by oxidative stress induced by the lipophilic which Ca2+uptake by the ER store and mitochondria is blocked, steady-state [Ca2]j is determined oxidant tert-butylhydroperoxide (tBHP) in the HEK293 expression system. HEK293 cells stably by the balance of Ca2+ influx through CRAC channels and efflux via PMCA. Thus, under expressing the Trp3 isoform displayed a moderate basal, nonselective cation conductance which constant-[Ca2+]i conditions IcRAC can be used to calculate the ongoing PMCA-mediated flux was significantly larger than that of vector transfected controls. Exposure of Trp3 expressing cells provided that (l) Ca2+ entry is only through CRAC channels and efflux is only via PMCA; (2) to 400 pM tBHP for one hour caused a increase in the cation PMCA is not and dramatic Trp3-mediated electrogenic; (3) PMCA is not voltage-dependent. Our experiments indicate that conductance, resulting in large Na' inward currents. This oxidant-induced cation conductance was all three conditions are met and validate the technique for measurement of PMCA activity. The not observed in vector-transfected control cells. Pretreatment of cells with the phospholipase C Ca2+ clamp will be used to measure Vmax and Km of Ca+ tranwsport by the unmodulated PMCA inhibitor and the U73122 (4 pM) prevented tBHP-induction of membrane currents in Trp3 expressing Ca` sensitivity and kinetics of pump modulation. cells. The oxidant-induced activation of Trp3 currents was mimicked by administration of 1- oleoyl-2-acetyl-sn-glycerol (100 ILM). We demonstrate that the Trp3 protein forms cation channels that are able to respond to oxidative stress. Oxidative stress-induced stimulation of cellular phospholipase C activity is suggested to serve as alink between the cellular redox state and the activity of Trp channels. Supported by the FWF, SFB Biomembranes F708 and F715, P12667 and P12728.

1131 - Pos 1132- Pos FUNCTIONAL INTERACTION BETWEEN TRPI AND TRP3 PROTEINS IN THE ARACHIDONIC ACID INHIBITS THE RECEPTOR-DEPENDENT AND STORE- HEK293 EXPRESSION SYSTEM DEPENDENT CAPACITATIVE Ca> INFLUX IN EHRLICH ASCITES TUMOR CELLS. Monlka Balzer-Geldsetzer', B. Lintschinger', C. Romanin2, X. Zhu3, K. Groschner4, 'Dept. of Elena N. Dedkova', Valery P. Zinchenko', Lothar A. Blatter2, 'Institute of Cell Biophysics, Pharmacol. and Toxicol., University of Graz, Universitatsplatz 2, Graz, Styria A-8010 Austria, Russian Academy of Sciences, Pushchino, Russia, 142292, 2Department of Physiology, Loyola 2Dept. of Biophysics, University of Linz, 3Dept. of Neurobiotechnol., Ohio State University, University Chicago, IL 60153, USA 4Dept. of Pharmacol. and Toxicol., Karl-Franzens University Graz, Universitgtsplatz 2, Graz, Styria A-8010Austria The initiation of many physiological processes, including activation of Ehrlich ascites tumor cells Variations in the isoform composition of Trp channel complexes are considered as the basis of functional (EATCs), is accompanied by arachidonic acid (AA) production. Using fluorescent Ca2+-sensitive diversity among mammalian Trp channels. Here we tested the hypothesis of a specific probe Fura-2 functional interaction between and isoforms in the effect of the AA on receptor-dependent and store-dependent capacitative Ca` Trpl Trp3 HEK293 cells. Trp currentsderived by influx in EATCs was We observed expression of either isoform alone or of both investigated. that extracellular application of AA (1-20 WM) by coexpression isoforms were characterized with the produced a of ATP-induced patch-clamp technique. Overexpression of Trpl alone did not affect basal membrane conductance concentration-dependent suppression receptor-dependent and store- dependent capacitative Ca2+ entry in EATCs. It was found the IC50for AA inhibition of these of HEK293 cells but gave rise to a diacylglycerol-induced, nonselective conductance which was blocked by extracellular Ca2 Overexpression of Trp3 alone resulted in basal- and diacylglycerol- processes were 9 and 6,5 pM, respectively. The suppression of Ca> entry was mostly mediated by induced conductances. The Trp3-mediated currents were suppressed by extracellular Ca2> but AA, rather then its metabolites. independent of intracellular CaW+buffering. Coexpression of Trpl and Trp3 resulted in large basal- We found, that application of low doses of classical uncoupler of oxidative phosphorylation, and diacylglycerol-stimulated conductances which were again suppressed by extracellular Ca2+. protonophore FCCP inhibited the Ca2+entry similar to AA. Therefore, we compared the action of Trpl/Trp3 currents were, in contrast to Trp3 currents, strongly inhibited when the efficiency of AA and FCCP on uncoupling of oxidative phosphorylation, measured by NAD(P)H fluorescence. intracellular Ca2' buffering was reduced by exchanging 10 mM BAPTA by 5 mM EGTA in the We shown, that the AA uncoupled the oxidative phosphorylation similar to action of low pipette solution. concentration of FCCP. The correlation between the degree of uncoupling of oxidative phosphorylation and of Ca2+ in both under the Our results indicate the formation of functional which are sensitive to blocking entry EATCs, action of AA, and under the Trp l/Trp3 heterooligomers action of low concentration of FCCP was observed. Possible connection between Ca2' diacylglycerols and display adistinct sensitivity to intracellular Ca2+.We suggest Trpl as a crucial entry deteminant of the Ca2+sensitivity of Trp channels. inhibition and degree of uncoupling of oxidative phosphorylation in EATCs is discussed. Supported by the FWF, SFB Biomembranes F708and F715, P12667 and P12728.

192A MONDAY STORE-OPERATED CA2+ 1133-1138

1133 - Pos 1134 - Pos TRP4 PROTEINS ARE PART OF ENDOGENOUS CRAC CHANNELS IN SBAC CELLS. INTERACTION OF hTrpl WITH LIPID-RAFT DOMAINS IN HUMAN Stephan Philipp', Claudia Trost', Jan Warnat', Julia Rautmann', Nina Himmerkus', Gregor SUBMANDIBULAR GLAND CELLS: IMPLICATIONS IN STORE-OPERATED Cal+ Schroth', Oliver Kretz2, Adolfo Cavalie', Veit Flockerzi', Markus Hoth3, 'Dept. of Pharmacology ENTRY. and Toxicology, 2Dept. of Anatomy, 3Dept. of Physiology, University of Saarland, Homburg-Saar, Timothy P Lockwich, Xibao Liu, Brij Singh, Indu Ambudkar, NIH, Building 10, Room IA05, 66421 Germany Bethesda, Maryland 20892 From heterologous expression studies mammalian TRP proteins have been implicated to form The store-operated Ca2+ entry mechanism (SOC), which is activated by internal Ca2+ store store-operated Ca2' (SOC) channels. However, the molecular structure of any of the native SOC depletion, is present in non-excitable cells and allows sustained Ca2+ entry into the cell. Little is channels is not known. Here we analyze if TRP4 proteins contribute to endogenous SOC channels known about the molecular nature of this mechanism. Certain members of the Trp family of in bovine adrenal cortical (SBAC) cells. Northern blot analysis, inmmunoblots and proteins (e.g. Trpl) have been implicated in SOC. In this study, we show that endogenous hTrpl inmnunohistochemistry reveal that TRP4 transcripts and TRP4 proteins are highly expressed in in a human submandibular gland cell line, HSG, is partitioned into sphingolipid-cholesterol SBAC cells. The only other transcript expressed at a significant level is TRPI whereas transcripts enriched areas within the plasma membrane known as lipid raft domains (LRD's) by of TRP2, TRP3, TRP5, and TRP6 were not detectable. Following Ca2+ store depletion by demonstrating; 1). insolubility of hTrpl in 1% Triton X-100 at 4°C, 2). presence of hTrpl in the perfusion of InsP3 or external application of thapsigargin during whole-cell patch-clamp low density fractions of an optiprep gradient, 3). increased solubility of hTrpl in Triton X-100 experiments, currents could be activated. The SOC channels in endogenous store-operated SBAC following treatment with ,B-cyclodextran to deplete membrane cholesterol, and 4) co- cells were found to be inwardly rectifying and highly Ca2+ selective excluding Na+ in the presence immunoprecipitation of hTrpl and caveolin I (a known LRD HSG cell of Ca2'. much resemble Ca2+ release-activated component) from plasma external Thus, they very Ca2'+ (CRAC) channels membrane. Additionally, treatment of the HSG cells with ,B attenuated SOC described in mast cells and of TRP4 cDNA in cyclodextran originally lymphocytes. Expression antisense stimulated by either carbachol or thapsigargin (to directly empty the internal stores). These results orientation significantly reduced both the endogenous CRAC currents (0.78 p 0.09 to 0.34 p 0.05) suggest that the association/recruitment of hTrpl into lipid raft domains is required for the SOC and the amount of endogenous protein as detected with a TRP4 specific antibody. In conclusion mechanism in HSG cells. our results demonstrate that TRP4 proteins contribute to CRAC channels in bovine adrenal cortical cells in vivo.

1135 - Pos 1136 -Pos CHARACTERIZATION OF IARC, A NOVEL ARACHIDONATE-REGULATED NON- STORE-OPERATED NON-SELECTIVE CATION CHANNELS DIRECTLY CAPACITATIVE Ca2'+ ENTRY PATHWAY ACTIVATED BY CALCIUM INFLUX FACTOR (CIF) IN SMOOTH MUSCLE CELLS. Trevor J Shuttleworth, Olivier Mignen, University of Rochester Medical Center, 601 Elmwood Elena S Trepakovai, P Csutora2, M Gericket, R B Marchase2, R A Cohen', V M Bolotina', Avenue, Rochester, NY 14642 'Boston University Medical Center, 88 East Newton Street, Boston, MA 02118, 2University of We have recently questioned whether the capacitative, or store-operated model for receptor- Alabama, Birmingham, AL 35294 activated Ca2+ entry demonstrates the appropriate properties to account for the influx of Ca2+ seen at physiological agonist concentrations, where oscillatory Ca2+ signals are produced. Instead, we Depletion of intracellular Ca2' stores in smooth muscle cells (SMC) activates store-operated Ca2+ have identified a non-capacitative Ca2+ entry pathway that is regulated by arachidonic acid (AA) influx. The nature of the channels and which to be mediating such influx and the mechanism of their activation are appears specifically responsible for Ca2+ entry under these conditions. Using still unclear. Here we describe native channels which be for whole-cell patch clamp techniques we have now characterized the Ca2+-conducting might responsible properties of the current store-operated Ca2+ influx in SMC, and test the hypothesis that they can be directly activated by associated with this Ca2+ entry pathway. In HEK293 cells, low concentrations of AA activate a current that CIF produced during depletion of Ca2+ stores. Using SMC from mouse aorta, we show that ('ARC) shows certain features in common with the archetypal store-operated current thapsigargin (TG) induces Ca2+ influx and ICRAC, as well as with the (using fura-2) non-selective cation current (using endogenous store-operated current (ISOC) of HEK293 cells. These patch-clamp). In cell-attached patches TG activates non-selective cation include a high selectivity for Ca2+, marked inward Ca2+-independent rectification, inhibition by La3+, and channels of 3.1±0.6 pS. The same channels of 3.3±0.6 could be in development of a monovalent permeability on removal of extracellular divalent pS activated inside-out cations. However, membrane patches from SMC CIF derived from pmrl IARC also demonstrates features that it from by yeast (mutants lacking reticular Ca2+- distinguish ICRAc including absence of fast- ATPase) or from TG-activated platelets. These channels were also activated by specific HPLC- inactivation, and insensitivity to reduction in extracellular pH. More importantly, Iac can be activated in purified CIF fraction from platelets which induces Ca2+ influx when injected into Xenopus readily cells whose Ca2+ stores have been maximally depleted (e.g. by treatment with oocytes. To CIF, thapsigargin). therefore a novel Ca2+ bioassay permeabilized platelets were applied to the inside-out membrane IARC represents entry pathway which is entirely distinct from patches. addition of TG caused slow of those activated by store depletion, and is specifically activated at physiological levels of Subsequent depletion platelet stores and activation of the stimulation. channels in thethepatches.in Ca~~~2+, TG,IP3,IP4,GTPyS,ATP,cGMP, cAMP did not stimulate the channels, and heparin did not prevent channel activation by CIF. Our data indicate that activation of native store-operated cation channels in SMC can be induced by cytosolic factor derived from other cell types, and does not require direct interaction with the endoplasmic reticulum.

1137 - Pos 1138 - Pos ACTIVATION OF CAPACITATIVE CALCIUM INFLUX: CONFORMATIONAL ELECTROPHYSIOLOGICAL ANALYSIS OF TRP3 CHANNEL ACTIVATED BY A TRP COUPLING? BINDING DOMAIN OF IP3 RECEPTOR Elena S Trepakova, Kasey Lai, Victoria M Bolotina, Boston University Medical Center, 88 East Michael Xl Zhu, Zongming Zhang, Neurobiotechnology Center, The Ohio State Newton Street, Boston, MA 02118 University Recent evidence indicates that human Trp3 expressed in HEK 293 cells is activated through Conformational coupling of channels with reticulum store-operated endoplasmic (ER) has been conformational coupling by IP3 receptors. Direct interactions between Trp and 1P3 receptors are proposed as a mechanism of activation of calcium key capacitative influx (CCI). Recently we demonstrated by coimmunopreciptation and in vitro binding assays. Furthermore, a small showed direct activation of native region store-operated channels by calcium influx factor (CIF) in from the carboxyl-terminus of Trp and two sites from the amino-terminus of IP3 receptor isolated membrane excised from have patches smooth muscle cells (SMC) which did not require been determined to confer the interaction. We synthesized a peptide, F2v, containing the first coupling of the channel to the stores. resolution Trp Using high imaging of individual cells we binding domain of the human type 3 IP3 receptor (E681 to D698) and tested its effect on demonstrate here that inhibition of CCI following treatment with Trp3 phosphatase inhibitor calyculin channel activities. In whole-cell recordings, intracellular perfusion with 5 mM EGTA elicits a A (calyA) does not correlate with ER a displacement by dense cortical layer of F-actin under slow appearance of a non-selective cation current in a nominally Ca2+-free bath membrane in mouse SMC solution. plasma aortic (in short term culture). Indeed, calyA inhibited Inclusion of F2v in the pipette solution leads to a faster development and two-fold increase of CCI the thapsigargin-induced by 70% reducing the peak Ca2+ rise from 633+118nM to 187+46nM current. The non-selective cation current displays a non-linear current-voltage relationship and is (n=25). However, it did not cause rearrangement of F-actin (stained with phalloidin/rhodamine) highly sensitive to increase in extracellular Ca2+. Chelating external EGTA results towards the Ca2' by in a plasma membrane. Instead, actin collapsed to the center of the cells forming further increase of the current and a linear current-voltage relationship in F2v perfused cells condensed bundles which did not interfere with ER but distribution beneath plasma membrane, as was not in cells perfused without peptide or with a control peptide that does not bind Trp. Thus, the monitored using ER-tracker. Moreover, in some control SMC (at early stages of their spreading on Trp binding domain of IP3 receptor interacts with Trp3 and induces a conformational change that the glass) F-actin formed a tight cortical layer beneath plasma membrane, which could displace facilitates the influx of Ca2'. ER from plasma membrane, but did not prevent normal CCI in response to TG. Thus, our data in SMC are inconsistent with the "conformational coupling" mechanism of regulation of CCI, although are compatible with the possibility of secretion-like delivery of CIF from the stores to plasma membrane.

193A 1139-1144 MITOCHONDRLAL STRUCTURE/FUNCTION MONDAY ~~ ~ ~ ~ MNA

1139 - Pos 1140 - Pos TRANSMON TO CUBIC MORPHOLOGY OF MITOCHONDRIAL INNER ROLE OF CRISTAE MORPHOLOGY IN REGULATING MITOCHONDRL4L MEMBRANES IN FASTING AMOEBA CORRELATES WITH ONSET OF CYANIDE- ADENINE NUCLEOTIDE AND H+ DYNAMICS. RESISTANT RESPIRATION. Ion I. Morarh', Boris M. Slepchenko', Carmen A. Mannella5, Leslie M. Loew', 'University of Deng, Dora D'Arcangelis, Carmen A. Mannella, Wadsworth Center, Empire State Plaza, Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3505, 2Wadsworth Box 509, Albany, NY 12201-0509 Center, Empire State Plaza, Box 509, Albany, NY 12201-0509 Mitochondria in the amoeba Chaos carolinensis undergo a dramatic change in intemal Mitochondrial morphology is highly dependent on the level of respiratory activity. It is still organization during fasting. The inner membrane infoldings (cristae) change from random tubular unclear whether the transition between the "orthodox" and "condensed" states plays a significant to ordered cubic morphology, starting within 1 day of fasting and increasing in frequency over role in regulating mitochondrial function or is just an epiphenomenon. Recent 3D reconstructions days (e.g. Deng and Mieczkowski, 1998, Protoplasma 203:16). Whole-cell polarographic from high voltage transmission electron micrographs provided new insights into cristae measurements indicate that the respiration of fed cells is fully inhibited by cyanide. Fasted cells morphology, suggesting that gradients in H+ concentrations may occur in the intracristae space. slower respiration after only I day, a significant fraction of which is cyanide resistant but We used the Virtual Cell modeling system to test this hypothesis. Both 2D and 3D models inhibited by salicyl hydroxamate (SHAM). Also, starved cells display considerable cyanide- showed neither steady state nor dynamic gradients in [HI] even at maximum pumping rates. sensitive oxygen generation upon addition of hydrogen peroxide, suggesting enhanced catalase However, the data suggested that significant gradients in adenine nucleotide concentrations, in activity. is possible that during starvation, Chaos switches from carbohydrate- to lipid-based particular ADP, may occur. We therefore included full kinetic models of the ATP/ADP antiporter energy sources, turns on protective mechanisms (nonphosphorylating respiration and catalase) and the FOF1 ATP synthase in the simulations. Depending on the number and size of intracristae formation of reactive oxygen species (Skulachev, 1996, Quart Rev Biophys 29:169). sacs, the system displayed a wide range of steady state spatial distributions in ADP concentrations, the paracrystalline transition in inner membrane morphology (currently being studied by both within the matrix and the intracristae space. Static and dynamic gradients in ADP tomography) may be due to expression of the SHAM-sensitive alternative oxidase or to concentrations were present, as high as 80% drops from cytosolic levels ([ADP],,,,., < 10 FM). lipid composition is under investigation. (Supported by CAMURUS Lipid Research Surprisingly, the consequence of the observed distributions is that the "condensed" state, Foundation, Sweden and NIH grant RR01219.) characteristic of highly active mitochondria, appears to be energetically less efficient, having a significant amount of inner membrane surface working under conditions unfavorable to ATP synthesis. However, this state is much more responsive to large changes in energy demands by the cytosol. (Supported by NIH grants GM35063 and RRI3 186)

Pos 1142 - Pos NEGATIVE CONTRAST IMAGING OF MITOCHONDRIA BY CONFOCAL STRUCTURAL MODIFICATIONS OF THE TWO PROTEIN IMPORT COMPLEXES MICROSCOPY OF MITOCHONDRIA CAUSE FUNCTIONAL ALTERATIONS OF TIM AND TOM Ting Qian, John J. Lemasters, University of North Carolina at Chapel Hill CHANNEL ACTIVITIES. appear as dark voids in confocal images of calcein-loaded hepatocytes. These voids Sonia Martinez-Caballero', C. Muro2, Kathleen W Kinnally2, Maria Luisa Campo', 'U. disappear after onset of the mitochondrial permeability transition (MPT). Here our aims were to: Extremadura, Caceres, Spain, 2Wadsworth Center 1) assess if the voids are due to fluorescence quenching, 2) determine what factors influence the The TOM and TIM complexes of the outer and inner mitochondrial membranes mediate the appearance of dark voids, and 3) develop a new approach to positively label mitochondria too import of proteins to mitochondria, respectively. Patch-clamping experiments reveal two channels small image by negative contrast in order to visualize the MPT. One-day cultured rat in mitochondria whose activities are regulated by synthetic signal peptides. The sequences of these hepatocytes were loaded with calcein AM (1 ,M) for 15 min at 370C. Confocal imaging revealed signal peptides correspond to the targeting regions of mitochondrial precursor proteins. Data will mitochondrial voids. Subsequent addition of TMRM (250 nM) produced red mitochondrial be presented that corroborates the identification of the two channel activities, previously named fluorescence not change the dark voids in the calcein fluorescence. Hepatocytes were also MCC and PSC, as the protein translocation pores of the TIM and TOM complexes, respectively. calcein AM at 4°C for 60 min. After cold ester-loading, calcein was distributed The concentrations of two signal peptides (yCOXIV51.t3)and yCOXIV(5.22)) needed to modify the uniformly in the cytosol and the mitochondria, and voids were absent. Subsequent TMRM did not flickering rate (transitions/sec) of the Tom and Tim channel activities, were determined in cause voids to appear. Dark voids were also observed for 1-Am latex beads suspended in calcein- proteoliposomes containing yeast mitochondrial outer and inner membrane. This peptide containing agar. Misalignment of the confocal pinhole caused the voids to disappear. A cold ester- sensitivity was then used as a functional assay to evaluate the effect of structural changes in the loading/warm incubation procedure produced positive labeling of mitochondria with calcein, TOM and TIM complexeson the channel activities. Various protocols of proteolytic digestion of was not quenched by TMRM and was lost after the MPT. In conclusion, negative contrast mitochondria and mitoplasts prior to reconstitution were used to generate structural changes in the imaging of mitochondria is not an artifact but requires warm loading of calcein AM and complexes. Loss of Tom22p, but not Tom20p or Tom7Op components, significantly reduced the optimal microscope alignment. A cold loading/warm incubation protocol can also be used to label peptide sensitivity of the Tom channel. These results indicate Tom22p is the receptor of the outer mitochondria too small for negative contrast imaging. membrane for signal peptides that are cationic and alpha-helical. In parallel studies, proteolytic digestion of the TIM complex that altered Tim23p, but not several other components (Tim44p, Timl7p, Hsp7O), rendered the Tim channel virtually insensitive to signal peptides. This finding indicates Tim23p is required for normal Tim channel activity and that Tim23p is the receptor of the Tim complex for these signal peptides. Supported by DGICYT PB95-456 and JE IPR98C038 to MLC, and NSF MCB9513439 and MCB-9816950 to KWK.

Pos 1144 - Pos HETEROGENEITY OF CARDIAC MITOCHONDRIAL MEMBRANE POTENTIAL: FATTY ACIDS ACTIVATE CALCIUM EFFLUX FROM YEAST MITOCHONDRIA REGULATION BY RETRO-RETINOIDS. Patrick C. Bradshaw, Dennis W. Jung, Douglas R. Pfeiffer, Ohio State University Irina Korichneva, U. Hammerling, Memorial sloan-Kettering Cancer Center, 1275 York Avenue, Isolated mitochondria from the yeast Saccharomyces cerevisiae degrade their York, NY 10021 phospholipids by a constitutive process and accumulate free fatty acids at a rate of -0.3 disfunction, a key component of apoptotic program in many cell types is important nmol/min/mg protein. While lacking a Ca2+uniporter, these mitochondria accumulate and retain apoptosis as well. The disruption of the mitochondrial membrane potential ( AT. ) Ca2' via the electrogenic ionophore ETH129, but only in media containing BSA. When ETH129 following the opening of permeability transition pore relates to the release of the substances is absent, membrane potential (AT) is similar in the presence and absence of BSA, as determined activating caspases, to the generation of reactive oxygen species (ROS), and to the uncoupling of by the safranin technique. This finding and actions of fatty acids in the presence of phosphorylation. We used a dual-emission potential-sensitive probe 5,5',6,6'-tetrachloro- carboxyatractyloside indicate that uncoupling by fatty acids does not explain the BSA requirement 1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and confocal laser scanning for Ca2+accumulation. When BSA is present, A' decreases slightly during Ca2+accumulation to monitor A't in microscopy primary culture of neonatal rat cardiac cells. Cardiac myocytes had and recoverswhen accumulation is complete. The decrease is marked when BSA is absent and no higher ATn, compared to fibroblasts from the same culture.The mitochondria of individual recovery is seen unless BSA or a Ca2+ chelator is subsequently added. The electroneutral myocyte displayed heterogeneity with both high and low AT.. Anhydroretinol (AR), a ionophore ionomycin does not substitute for ETH129 to depress ATwhen BSA is absent, while proapoptotic retro-retinoid, induced at micromolar doses the decrease in cardiac AT. within other cationstransported by ETH129 will substitute for Ca2+in this regard. These findings However, the hydroxylated molecules, retinol and 14-hydroxy-retro-retinol (14HRR) indicate that ETHI29 and an endogenous cation release carrier create a futile cycle of Ca"+ known to promote cell survival did not affect AT.. In retinol-depleted cells, then accumulation and release that depresses A'. The endogenous carrier is activated by small reconstituted with either retinol or AR, or left untreated, once again basal AT.' was significantly amounts of free fatty acids derived from endogenous phospholipids, accepts Ca2+but is somewhat AR-loaded myocytes as compared to control and retinol-loaded cells. Like AR, oxidative nonspecific, and is of high activity. (Supported by the American Heart Association and the hydrogen peroxide (H202) depolarized cardiac mitochondrial membrane within Wallace Research Foundation). effect was reversed by retinol and augmented by AR. We discuss the possible by which retinoids control mitochondrial function, and the role of mitochondria in damage in cardiac tissue.

194A MONDAY MITOCHONDRIAL STRUCTURE/FUNCTION 1145-1150

1145- Pos 1146 - Pos SIGNAL PRESEQUENCES INDUCE A PERMEABILITY TRANSITION AND CASPASE CLEAVAGE PROMOTES BCL-XL-DEPENDENT PORE FORMATION CYTOCHROME C RELEASE IN ISOLATED LIVER AND BRAIN MITOCHONDRIA Gorka Basalez', Jun Zhang2, B. Nelson Chau2, Vadim A. Frolov, Grigory I. Maksaev3, J. AND PERMEABILIZED NEURAL CELLS. Demetria Galanis', J. Marie Hardwick2, Joshua Zimmerberg', 'LCMB, NICHD, NIH, 10 Center YulIa E. Kushnareval, Brian M. Polster', Gary Fiskum', Patricia M. Sokolove', Kathleen W. Drive, Bethesda, MD 20892-1855, 2John Hopkins Schools of Public Health and Medicine, Kinnally2, 'University of Maryland School of Medicine, 2Wadsworth Center, New York State Baltimore, MD, 21205, 3Frumkin Institute of Electrochemistry, RAS, Moscow Department of Health The cyclosporin A (CsA)-inhibitable mitochondrial permeability transition (MPT) has been Bcl-XL is an anti-apoptotic member of the Bcl-2 protein family, which localizes to the outer implicated in the release of cytochrome c (CytC) which triggers apoptosis. Mitochondrial signal mitochondrial membrane and has been reported to form ion channels. Caspases can cleave Bcl-XL presequences have been reported to induce a permeability transition in isolated liver mitochondria releasing a carboxyl-terminal fragment (tN76Bcl-xL) with pro-death activity. In the present study, that is distinct from the classical CsA-sensitive we MPT. Here compared the effects of signal we used phospholipid bilayer membranes to study the membrane-perturbing effect of AN76Bcl-xL, peptides on the permeability of mitochondria from different sources and tested the hypothesis that full-length BCI-XL and a throughout construct lacking a putative membrane-anchoring domain, these physiological effectors can trigger CytC release. Our data demonstrate that: (1) Under induces much more than the physiologically relevant conditions, human cytochrome oxidase subunit IV signal peptide induces AC2OBcl-xL. AN76Bcl-xL liposome leakage potently full-length protein over a broad pH range, despite similar binding of the two proteins to phospholipid swelling, a decrease in the membrane potential and CytC release in mitochondria isolated from vesicles. at liver and brain and in digitonin-permeabilized PC12, GTI-7 and SYSY cells; however, brain AC20Bcl-xL only forms pores low pH, and is severely impaired in binding liposomes at In but not induces mitochondria and permeabilized cell mitochondria exhibit less swelling than liver mitochondria. physiological pH. planar lipid membranes, AN76Bcl-xL, Bcl-XL, iepgular (2) CytC efflux is observed when swelling conductance changes and decreases planar membrane lifetime. This effect is similar to the one is suppressed by polyethylene glycol (MW 3000) observed with Bax that both suggesting that a specific mechanism rather than disruption of the outer membrane is involved in suggesting proteins may perturb lipid bilayers by the same the release of CytC. (3) Dibucaine and propranolol inhibit membrane depolarization, swelling, and mechanism. Interestingly, the membrane lesion caused by AN76Bcl-xL is large enough to allow for the of c. CytC release, while CsA, EGTA and bongkrekic acid are without effect. Signal peptides can passage high molecular weight dextrans and cytochrome Furthermore, when therefore evoke CytC release by a mechanism that is independent from the classic MPT. (Support: overexpressed, AN76BcI-xL also induces cytochrome c release from mitochondria of BHK cells. NS34152 to G.F. and GM57429 to K.W.K.) Taken together, these results indicate that caspase cleavage releases a latent pore-forming function of Bcl-XL. The pore-forming function is likely responsible for its pro-apoptotic phenotype and may serve to amplify the apoptotic cascade.

1147- Pos 1148- Pos THE MITOCHONDRIAL PERMEABILITY TRANSITION, HYPERCONTRACTURE Ca2'+NDUCED MITOCHONDRIAL APOPTOSIS IN CARDIAC CELLS AND CELL DEATH IN ISOLATED CARDIAC MYOCYTES. Pal Pacher, Gyorgy Hajnoczky, Thomas Jefferson university Paavo Korge, James N Weiss, UCLA School of Medicine, 675 Young Dr MRL, South, 3-645, We previously showed that mitochondrial matrix evoked Ca2+ pulses or Los Angeles, CA 90095-1760 [Ca2j] signals by by IP3- mediated cytosolic [Ca2+] ([Ca2+],) spikes trigger opening of the permeability transition pore During reperfusion or reoxygenation, cardiac myocytes undergo hypercontracture and cell death. (PTP) and in tum activate the apoptotic machinery in HepG2 cells exposed to proapoptotic agents. We examined whether hypercontracture and cell death could be caused by mitochondria Similar mechanisms could be particularly important in the heart since large elevations of [Ca2+]c undergoing the mitochondrial permeability transition (MPT) by testing the effects of the MPT- occur during each heartbeat in the myocytes. Thus, we studied the effects of proapoptotic agents inducer phenylarsine oxide (PAO). Isolated rabbit ventricular myocytes were loaded with calcein (ceramide(C2) and ethanol(EtOH)) on mitochondrial membrane potential (AT.) responses given at low temperature followed by warm treatment to isnage mitochondria or with Rhod-2 at 37°C to to [Ca2j], signals in permeabilized H9C2 myotubes. AT,,, and [Ca2+]c were simultaneously measure cytoplasmic Ca2' and studied with confocal microscopy. PAO promoted near measured using TMRE and fluo3 confocal imaging. synchronous MPT (detected by calcein efflux from mitochondria), mitochondrial Ca2' efflux Preincubation of the cells with C2 or (detected by Rhod-2), severe hypercontracture, and LDH release indicating (5 min) EtOH (48h) did not change the steady state AT. or irreversible but Ca2+-induced Ruthenium red sarcolemmal injury. The myofilament uncoupler 2,3-butanedione monoxime or prior [Ca2'], potentiated depolarization. prevented and cyclosporin-A ATP inhibited the Ca2+-induced in C2 and EtOH cells. depletion with FCCP or antimycin A prevented hypercontracture, but not cell death. BAPTA-AM largely depolarizations pretreated Reversal of Ca +-induced was achieved when a Ca EGTA was and the MPT inhibitor cyclosporin A or reagents preventing disulfid formation N-ethylmaleimide depolarization 2+-chelator, administered. Interestingly, Ca`+-induced depolarization appeared as a wave in most of the and dithiothreitol delayed or prevented both. In isolated cardiac mitochondria, PAO promoted propagating Waves of were also MPT and Ca2+ efflux under comparable conditions. Thus, near-synchronous induction of MPT myotubes. depolarization observed in cells pretreated with thapsigargin and to the reticular Ca2+ stores. releases sufficient Ca2+ from mitochondria to 1) cause rapid hypercontracture provided that caffeine/ryanodine deplete sufficient ATP for cross-bridge cycling is available, and 2) promote irreversible sarcolemmal These results suggest that [Ca2+] spikes may lead to a transient activation of PTP in permeabilized injury by a Ca2+ -dependent mechanism unrelated to hypercontracture. During reperfusion or myotubes exposed to apoptotic agents. Organization of PTP opening into waves may utilize a reoxygenation, when conditions promoting MPT are present, this may be an important mechanism factor released from the mitochondria. of cardiac injury.

1149 Pos 1150 - Pos CHANGES IN PERMEABILITY OF MITOCHONDRIAL Ca2'+ UPTAKE SITES DURING LOCAL CALCIUM SIGNALING BETWEEN RYANODINE RECEPTORS AND IP3-INDUCED Ca'+ RELEASE MITOCHONDRIA IN HEART CELLS Gyorgy Csordas, Gyorgy Hajnoczky, Thomas Jefferson university Pal Pacher', Gabor Szalai', Gyorgy Csordas', Hantash M Basil2, Andrew P Thomas2, Gyorgy Mitochondrial Ca2+ uptake sites (uniporter) are activated by high local [Ca2+] in the junctions of Hajnoczky', 'Thomas Jefferson university, 2UMDNJ - New Jersey Medical School ER and mitochondria during IP3 receptor-mediated Ca2+ signals, but the mechanism underlying the Cytosolic [Ca2'] ([Ca2'].) signals relayed to the mitochondria may play a fundamental role in activation is still not understood. coordination of ATP supply with energy demand in the heart. Here we study the organization of To probe the permeability of the uniporter we established a method using Mn2+-quenching of the Ca2+ coupling between ryanodine receptor Ca2+ release channels (RyR) and mitochondrial Ca2+ fura2FF compartmentalized in mitochondria in mast We permeabilized cells. determined that the uptake sites. We use differentiated cardiac myoblasts (H9c2) that we find to express L-type Ca2+- low affinity Ca2+-tracer fura2FF (Kd=35pM) is just twice less sensitive to Mn2+-quenching than channels (cardiac and skeletal muscle) and RyR. Confocal and fluorescence imaging of fura2 of Therefore, mitochondria [Ca2+],, (ECso

195A 111S-1156 MEMBRANEMEMEBRANE TRANSPORT MONDAY

1151 - Pos 1152 - Pos TRANSITION STATE ANALYSIS OF THE COUPLING OF DRUG TRANSPORT TO MODULATION OF K+ TRANSPORT IN YEAST MITOCHONDRIA. ATP HYDROLYSIS BY P-GLYCOPROTEIN Vicente Castrej6n, Antonio Pefla, Salvador Uribe, Inst de Fisio Celular. Dept. Bioquim. Univ Marwan K Al-Shawl, Mark K. Polar, Robert A. Figler, University of Virginia Health Sciences Nacional Autonoma de M6xico (UNAM). M6xico Center, Dept. Molecular Physiology, 1300 Jefferson Park Avenue, Charlottesville, VA 22908 K+ is the most abundant cation in mitochondrial matrix. In yeast mitochondria K+ increases Steady state thermodynamic analyses of ATP hydrolysis in the absence and presence of transport oxygen consumption and increasea the FlFOATPase activity. In mammalian mitochondria K+ substrates were performed using purified human and Chinese hamster P-glycoproteins (P-gps). enters trough an uniport and exits through an antiport K+/H+; Mg++ inhibits both systems. Between 23-35 °C, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis Quinine inhibit the uniport partially and the antiport strongly. In yeast mitochondria using ethanol of saturating MgATP (k,,,) from which we calculated the enthalpic, entropic and free energy terms as substrate, the modulation by Mg++, quinine and polyvalent anions (Pi, AsO4 and S04) on the for the rate limiting transition state. Linearity of the plots indicated that the same rate limiting step K+ transport was studied. Swelling and 86Rb uptake was evaluated. Swelling and 86Rb uptake was being measured over the temperature range employed. This conclusion was verified through were inhibited by FCCP. Swelling but not the transport of 86Rb was inhibited by 4 mM Pi in a the utilization of Linear Free Energy Relationship (LFER) analysis. We established that there was mersalyl sensitive fashion. At 4 mM Pi 86Rb transport was inhibited by Mg++. However, the a single LFER for drug activation, indicating that the steady-state transition state is common to inhibition on the 86Rb transport and swelling in mitochondria incubated in 0.4 mM Pi, was seen both P-gps and to all the transport drugs employed. In the absence of drugs, uncoupled ATP only at high concentrations of Mg++. To inhibit 86Rb transport and swelling higher hydrolysis had different LFERs for human and hamster P-gps which were also different from the concentrations of quinine were necessary when 0.4 mM Pi was usedthan at 4 mM Pi. Polyvalent coupled LFER above. It is concluded that basal ATPase activity associated with P-gp is not a anions like AsO4 and S04 modulate K+ permeability like Pi. This effect was not observed with consequence of an endogenous lipid or other endogenous substrate, rather, it is an intrinsic S03 or N03. The results suggest that in yeast mitochondria 86Rb transport is energy dependent mechanistic property of the enzyme. The coupling mechanism of the colchicine selected human and that this transport is similar to the system found in mammalian mitochondria in its sensitivity mutant G185V was also probed by thermodynamic analysis. We confirmed that the G185V to quinine, Mg+H and mersalyl. However at low Pi concentration K+ transport exhibits a different enzyme had altered ATPase activity. Supported by PHS grant GM52502. behaviour, probably due to channel opening.

1153 - Pos 1154 - Pos FT-IR ANALYSIS OF THE BACTERIAL MEMBRANE TRANSPORT PROTEIN- COPPER BINDING TO THE HUMAN COPPER-TRANSPORTING ATPASES: LACTOSE PERMEASE OF ESCHERICHIA COLI STRUCTURAL BASIS AND PHYSIOLOGICAL IMLICATIONS. Jason S. Patzlaff, Robert J. Brooker, Bridgette A Barry, University of Minnesota, 1479 Gortner Matthew J. Cooper', Martina Ralle2, Ninian J. Blackburn2, Hans-Peter Blchinger3, Svetlana Lab, St Paul, Minnesota 55108 Lutsenko', 'Oregon Health Sciences University, Biochemistry and Molecular Bio, 3181 SW Sam The lactose permease is an integral membrane transport protein that functions as a proton and Jackson Park Road, Pordand, OR 97201, 2Oregon Graduate Institute, 3Shriners Hospital lactose symporter. The protein translocates lactose and protons across the cell membrane with a Wilson's and Menkes' disease proteins are human copper-transporting P-type ATPases which play 1:1 stoichiometry. Lactose permease catalyzes the accumulation of lactose against a concentration key roles in intracellular distribution of copper and in regulation of copper metabolism. Both of gradient using the free energy contained within a proton electrochemical gradient as a driving these proteins contain a large N-terminal domain composed of six repetitive sequences of about 70 force. Lactose permease is encoded by the lacY gene of Escherichia coli and consists of 417 residues, each of which includes the conserved metal-binding motif GMTCXXC. We have amino acid residues. In previous studies we have used FT-IR spectroscopy to analyze a purified expressed and purified the N-terminal domains of Wilson's and Menkes' disease proteins as monodisperse lactose permease showing it is stably folded, a-helical and highly accessible to maltose-binding protein and His-Tag fiusions. Using these preparations we have demonstrated that deuterium exchange (Patzlaff, J. S., Moeller, J. A., Barry, B. A., and Brooker R. J. Biochemistry, the N-terminal domain of each protein binds copper specifically, with a stoichiometry of about 6 1998, 37, 15363-15375). Difference FT-IR spectroscopy has been shown to be a useful tool for copper per domain. X-ray absorption spectroscopy indicates that copper is bound in reduced form the study of protonation and electron transfer events in a variety of membrane proteins. Currently via a novel 2-coordinate copper(I)-cysteinate configuration. Circular dichroism spectroscopy we are using difference FT-IR spectroscopy to investigate the mechanism(s) of proton and lactose reveals that the copper-binding domain is well-folded and composed of the following secondary translocation performed by the lactose permease (Supported by NIH GM53259 and GM08347- structure elements: 19.5% a-helix, 26% n-sheet, 21% 3-turn, and 33.5% random coil. Further, 09). we demonstrate in situ that addition of as little as I FM copper to cultured cells induces "hyperphosphorylation" of Wilson's disease protein, which is reversed when copper is removed. This copper-dependent phosphorylation is associated with redistribution of Wilson's disease protein from the trans-Golgi network into vesicular compartments, and constitutes an important element of a copper sigpaling pathway. Supported by NIH grant DK55719 to S.L.

1155- Pos 1156- Pos NEURONAL VESICULAR UPTAKE OF MEMBRANE-PERMEANT SUBSTRATES AND CREATINE KINASE IN HUMAN RETINAL PIGMENT EPITHELIUM THEIR APPLICABILITY AS CARDIAC SYMPATHETIC PET TRACERS. Brian G Kennedy', Nancy J Mangini2, 'Indiana University School of Medicine, 3400 Broadway, Victor G Romanenko, Rani Gebara, David Njus, Wayne State University, 5407 Gullen Mall, Gary, IN 46408, 2University of Illinois at Chicago College of Medicine Detroit, MI 48202 The retinal pigment epithelium (RPE) functions as the blood retinal barrier, controlling water, ion and nutrient transport between the sensory retina and its choroidal blood supply. RPE transport Meta-hydroxyephedrine (mHED) and phenylephrine are structurally similar to natura activity and consequent ATP consumption, vary with retinal light exposure. Since creatine kinase catecholamine neurotrammitters and can be transported into noradrenergic synaptic and secretory (CK) is lmown to function as part of a critical energy buffering system in tissues with high yet vesicles. The relatively high hydrophobicity of these neurotransmitter analogues makes them fluctuating energy demands, the present work was designed to assess the importance of CK in significantly membrane-permeant, however, and attenuates their accumulation in the neuronal human RPE (HRPE) energy metabolism. The bulk of CK activity in HRPE, determined both by vesicles. Using an in vitro method, we evaluated membrane permeability and vesicular uptake antibody inhibition studies and non-denaturing electrophoresis, is mediated by the so-called brain parameters of phenylephrine and three stereoisomers of mHED. All the molecules appeared to be (B-CK) isoform. However, by immunoblotting, immunofluorescence and non-denaturing good substrates of the vesicular monoamine transporter with Km's comparable to and Vmax's electrophoresis the muscle (M-CK) isoform was also detected. By several measures, the M-CK somewhat lower than that of natural catecholamine neurotransmitters. The combination of high isoform was found to be membrane associated. Furthermore, it was localized, predominantly, at specificity for the target neuronal vesicular pool and moderate permeability through cell the apical surface of the RPE cell. Physiologically, phosphocreatine could preferentially fuel membranes makes the studied compounds suitable for use as positron emission tomography (PET) plasma membrane Ca transport activity, suggesting a possible coupling between CK and tracers for dynamic visualization of the cardiac sympathetic nervous system. For phenylephrine CaATPase on the plasma membrane. (Supported by NIH RO1 EY-11308 and by a grant from and the mHEDs, we conclude that the vesicular pool will contribute less to the total uptake Fight for Sight, Research Division of Prevent Blindness America). capacity of heart nerve tissue than in the case of norepinephrine. This confirms a hypothesis made by DeGrado et al. (J. Nucl. Med. (1993), 34, 1287-1293) on the basis of PET imaging of perfused rat hearts. Quantitative analysis of PET data is complicated by the many compartments into which the tracers may partition. Our method can be applied to determine uptake and clearance rate constants for the compartmental analysis of PET tracers and potentially to determine how different tracers assess different cardiac functions.

196A MONDAY MEMBRANE TRANSPORT 1157-1158

1157 Pos 1158- Pos ACUTE EFFECTS OF THE NEUROTOXIN 3-NITROPROPIONIC ACID ON CREATINE OLIGOMERIC STATE OF WILD-TYPE AND CYSTEINE-LESS YEAST AND TAURINE TRANSPORT. Hemanta K Sarkar, Shyamala Vinnakota, and Cynthia MITOCHONDRIAL CITRATE TRANSPORT PROTEINS. Edwards. Baylor College of Medicine, Houston, Texas 77030. Ronald S. Kaplan, R. Kotaria, J. A. Mayor, FUHS/The Chicago Medical School, 3333 Green Hemanta K. Sarkar, Shyamala Vinnakota, Cynthia Edwards, Baylor College of Medicine, One Bay Road, North Chicago, IL 60064 Baylor Plaza, Houston, Texas 77030 Experiments have been conducted to determine the oligomeric state of the mitochondrial citrate In animal models, systemic chronic injection of 3-nitropropionic acid (3-NP) results in transport protein (CTP) from the yeast Saccharomyces cerevisiae. Both wild-type and cysteine- neurodegeneration similar to that found in the brain of Huntington's disease afflicted patients. less CTPs were overexpressed in E. coli and solubilized with sarkosyl. The purity of the The 3-NP is thought to impair energy production by inhibifing the funcfion of the mitochondrial solubilized material is approx. 75%. Upon incorporation into phospholipid vesicles a high specific enzyme succinate dehydrogenase, which subsequently depletes cellular ATP levels and leads to transport activity was obtained with both the wild-type and Cys-less CTPs, thereby demonstrating excitotoxicity. Since the chemical structure of 3-NP is very similar to 0-guanidinopropionic acid, the structural and functional integrity of the preparations. Two independent approaches were an inhibitor of both creatine and taurine transport, we examined the effects of 3-NP on the utilized for determination of native molecular weight. First, native gel electrophoresis was carried endogenous creatine and taurine uptake in cultured cells. out in the presence of a low concentration of the negatively charged detergent sarkosyl in order to: Addition of 3-NP during uptake studies inhibited both Nae/Clr-dependent creatine and taurine i) impart a charge shift to the CTP and the protein standards; and ii) to promote protein solubility. uptake in a dose-dependent manner with IC50 values of 3.1 mM and 2.2 mM, respectively. Further Second, CTP molecular weight was determined via non-denaturing size-exclusion analyses showed that while the creatine uptake was inhibited non-competitively by 3-NP, the chromatography. Both methods clearly indicate that following solubilization, both the wild-type taurine uptake was inhibited in a competitive manner. In parallel experiments, 3-NP did not alter and Cys-less CTPs exist exclusively as dimers. This finding has important implications for the the glucose uptake mediated by the facilitated glucose transporter. Our results shed new insights structural basis underlying the CTP translocation mechanism which will be discussed. Supported into the mechanism underlying the mode of action of 3-NP. [HKS thanks USDA/ARS (No. 6250- by NIH grant GM54642 to R.S.K. 51000-033) for partial support].

MONDAY OXIDATIVE iPHOSP'HORYLATION 1159-1162

1159 - Pos 1160 - Pos IN SILICO REGULATION OF OXIDATIVE PHOSPHORYLATION IN MUSCLE CELLS SITE-DIRECTED SPIN LABELING OF THE y SUBUNIT OF Escherichla coli FoFt ATP IN HEALTH AND DISEASE SYNTHASE Olav Kongas', Marko Vendelin2, Valdur Saks3, 'Institute of Cybernetics at Tallinn Technical Scott H. Andrews, Vincent B. Herlihy, Robert K. Nakamoto, Molecular Physiology & Biological University, Akadeemia 21, Tallinn, 12618 Estonia, 2Institute of Cybernetics, Tallinn Technical Physics, University of Virginia, PO Box 1001 1, Charlottesville, VA 22906-0011 University, Akadeemia 21, Tallinn, 12618 Estonia, 3Laboratory of Bioenergetics, Joseph Fourier The F0F, ATP synthase uses a rotational mechanism to couple translocation of protons to University synthesis or hydrolysis of ATP. A segment of the y subunit which interacts with e and c subunits During the last decades, the mechanism of regulation of ATP synthesis in the mitochondria to is critical for energy coupling between the soluble catalytic F, sector and the membranous balance ATP consumption in cardiac myofibrils has been much debated. The possible candidates transport Fo sector. Mutations, such as yGlu2O8, disrupt the coupling interactions. The crystal are the parallel activation and the feedback regulation mechanisms. The aim of this presentation is structure of the bovine mitochondrial F, (Abrahams, J.P., Leslie, A.G.W., Lutter, R., Walker, J.E. to demonstrate by mathematical modeling that it is not necessary to introduce the parallel Nature 370, 621-628, 1994) lacks most of the y subunit, including the portion involved in activation mechanism to keep myocytes metabolically stable. We have built a reaction-diffusion coupling, and all of the subunit. In order to provide structure and molecular dynamic model describing transport, and energy production, consumption of the high phosphates in information, we have begun a site-directed spin labeling analysis of this critical portion of the myocytes. The model has been tested comparing the simulation results with experimental data; it enzyme. describes quantitatively: (a) respiration activation with ADP and creatine in skinned cardiac fibers; Single cysteine mutations are being introduced between yHis200 to and (b) metabolite fluxes and metabolic stability over five times of workload change in perfused yAla236 expressed in a cysteine-less background (Kuo, P.H., Ketchum, C.J., Nakamoto, R.K. FEBS Lett. hearts; (c) linear dependence of respiration rate on workload and fall of maximum respiration rate 426, were from 160 to 46 mmol/kg dw/min (rat heart) by inhibiting creatine kinase. We conclude that the 217-220, 1998). yS202C and yD204C modified by methanethiosulfonate nitroxide. The EPR spectra indicate mobile, solution a requirement for the metabolic stability can be met by the feedback regulation over a wide range of accessible side chains at these positions, suggesting location along the periphery of a structure. respiration rates. The cytoplasmic factors mostly influencing the respiration rate are inorganic globular Future experiments will provide information phosphate and substrates for creatine kinase and adenylate kinase systems dependending on the on the dynasnics of y subunit interactions with e and Fo subunits. This work was supported by metabolic, functional, and enzymatic states of the cell. PHS Grant GM-50957. S.H.A. is the recipient of a CVRC Post-Doctoral Fellowship, NIH HL07284.

1161 - Pos 1162- Pos METABOLIC CHANNELLING IN CARDIAC CELLS: DEPENDENCE OF KINETICS pH EFFECTS ON Na+ INITIATED DECREASE OF RESPIRATION AND OXIDATIVE OF RESPIRATION REGULATION ON SOURCE OF ADP. PHOSPHORYLATION IN CONTROL AND DIABETIC HEART MITOCHONDRIA. Valdur A. Saks', T. Tiivel', P. Sikk2, T. Kaambre2, L. Kay2, M. Tonkonogi2, E. Seppet2, K. Andrly M Babskyl', Nicolai M Doliba', Natalya M Doliba', Andrey B Savchenko', Suzanne L Sahlin2, 'J. Fourier Univ. & Institute of Chem. Biol. Phys. Tallinn, Estonia, 2Univ. of Tartu, Wehrli2, Mary D Osbakken', 'University of Pennsylvania, 352 Anatomy/Chemistry Bldg., 37th & Estonia, Karolinska Inst., Stockhoim, Sweden Hamilton Walk, Philadelphia, PA 19104, 5Childrens Hospital of Pennsylvania We find that Na+ (1-10 mM) can decrease State 3 respiration and Rate of Oxidative Mechanism of regulation of mitochondrial respiration was studied in permeabilized cardiac cells Phosphorylation (ROP) in isolated heart mitochondria (Mito) and that Diabetic Mellitus (DM) by using oxygraphy in combination with HPLC to assay adenine nucleotides in the medium. Mito are more sensitive to Na+ than Control (Con) Mito. The goal of this work was to determine Oxidative phosphorylation in mitochondria was supported by ADP generated by two different the optimum pH for the Nae effect and to evaluate effects of H+ change on Na+ induced depression systems: (I) exogenous ADP was added in different initial concentrations in presence of of bioenergetics in Con and DM Mito. Mito were isolated from hearts of Con and streptozotocin- hexokinase (4 IU/ml) and glucose (11 mM); (II) endogenous ADP was produced by intracellular induced (4 weeks) DM rats. Mito respiration and ADP phosphorylation were studied using routine ATPases activated by adding ATP in different concentrations. In both systems experiments were polarographic techniques with ca-ketoglutarate as substrate. We exposed isolated mitochondria to carried out in absence and in presence of creatine (Cr, 20 mM). Apparent Km for ADP in the different H+ levels by changing pH (6.7, 7.0, 7.2, 7.4, 7.6). In CON, the largest effects of Na+ were medium in regulation of respiration rate was calculated. Values were strikingly different: in (I) found at buffer pH=7.4; State 3 decreased 19% (p<0.05); ROP decreased 18.3% (p<0.05). Km(ADP) was 341 ± 59 FM, while in (II) it was 46 ± 5. In (I) addition of Cr decreased the Km Increasing pH above 7.4 did not lead to significant increases in the Na+ effect. On the other hand, (ADP) by factor of 2-3. In presence of pyruvate kinase and phosphoenolpyruvate in (II) ADP was decreasing buffer pH (7.2, 7.0, and 6.7) led to decreased Nae effect in both DM and CON. kept constant at 14 FM, but respiration rate was increased 2.5 times by Cr. In (I) Km(ADP) was However, in DM mitochondria, pH needed to be decreased to lower levels to abolish the Na+ decreased to 98±8 &M by short selective treatment with proteases. These data are taken to show effect; i.e., lowing pH to 7.0 did not abolish the Nae induced changes on State 3 which is in diffusion restrictions for adenine nucleotides within cardiomyocytes, metabolic channeling of contrast to what we found in CON. In summary, the magnitude of Na+ induced changes on endogenous ADP within the cells and importance of local ADP production by mitochondrial bioenergetics are related to pH in both CON and DM Mito. However, DM Mito appeared to be creatine kinase in the intermembrane space of mitochondria. less sensitive to pH effect, possibly due to disease-related down-regulation of the Na+lH+ exchanger.

197A 1163-1169 OXIDATIVE PHOSPHORYLATION MONDAY

1163 - Pos 1164- Pos DETERMINATION OF CATALYTIC ROTATION RATES OF THE MUTANT MODELS OF REGULATORY ENZYMES IN THE MITOCHONDRIAL CITRIC ACID Escherichia coU ATP SYNTHASE BY PHOSPHORESCENCE ANISOTROPY CYCLE OF HEART CELLS Matthew D. Zimmerman, Vincent B. Herlihy, Robert K. Nakamoto, Molecular Physiology & Stephen James Dudycha', Mohsin S. Jafri2, 'The Johns Hopkins University, 720 Rutland Ave., Biological Physics, University of Virginia, P.O. Box 10011, Charlottesville, VA 22906 Baltimore, MD 21205, 2The University of Texas at Dallas, Richardson, TX 75083 The FoF, ATP synthase uses a rotational mechanism to couple the translocation of The citric acid cycle is the final step in glucose and fatty acid metabolism. Its products drive protons to the synthesis or hydrolysis of ATP. Replacement of the yMet23 with Lys was shown to oxidative phosphorylation and ATP production in heart cells. Better understanding of the effects cause an increase in the energy of interaction between the catalytic P subunits and the rotor y of changing [Ca2e] due to pacing, increased acidity from ischemia, changing respiratory state, and subunit (Al-Shawi, M.K., Ketchum, C.J., Nakamoto, R.K. J. Biol. Chem. 272, 2300-2306, 1997). the availability of carbon substrates and intermediates within the cycle can be achieved through We earlier proposed that this effect will impede the rotation of the y subunit which defines the rate the development of a detailed model of the citric acid cycle. Several regulatory enzymes of the limiting catalytic step. To test this hypothesis, we will monitor the rotation rate of the y subunit citric acid cycle have been modeled to capture their behavior under these conditions to help using phosphorescence anisotropy. detennine the effects on the cycle. The models of isocitrate dehydrogenase (IDH) (EC 1.1.1.41) Single cysteine mutations at positions yLys201 to yAsp2O4 were expressed in a cysteine- and the 2-oxoglutrate dehydrogenase complex (ODHC) (EC 1.2.4.2, EC 1.6.4.3, and EC 2.3.1.61) less background (Kuo, P.H., Ketchum, C.J., Nakamoto, R.K. FEBS Lett. 426, 217-220, 1998). are affected by [Ca2'], capturing pacing effects and regulating the rest of the cycle through The complexes were purified and modified by eosin-5-maleimide. All four mutant complexes substrate concentration effects. IDH, ODHC and malate dehydrogenase (MDH) (EC 1.1.1.37) are labeled and on the subunit without ATP sensitive to pH, [NADH]/[NAD+], and [ADP], capturing ischemia and respiratory state of the stoichiometrically exclusively y affecting hydrolytic mitochondria. Finally citrate synthase (CS) (EC 4.1.3.7) is the start and end point of the cycle, activities. The labeled yS202C complex was immobilized to beads via a FLAGd epitope tag on the of the subunits used in time-resolved introducing acetate from organic fuel catabolism to the cycle, and recycling oxaloacetate. amino termini P phosphorescence anisotropy Parameter optimization of these models is based on published experimental data from in vitro measurements. This work was supported by PHS grant R01-59057. MDZ is supported by NIH predoctoral fellowship GM08323. assays.

1166 - Pos 1167 - Pos INHIBITION OF CREATINE KINASE CAUSES FAILURE OF ENERGY FREE RADICAL MECHANISMS OF MITOCHONDRIAL DYSFUNCTION DURING HOMEOSTASIS IN INTACT RAT CARDIAC TRABECULAE. CARDIAC ISCHEMIA/REPERFUSION. Rolf Brandes, Loyola University Chicago, Department of Physiology, 2160 S. First Ave., Kenneth M. Humphries, David T. Lucas, Pamela A. Szweda, Luke 1. Szweda, Case Western Maywood, IL 60153 Reserve University The relative importance of creatine kinase (CK) in the regulation of cardiac energy homeostasis Reperfusion of cardiac tissue results in increased rates of mitochondrial free radical production. and oxidative phosphorylation (Ox.Phos) is not clearly understood. As we have shown Subsequent peroxidation of membrane lipids leads to the formation of numerous cytotoxic previously, the mitochondrial redox potential is regulated by (Ca2+]. and another Ca2+- products which can interact with and inactivate enzymes. 4-Hydroxy-2-nonenal (HNE) is thought independent signal (possibly ADP). The aim of the current study was to investigate how this to be the most reactive of these compounds under physiological conditions and has been shown to regulation mechanism depends on CK activity by inhibiting it with 2,4-dinitrofluorobenzene increase in concentration during myocardial reperfusion. We therefore propose that cardiac (DNFB). Fluorescence of NADH and Rhod-2 (loaded in cytosol and mitochondria) was used to reperfusion leads to loss in mitochondrial function mediated, in part, by HNE modification of assess mitochondrial redox potential and simultaneously specific mitochondrial proteins. To test this hypothesis, we performed in vitro studies with intact [Ca2i,. & [Cas2], respectively, in pyruvate perfused cardiac mitochondria to evaluate the effects of HNE on mitochondrial respiration and ATP trabeculae. Fig shows that with fully functioning CK (Ctrl), synthesis. Under conditions expected during cardiac reperfusion, HNE inhibited NADH-linked increased work (by increasing pacing frequency from 0.25 Ctr. 25 FM DNFB ADP-dependent mitochondrial respiration by selectively inactivating a-ketoglutarate to 2 Hz) causes an initial fall of [NADH]J (due to increased Ox.Phos followed slow dehydrogenase (KGDH), an enzyme critical for the supply of NADH for electron transport. rate) by (Ca2'-dependent) recovery / Mechanistic studies revealed that loss of KGDH activity was due to reaction of essential lipoic to a new steady-state level. When work was returned to acid residues with HNE. This information was used to develop immunochemical methods for control, [NADH]m overshot its original value before evaluating the occurrence of this mechanism in an isolated rat heart model of returning. In contrast, with CK inhibition the recovery was fell ischemia/reperfusion. Using these methods, we provide the first direct evidence that losses in blunted, [NADH], during sustained stimulation, and , 2 2 Hz NADH-linked mitochondrial respiration during cardiac ischemia/reperfusion are in there was no mediated, part, overshoot. With increasing [DNFB] (10 to 30 by reaction of HNE with lipoic acid residues bound to the E2 subunit of KGDH. pM) the fall was more severe, and above -25 gM, the fall was accompanied by increased diastolic force (FJ.). The fall of [NADH]. (or rise of was not associated with altered Rhod-2 fluorescence. Fe,) Conclusion: inhibition of - CK causes increasing rate of Ox.PhosIncreasingand may eventually- lead to ATP depletion. m{ue)

1168- Pos 1169- Pos INTERACTION OF DERIVATIVES WITH MITOCHONDRIAL SELF-ORGANIZATION ELECTRON TRANSFER5-HALO-UBIQUINONESYSTEM. IN OSCILLATORY AND EXCITABLE SYSTEMS Marcus J.B. Hauser, V. S. Zykov, S. C. MUller, Otto-von-Guericke-Universitit Magdeburg, Tong Xie, Lian-Quan Gu, LindaYu, Chang-An Yu,Oklahoma State University, Biochem. & Universitltplatz 2, Magdeburg, 39106 Germany Molecular Biology, Stillwater, OK 74078 To the Self-organization was investigated in chemical, biochemical, and cellular systems like the study Q:protein interactions in the mitochondrial electron transfer chain, ubiquinone Belousov-Zhabotinsky reaction, the peroxidase-oxidase reaction, and cultures of the slime mold derivatives that have a or bromine chlorine atom at the 5-position and varying side chain lengths at Dictyostelium discoideum. The Belousov-Zhabotinsky (BZ) reaction serves as a convenient the 6-position of the benzoquinone ring were synthesized and characterized. 5-Bromo- and 5- derivatives have experimental laboratory model for the investigation of a rich varity of temporal and spatiotemporal chloro-Q redox midpoint potentials of 142 mV and 148 mV, respectively. These dynamics (oscillations and waves). Temporal dynamic behavior was also studied in a derivatives are simple 5-halo-Q reduced by ascorbate alone or by succinate in the presence of succinate- biochenical system, the peroxidase-oxidase reaction, which comprises a single enzyme and its ubiquinone reductase (SQR). The reduced 5-halo-Q (5-halo-QH2) can be oxidized by cytochrome substrates. Upon in the concentration of NADH various c alone or via changes dynamic states were induced. ubiquinol-cytochrome c reductase (SQR). In the presence of cytochrome c and Two qualitatively different transitions between periodic oscillations and chaotic regimes cytochrome c oxidase is oxidized were (CcO), 5-halo-QH2 by oxygen. However, no proton pumping observed. When diffusion is coupled with an excitable or oscillatory reaction, traveling waves may activity of QCR is when 5-Br- or is used as substrate. This with the results observed 5-CI-Q5Cjo H2 together emerge. Such behavior was investigated in the BZ reaction as well as in the cell aggregation of showing oxygen uptake by a mixture of SQR, CcO, and cytochrome c in the presence of 5-Br- or amobae. Circular and spiral-shaped waves are readily induced in an excitable preparation of the , and decreasing mitochondrial state 3 respiratory these 5-CI-QoC15that these by 5-halo-Q BZ reaction and were studied in mechanistic detail.In particular, the trajectories of spiral tips and derivatives, suggests halo-Q derivatives can act as electron mediator between SQR and transitions from simple rotating to complex meandering motion were analyzed. Similar circular cytochrome c, by passing QCR, in mitochondria thus decreasing itscoupling efficiency. This and spiral waves are work was in NIH shaped cAMP also observed during the morphogenesis of the slime mold supported part by (GM3072 1). Dictyostelium discoideum. These waves provide information which guides the amoboid cells towards the site of spiralwave initiation by chemotactic response to cAMP gradients.

198A MONDAY VOLTAGE-GATED CA CHANNELS I 1170-1175

1170-Pos 1171 -Pos IDENTIFICATION OF MULTIPLE HUMAN QIG ISOFORMS OF T-TYPE CALCIUM CHARACTERIZATION OF Ca'+ CHANNEL SUBTYPES IN THE SUPRAOPTIC CHANNELS WITH DISTINCT FUNCTIONAL PROPERTIES. NUCLEUS. Arnaud Montell, Jean Chemin, Emmanuel Bourinet, Joel Nargeot, Philippe Lory, IGH-CNRS, Masoud M. Zarel', Ligia Toro2, Enrico Stefani3, 'Dept. of Anesthesiology, 2Depts. of 34396 Montpellier, France Anesthesiology and Molecular & Medical Pharmacology, Brain Res. Inst., 3Depts. of and Brain Res. Los CA 90095-7115. In humans, the coding region of the gene encoding theaIG subunit of T-type calcium channels, Anesthesiology Physiology, Inst., UCLA, Angeles, CACNA1G, encovers about 75 kb at chromosome 17q22. Two cDNAs composed of 34 exons The supraoptic nucleus (SON) contains magnocellular neurons that release VP or OT hormones were first identified (Monteil et al, submitted). These two isoforms,a,o, anda00.b, are generated into the circulation. Since Ca2' channels are known to regulate hormonal secretion, we by alternative splicing and exhibit distinct III-IV loop. They give rise to typical T type currents investigated the molecular distribution of Ca2+ channel subtypes in the SON. We used 301tm slices when expressed in HEK 293 cells. Several additional exons were predicted using exon fromrats and labeled them with polyclonal antibodies against 01A, OLC,CXD, anda0E subunitsof determination computer programs and further identified in RT-PCR and cDNA cloning Ca2' channels, using immunocytochemistry. To measure labeling intensity in cell bodies (C), experiments. Here we describe the molecular and the functional properties of novel humana0G dendrites (D), and synapses (S), tissues were co-labeled with NCAM, MAP-2 and synaptophysin isoforms that are generated by the use of aditionnal exons. These exons encode insertions in II-III antibodies. Our results show that the aic subunit is evenly distributed in cell bodies, dendrites, loop, III-IV loop, and C-terminal regions. The various exon combinations of thea,G subunit were and synapses (ratio:lC:l.ID:IS). In contrast, the a,D subunit is highly expressed on dendrites, engineered and examined for their functional properties. As compared to Ca2+ currents generated and correlate with synapses (IC:2D:1.7S). a,A subunit expression is punctuated and expressed bya0G.,, X.Gb induced a leftward shift of 6 mV in the VO.5 of the activation. The presence of the 11- more in cell bodies (IC:0.6D:0.8S). Thea,E subunit is highly expressed at dendrites and correlate III loop insertion, but not the C-terminal insertion, is also responsible for a shift of the voltage- with synapses (IC:2.2D:1.5S). Furthermore, the 0LE subunit shows high expression at synaptic dependent activation and inactivation (-5 mV in the V0.5). The role of the III-IV insertion is also locations that surround the cell bodies. No differences in cell body Ca2+ channel expression were under investigation. Parallel experiments are undergoing in order to determine the expression observed in VP vs. OT neurons. Distinct subunit expression of Ca2' channels may play important pattern of these various QIG isoforms in native human tissues. roles in SON function. For example, the dendritic expression of thealD subunit may be important in receiving information from cerebral spinal fluid and blood vessels providing means for input to the SON. Supported by NIH. LT(AHA-E.I.).

1172 -Pos 1173 - Pos CHRONIC TREATMENT OF MICE WITH LAMBERT-EATON MYASTHENIC TEIREE NEW FAMILIAL HEMIPLEGIC MIGRAINE MUTANTS AFFECT alA SYNDROME (LEMS) PLASMA INDUCES DIHYDROPHYRIDINE SENSITIVITY OF CALCIUM CHANNEL KINETICS NEUROMUSCULAR TRANSMISSION. Richard L. Krauss', M. J. Sinnegger', A. Koschak', E. Wappl', P. Carrera2, H. Glossmann', J. Michael T FUnk, William D. Atchison, Michigan State University, B331 Life Sciences Bldg, East Striessvig2, 'Department for Biochemical Pharmacology, University of Innsbruck, Austria, Lansing, Michigan 48824-1317 2Clinical Molecular Biology Laboratory, Hospitale San Raffaele, Milan, Italy LEMS is a neuromuscular disorder in which autoantibodies apparently target the voltage-gated Missense mutations in the pore-forning human aoIA subunit of neuronal P/Q-type Ca2' channels Ca2+ channels involved in release of acetylcholine from nerve terninals. Transfer of LEMS to are associated with Familial Hemiplegic Migraine (FHM). We studied the functional mice by repeated administration of plasma from LEMS patients reduces the amplitude of the P/Q consequences of three recently identified FHM-mutants, R583Q, D715E and V1457L introduced type current, and unmasks a dihydropyridine-sensitive Ca current. The present study was into rabbit alA after expression in Xenopus laevis oocytes. designed to determine if this component contributes to acetylcholine release. Mice were injected The potential for half-maximal channel activation was shifted by > 9mV to more negative for 30 days with LEMS or control plasma. Acute studies involved incubating the diaphragm from potentials in all three mutants. The potential for half-maximal channel inactivation was shifted by mice with plasma for 2 or 24 hrs. Endplate-potentials and miniature endplate-potentials were > 7 mV in the same direction in R583Q and D715E. Biexponential Ba2+ current inactivation recorded from neuromuscular 0.5 Hz diaphragm junctions. During stimulation, quantal content during 3 second test pulses was significantly faster in D715E and slower in V1457L than in (m) was reduced by 57% with chronic LEMS treatment compared to treatment with control wildtype. In contrast to D715E, mutations R583Q as well as V1457L delayed the time course of plasma. After 2 and 24 hrs of treatment with LEMS m was reduced by 46% and 48%, plasma, recovery from channel inactivation. R583Q (30.2 + 4.5%; p<0.01) and D715E (30.1 ± 1.4%; respectively. Nimodipine did not significantly affect m of muscles treated acutely with either p<0.01) but not V1457L (18.7 + 3.3%), displayed significantly higher accumulation of channels in control or LEMS plasma or following chronic treatment with control plasma. However, following + inactivation during a 1 Hz train of 15 100 ms pulses than wildtype (18.2 1%). chronic treatment with LEMS plasma, nimodipine reduced m by 43%. Thus, dihydropyridine- Our data demonstrate that the three new identified FHM mutations D715E sensitive Ca2+ channels become involved in transmission at the mouse neuromuscular R583Q, and V1457L, synaptic like the mutations and affect junction after chronic treatment with LEMS plasma. (Supported by NIH grant ES05822 and Viets previously reported T666M, V714A, 11819L, P/Q-type Ca2+ channel We therefore that a to M.Flink from the gating. propose altered channel gating represents common pathophysiological Fellowship Myasthenia Gravis Foundation.) mechanism in FHM. Supported by grants from the Fonds zur Forderung der Wissenschaftlichen Forschung (P- 12641 to JS, P-12689 to HG) and the Osterreichischen Nationalbank (to JS). MJS is recipient of a Hertha- Firnberg fellowship.

1174 Pos 1175 Pos EXPRESSION AND IGF-1 REGULATION OF CARDIAC CALCIUM CHANNEL a26 DIFFERENTIAL MODULATION OF PRESYNAPTIC CALCIUM CHANNELS BY SUBUNITS DIFFRENT Gp ISOFORMS Po-Ju Chu, P. M. Best, University of Illinois, Department of Molecular and Integrative Michelle l. Arnot, S E Jarvis, A. M. Beedle, Gerald W Zamponi, University of Calgary, 3330 Physiology, Urbana, IL 61801 Hosptial Dr NW, Calgary, AB T2N 4N I Canada Calcium channel a26 subunits regulate current amplitude and activation/inactivation kinetics. The activities of presynaptic calcium channels are subject to modulation by direct binding of G Three 026 are in mammals. Here we genes expressed report the detection of all three a26 mRNAs protein 1t subunits. This study examined the relative effects of different Gp subtypes on in rat cardiac tissues and atrial myocytes by RT-PCR. The a28-1 mRNA detected in rat atrial transiently expressed N-type (aII3, pIb, a2-6) and P/Q-type ((IA, Ilb, a2-6) calcium channels by tissue is the isoform "e". Several partial clones of atrial a26-2 and 026-3 cDNAs were generated using the degree of prepulse relief as an indication of G protein inhibition. Upon coexpression of by RT-PCR. After alignment, the DNA sequences from those partial clones span most of the a26-2 the N-type calcium channel with the GpI72 subunit, current amplitude is increased by 90% and and have 90% and and a26-3 coding regions 95% homology with their counterparts in human following the prepulse. In contrast, in the presence of Gp3,2, the prepulse effect is reduced to a 30 mouse, respectively. RNase protection assay reveals that the expression level of both a26-2 and % increase. For P/Q-type calcium channels, coexpression with either GpI.2 or Gp3y2 the degree of a26-3 is higher in atrium than in ventricle. To analyze the effect of IGF- I on the expression level prepulse relief was identical (20% enhancement following the prepulse). These results of a26 mRNAs, we measured the relative abundance of various a26 mRNAs obtained from demonstrate that the N-type channel is differentially modulated by the G protein ,BI and ,B3 cultured atrial myocytes with or without IGF- I. The data show that both 026-2 and a26-3 mRNAs subunits, whereas modulation of the P/Q- type channel does not disciminate between the two Gp are increased upon IGF-I treatment; however, a26-1 mRNA does not significantly change. LVA isoforms examined. These results indicate that the molecular determinants of GP interaction calcium current density in cultured atrial myocytes is selectively increased by treatment with IGF- differs between N-type and P/Q-type calcium channels. These differences may underlie selective 1. Therefore, these results indicate that the expression level of the a26-2 and a28-3 subunits is control of the presynaptic calcium channels by the various Gp isoforms. correlated with the increase of LVA calcium current density in IGF-I treated atrial myocytes. (Supported by NIH AR44352 to P.M.B. and AHA predoctoral fellowship to P.-J.C.)

199A 1176-1181 VOLTAGE-GATED CA CHANNELS I MONDAY

1176- Pos 1177 - Pos A MUTATION IN THE P INTERACTION DOMAIN OF THE Ca2+ CHANNEL aic MODULATION OF SLOW INACTIVATION IN CLASS A CA2+ CHANNELS BY 13- SUBUNIT AFFECTS ISRADIPINE-BINDING AND P ASSOCIATION. SUBUNITS Monlka Hitzl, Jlorg Striessnig, Birgit Neuhuber, Bernhard E. Flucher, Dept. of Biochemical Stanulav Sokolov, Regina G. WeiB, Steffen Hering, Biochemische Pharmakologie Pharmacology, Peter-Mayr-Str. 1, Innsbruck, A-6020 Austria Class A calcium (Ca2) channels affect the synaptic function in the mammalian nervous system by The L-type calcium channel is composed of the pore-forming a, and the accessory and a28 mediating the release of neurotransmitters. It is believed that the fine tuning of voltage-dependent subunits. The P subunit modulates membrane expression, single-channel properties and ligand Ca2+ entry into neurons is mediated by the ,8-subunit composition of class A Ca2+ channels. In binding of the calcium channel. A conserved interaction motif has been identified in the I-II order to elucidate the 1-subunit-modulation of slow inactivation we expressed a cz,A-subunit (BI- or in and the kinetics of cytoplasmic loop of the a, subunit. Mutation of a single residue (Y467S) of this motif impeded 2) together with either P1C-,02C.-,3- P4-subunits Xenopus oocytes analysed membrane targeting but not current modulation of alc. Here we compared (+)_[3H]isradipine slow inactivation of the corresponding Ca2+ channel currents in two microlectrode voltage clamp binding properties and association with 01, in wild type alc and cx1cY467S. Different experiments. A novel "back extrapolation technique" was developed to estimate the on- and off- combinations of channel subunits were expressed in tsA201 cells and were analyzed with rate constants of slow inactivation in channels with different P-subunit composition. We report radioligand binding and immunoprecipitation assays. The Y467S substitution lowered the affinity here, that a reduction of fast inactivation by coexpression of Q1A- with the P2.-subunit simultaneously accelerates the channel state transitions into slow inactivation. Slowing fast and B,,,. of (+)_[3H]isradipine. However, coexpression of myc-epitope-tagged 13i. (P1. -myc) inactivation of channels by mutating two inactivation determinants in segmnent IIIS6 to increased both parameters, demonstrating that functional interactions between a1cY467S and 1,.- a,A/P3 alanine had a similar effect that channels in the open state are more myc still occurred. Furthermore, anti-myc antibodies coprecipitated P,.-myc and alCY467S, as (IFI1612/1613AA) suggesting shown with (+)-[3H]isradipine binding and Western blot analysis. Our experiments indicate that willing to enter slow inactivation. Accelerated slow inactivation in class A Ca+ channels with 13- subunit compositions promoting a slow current decay may help to maintain neuronal enhanced ligand-binding of aCic on coexpression of 13 is mediated through the 13 interaction domain CaW+ in the I-II loop and substitution of the conserved tyrosine in position 467 with serine reduces but homeostasis. The indirect P-subunit-modulation of slow inactivation appears to regulate long term in class A Ca2+ FWF 2649-MED. does not completely hinder the formation of alc/p,. complexes. (Suppoated by FWF grants P12653- changes channel availability. Supported by P1 MEDbtBEYF. andP12641-MEDboJS.)

1178- Pos 1179 - Pos CALCIUM CHANNEL p SUBUNrrS BIND REVERSIBLY TO alB CALCIUM GATING OF HUMAN CARDIAC L-TYPE CALCIUM CHANNELS WITH CA2 AS CHANNELS: CONCENTRATION-DEPENDENT EFFECT ON ACTIVATION AND G- CHARGE CARRIER: NO EVIDENCE FOR REGULATION BY CALCINEURIN PROTEIN MODULATION. Stefaa Herzlg', Renate Handrock2, Nils Haake3, Stephan Hirt3, Andreas Jager, 'University of Carles Canti, Y. Bogdanov, A. C. Dolphin, University College London, Gower St., London, Cologne, Gleueler Strasse 24, Cologne 50931, Germany, Gleueler Strasse 24, Cologne 50931 WC1 E 6BT United Kingdom Germany, 2University of Cologne, 3University of Kiel The P subunits of voltage-dependent Ca2+ channels regulate their biophysical properties. Here we focused on the voltage-dependent involvement of the P subunit isoforms (PI3b, P2a, 133 and 14) in Single L-type channel activity is increased in human heart failure (Schr6der et al., 1998, the process of activation and G-protein modulation of alB Ca+channels. All subunits Circulation 98: 969-976). We suggested as a mechanism decreased activity of channel-associated hyperpolarised the mid point for current activation, and antagonised its G1y-mediated protein phosphatases. Recent animal data suggest a role of calcium-dependent protein phosphatase depolarisation. Co-expression of subunits also enhanced the rate of voltage-dependent 2B (calcineurin) in some heart failure models (see Walsh 1999, Circ. Res. 84: 741-743). - - reduced channel facilitation by a 5-20 fold increase of the G1y dimer dissociation rate at +100 mV. In the case of Therefore, we hypothesized that given calcineurin regulates human channels could be due to absence of Ca2' under our P3, which was the most effective, we investigated the concentration-dependence, by injecting a dephosphorylation artifactual, previous recording conditions. range of concentrations of P3 cDNA. All the effects were dependent in a saturable manner on the 133 concentration, suggesting a reversible binding of the P subunit. We interpret our data in terms of a model whereby Gpy and Ca2+ channel P subunits bind to an overlapping site on the calcium To investigate the effects of Ca2+ entering through human ventricular L-type channels, recording channel. G1y subunits would be bound to alB at hyperpolarised potentials and would unbind at pipettes contained 30mM CaC12 and 60mM BaCl2 (n=5). Ca2' permeation led to near-complete depolarised potentials. We hypothesise that a ternary alB*P1G1y complex can form, but is time-dependent inactivation over 150ms at +2OmV, and to reduced current amplitudes (7.7±1.2 pS unstable, tending to dissociate to atl Boo at depolarised potentials. An elevation of Ca2+ channel slope conductance, n=4), compared with 70mM BaCl2 alone (see Schr5der et al., 1998, Handrock subunit concentration will therefore enhance the rate of Gpy dissociation by increasing the et al., 1998, Cardiovasc. Res. 37: 445-455). However, endogenous calcineurin did not reduce formation of the ternary complex. channel activity: 1. channel availability was high (70±5%, n=5), comparable to values found previously with Ba2+. 2. the life-time of the available state (estimated by sweep histogram analysis) was still long (r=2.24±0.39 s, n=3). 3. Ciclosporin, a calcineurin inhibitor, reduced channel activity (n=3). We conclude that increased single channel activity in human heart failure is preserved with Ca2' permeation, irrespective of possible calcineurin activity.

1180- Pos 1181 - Pos FUSIONS OF EYFP TO WILD-TYPE AND MUTANT CALMODULINS PRESERVE CALMODULIN AFFECTS FAST Ba+ INACTIVATION OF L-TYPE Ca CHANNELS. INTERACTIONS WITH CA2+-DEPENDENT INACTIVATION OF L-TYPE CALCIUM Carla D. DeMaria, H. L. Agler, D. T. Yue, Depts. Biomed. Engr. and Neurosci., Johns Hopkins CHANNELS. Univ., Balto., MD 21205. Michael G. Erickson, B. Z. Peterson, D. T. Yue, Johns Hopkins Sch. Med., Dept. Biomed. Engr., L-type calcium channels inactivate through distinct current (Ca2+)-dependent and voltage- 720 Rutland Ave., Baltimore, MD 21205 dependent mechanisms. We have recently shown that the sensor for the faster, current-dependent Recently cahnodulin (CaM) has been revealed as the Ca2+ sensor for Ca2+-dependent inactivation inactivation is calmodulin (CaM) (Peterson, et al. (1999) Neuron 22:549). The nature of voltage- of L-type calciqgn channels. In particular, Ca2+-insensitive mutant CaM functions as a dominant dependent inactivation is less clear, because much of its characterization is based on studies of negative, as its over-expression ablates Ca2+-dependent inactivation (Peterson et al., 1999). The Ba + currents, which presumably inactivate by purely voltage-dependent means. However, recent dominant negative nature of the effect suggests that CaM is constitutively tethered to the channel gating-current studies suggest that Ba2+ may still trigger current-dependent inactivation, albeit less complex, possibly in a spatially restricted pocket. To explore the feasibility of using fluorescently- effectively than Ca+ (Ferreira et al. (1997), JGP 109:449); hence, Ba2+ currents may inactivate by labelled CaM to investigate such tethering, we asked whether Ca2+-dependent inactivation was still both mechanisms. Here, we directly tested for this possibility, by examining whether Ba2+ current affected by a series of fusion proteins that link CaM or mutant CaM to the enhanced, red-shifted inactivation was affected by mutant Ca2+-insensitive CaM, which was co-transfected with L-type mutant of the Green Fluorescent Protein, EYFP. The CaM-EYFP fusions have been efficiently channels (a,CP1ba28) in HEK 293 cells. Because such mutant CaM ablates Ca2+-dependent expressed in HEK293 cells and characterized by confocal microscopy and spectrofluorimetry. inactivation (Peterson et al., 1999), mutant CaM Interestingly, fusion of EYFP via a short, rigid amino-acid linker to the N-terminus of CaM did would also slow the decay of Ba2+ currents if not disrupt the dominant-negative knockout of Ca2+-dependent inactivation in whole-cell patch- current-dependent inactivation were in effect. The clamp recordings, implying that CaM-EYFP also tethers to the channel complex. This result slight, but unmistakable slowing in the decay of m tant CaM (stop to' establishes the feasibility of using CaM-EYFP's to explore tethering and sets initial constraints on averaged Ba2+ currents by mutant CaM confirms a /(6 __ the dimensions of any pocket harboring the tethered CaM. partial contribution of current-dpdent 1 sac inactivation. These results prompt re-evaluation of voltage-dependent inactivation, as it arises from wildtype CaM(n=5 cetl) studies of Ba2'+ current.

200A MONDAY VOLTAGE-GATED CA CHANNELS I 1182-1187

1182 -Pos 1183 - Pos SINGLE CHANNEL FUNCTION IN NEURAL CREST RELATED HEART DISEASE MUSCARINIC STIMULATION OF NEURONAL R-TYPE CALCIUM CHANNELS IS Carol A. Nichols, Tony L. Creazzo, Institute of Molecular Medicine and Genetics, Medical MEDIATED Gctq College of Georgia, Augusta, GA 30912 Melliti, U. Meza, B. Adams, Department of Biology, Utah State University, Logan, Utah Ablation of the cardiac neural crest during early embryonic chick development produces a severe 84)22 congenital heart defect known as persistent truncus arteriosus (PTA). PTA is characterized by We have previously demonstrated that Ca channels formed by the rabbit alE subunit (BII-2) are failure of the outflow tract to undergo septation. Chick embryos with PTA have defective both inhibited and stimulated through M2 muscarinic acetylcholine receptors (Meza et al., excitation- contraction coupling (ECC) as evidenced by reduced ejection fractions, reduced twitch J.Neurosci. 19:6806-6817, 1999). M2 receptor-mediated inhibition of alE depends upon PTX- force, and small Ca2' transients, L-type Ca2+ currents (IC.,L) (Creazzo, et al 1998 Ann. Rev. sensitive G proteins, whereas stimulation of alE through the same receptor does not. To Physiol) at embryonic day (ED) 11 and 15. The above work has been done primarily in left determine whether Gq is responsible for stimulating alE, we expressed cloned MI receptors ventricle (LV) or pooled ventricular myocytes. In most instances of PTA, the common truncus which preferentially couple to Gq proteins. Activation of Ml receptors produced larger overrides the right ventricle (RV) placing that ventricle under increased volume and pressure loads stimulation, and smaller inhibition, of alE than M2 receptors which preferentially couple to Gi/o cause (Nishibatake, et al, 1987). We hypothesize that the additional stress placed on the RV will proteins. PTX affected neither stimulation nor inhibition through Ml receptors. Stimulation was the RV to have a smaller than the LV by ED 15. Patch on IC.,L clamp experiments performed blocked by PLCfI or RGS2, which function as GTPase-activating proteins for Gaq. cultured myocytes shows that peak IC.L declines more steeply in the RV than in the LV of Furthermore, stimulation was occluded by constitutively-active Gaq* (Q209L). The ,IARKI experimental hearts from ED 11 to 15. We hypothesize that this decline in peak IC.L is due to a carboxyl terminus (a GIy sequestrant) blocked inhibition through Ml receptors without altering reduction in the open channel probability (P,). Analysis of single channel properties in the RV stimulation, indicating that Gaq itself carries the essential signal. Significantly, an alE subunit and LV of experimental embryos shows that the PO of the RV (1.50±0.02, n=I 1) is significantly that is insensitive to G protein-mediated inhibition (rbE-II) was prominently stimulated through less than the of the LV n=8) in experimental embryos at ED 15 (p<0.01). We are P. (3.0±0.01, both muscarinic receptors. These results demonstrate that neuronal R-type Ca channels are currently examining the P. in the RV and LV of experimental embryos at ED 11. Supported by strongly stimulated through a Gaq-coupled signaling NIH HL36059 and HL58861. pathway.

1184 - Pos 1185- Pos TISSUE DISTRIBUTION OF SPLICE VARIANTS OF THE L-TYPE VOLTAGE- A GRADED RELEASE MODEL OF COUPLING BETWEEN L-TYPE AND RYANODINE DEPENDENT CALCIUM CHANNEL a,c SUBUNIT IN RAT. SENSITIVE Ca5+ CHANNELS BASED ON HETEROGENEOUS CO-LOCALIZATION. Satomi Adachi-Akahane, Shoichi Kanda, Taku Nagao, University of Tokyo, 7-3-1 Hongo, V. E. Bondarenko, D. A. Coast, Randall L. Rasmusson, Penn State College of Medicine, 100 Bunkyo-ku, Tokyo, 113-0033 Japan North Academy Ave, Danville, PA 17822

As a first step for elucidating pharmacological and physiological importance of splice variants, we Inactivation of L-type Ca2+ channels (Ic,) in cardiac muscle depends on membrane voltage and investigated tissue distribution of splice variants of L-type Ca2+ channel atiC subunit in adult rats. Ca2' release from the sarcoplasmic reticulum. Release of calcium from the sarcoplasmic reticulum By RT-PCR and subsequent sequencing, we identified previously reported three alternative is, in turn, dependent upon the size of the peak of the inward calcium current through the L-type splicing sites atIS6, IIIS2, and IVS3, and two insertion/deletion sites at I-II loop, and linker region calcium channel. We have developed a model based on morphological considerations and recent between IVS3-S4. However, we did not identify any alternative splicing nor insertion/deletion at observations of calcium channel sparks data which reproduce the graded release phenomenon. carboxyl terminus as reported for human brain and heart, respectively. Therefore, we analyzed This model contains several elements. 1) Calcium channel inactivation is modeled as being three fragments including splicing sites, IS6-I-II loop, IIIS1-IIIS4, and IVSI-IVS4, by RT-PCR partially coupled to inactivation, with a fast calcium dependent component and a slower calcium and restriction enzyme digestion. In IS6 of ventricle, atria, and brain, only insensitive inactivation component. 2) Ryanodine release channels with relatively simple "MQDAMGYELPWVYFVSLVIF" type without insertion at I-tI loop could be found. In cooperative calcium dependent opening and inactivation processes are incorporated based on contrast, "VNDAVGRDWPWIYFVTLIII" type of IS6 was dominant and substantial fraction had previously published models of their behavior. 3) These two channel types communicate with a insertion in smooth muscle such as aorta, lung, and stomach. As for IIIS2, small restricted space which can transiently reach relatively high local calcium concentrations. 4) "FYFDIVFTTIFTIEIAL" type was dominant in brain, but "GNADYVFTSIFTLEIIL" type was Diffusion can occur rapidly out of this space into the cytosol which contains calcium binding dominant in smooth muscle and adrenal. Ventricle and atria had both types equally. proteins associated with contraction. 5) There are multiple release units distributed over the cell "HYFCDAWNTFDALIVVGSIVDIAITEVH" type of IVS3 with insertion at linker between consisting of a few L-type calcium channels and ryanodine sensitive channels connected by a IVS3-S4 was dominant in all of tissues except for adrenal. Electrophysiological and small subspace. 6) The total number of functional L-type channels per release unit is low but pharmacological properties of these variants are under investigation. (JSPS 4264) randomly distributed in a binomial fashion. Graded release occurs as a consequence of these physical factors.

1186- Pos 1187- Pos INTERACTION OF CALMODULIN WITH THE aic SUBUNIT IN THE CA2+- A SEQUENCE IN THE CARBOXYL TERMINUS OF THE alc SUBUNIT CRITICAL DEPENDENT INACTIVATION PROCESS. FOR TARGETING, PERMEATION, KINETICS AND RUN-DOWN OF CLASS C-TYPE Martina Gaali, H Kahr', P Pammer', R Gamrsjager', G F6rstner', K JF Kepplinger', K CA2+ CHANNELS. Groschner2, DR Abernethy3, N M Soldatov3, C Romanin', 'Institute for Biophysics, University of Klaus JF Kepplingeri, H Kahr', G Forstner', M Sonnleitner', H Schindler', T Schmidt', K Linz, Austria, 2lnstitute of Pharmacology and Toxicology, University of Graz, Austria, 3Institute Groschner2, N M Soldatov3, C Romanin', 'Institute for Biophysics, University of Linz, Austria, on Aging, NIH, Baltimore, MD, USA 2Institute of Pharmacology and Toxicology, University of Graz, Austria, 3Institute on Aging, NIH, Calmodulin (CaM) has been suggested as mediator of Ca2+-dependent inactivation of L-type Ca2' Baltimore, MD, USA channels via interaction with an IQ motif found in the C-tail of the alc subunit (Peterson et al., The role of C-terminus in the function of the alC channel was studied by comparing membrane et al., Zuhlke et al., 1999). Here we employed GST-pulldown technique, fluorescence Quin targeting, ion permeation, inactivation kinetics and run-down of the conventional aIC,77 channel microscopy and patch-clamp to examine the proposed interaction with CaM. Binding of CaM to (GeneBank # Z34815) with its mutants containing 81- (alC,86), 27- or 58-aa segments the N-terminal third of the C-tail of alc expressed as GST-fusion protein was confirmed in the (aIC,77L) (alC,77K) of the rapidly inactivating isoform (# Z34817) incorporated into positions 1572-1651, presence of 100 gM Ca2 . The use of GFP analogues CFP and YFP for labeling of CaM and the alc subunit, respectively, enabled us to observe co-localization of both components in the plasma 1572-1598 or 1595-1652, respectively. These pore-forming subunits were co-expressed in tsA201 membrane. The effect of overexpression of CaM mutant (CaMI,2 3,4) deficient in Ca2' binding was cells with a26 and 12a subunits and studied by fluorescence microscopy and patch-clamp. The compared with that of wild type CaM in single channel recordings on tsA cells transfected with a1C,77 channel was found to target predominantly plasma membrane and formed clusters, aic, a28 and 2,s. subunits. Open probability and mean open times of Ca2+ channels with Ca2' as charge carrier were 2.3- and 2.4-fold, respectively, higher in cells overexpressing CaM123,4, whereas alC,86 was mainly distributed over the cytoplasm. Single channel Ba currents of the although both were still smaller than with Ba2'. These results are in accordance with an a1C,77 channel inactivated markedly slower compared to the alC,86, a1C,77L, and alC,77K involvement of CaM, but suggest the contribution of other alc tail motifs to Ca2+-dependent channels. Unitary conductance of the latter three channels was significantly smaller than that of inactivation (Soldatov et al., 1998). Results supporting this idea were obtained with arC,77L mutant the alC,77. All four channels showed two open states. The aIC,77 and alC,77L channels had which contains an intact IQ motif, but lacks Ca2+-dependent inactivation. (supported by FWF comparable longer open time constants which were almost twice greater than those of a I C,86 and P12728, P12667 and AHA) alC,77K channels. Unlike alC,77, xlC,77K and alC,77L, the alC,86 channel demonstrated little if any run-down. Our results indicate a pivotal role of the 1572-1651 sequence of the aIc subunit C-terminal tail in membrane targeting, ion permeation, kinetics and run-down of class C Ca channel activity. (Supported by FWF P12728, P12667 and AHA)

201A 1188-1193 VOLTAGE-GATED CA CHANNELS I MONDAY

1181- Pos 1189 - Pes EFFECTS OF GENISTEIN ON -ADRENERGIC RECEPTOR STIMULATION OF ICa ATTENUATION OF G-PROTEIN-MEDIATED INHIBITION OF N-TYPE CALCIUM IN CELLS FROM THE 5 DAY INFARCTED HEART CHANNELS BY RECOMBINANT EXPRESSION OF CAVEOLIN-3 IN NG 108-15 Takuya Yagi, Penelope A Boyden, Columbia University, 630W 168th St, New York, NY 10032 CELLS. Previously we showed a reduction in density of L-type Ca2+ currents(Ic,) in cells from epicardial M. Toselli', M. Parenti2, E. Fiorillo', L. Taglietti', Vaani Taglietti', 'University of Pavia, border zone of S day infarcted heart(lZs) compared to those from noninfarcted heart(NZs). Further 2University of Milano we showed a hyporesponsiveness of tc to 3-adrenergic stimulation. To determine the role of Caveolae and their principal component caveolin have been implicated in playing a major role in tyrosine kinase(PTK) activity in this altered adrenergic response, we studied the effects of G protein-mediated transmembrane signaling. To ascertain this, we stably expressed an isoform of genistein(G), a PTK inhibitor, on basal 'C. in the absence and presence of isoproterenol(ISO) using caveolin, caveolin-3 (Cav3), in the NG 108-15 cell line, lacking endogenous caveolins. Upon in whole-cell patch-clamp techniques. Genistein dose-dependent reduction of Ic. was similar in NZs vitro differentiation, NG108-15 cells express N-type Ca-channels, that are inhibited through and IZs(SO M:NZs;16±3%(n=6), IZs;13±l%(n=14)) and not mimicked by daidzein(50pM), an activation of G protein-coupled receptors. Therefore here the N-type Ca-channel was used as a inactive analog of G, although basal "c. of NZs(5.9±0.5pA/pF) and IZs(3.3±0.2pA/pF) differed. probe to investigate, by the use of the patch-clamp method, whether Cav3 has any regulatory Subthreshold ISO(I-5nM) produced little or no change in Ic.(3-4%) in both cell groups, but the action on Ca-channel coupled G proteins. The 6-opioid agonist [D-Pen2,D-Pen5]-Enkephalin same in presence of G increased peak Ic. by 22±6%, ll±2%(NZs(n=6), IZs(n=13),p=0.04) (DPDPE) reversibly decreased N-type Ba-currents both in NG108-15 cells and in the Cav3 respectively. Further, with G wash, ISO increased Ic. further, presumably due to removal of G's expressing clones. In both cell types inhibition had qualitatively similar features, however the nonspecific inhibitory effect on Ic.. In sum, 1) basal 1c6 is sensitive to G suggesting persistent PTK percentage of current inhibition was significantly lower in the cells exprssing Cav3 than in control activity contributes to C. in NZs and IZs, 2) in G, there is enhanced sensitivity of 'C. to ISO in cells (35% against 64% of inhibition respectively). Similar results were also obtained by direct NZs and this effect exists in IZs, 3) thus, hyporesponsiveness of 1c. to ISO in IZs is not due to activation of endogenous G proteins with GTPyS. Our results demonstrate that Cav3 can augmented PTK activity. participate in G protein-coupled signaling events, like modulation of voltage-gated Ca-channels, presumably with a GDI-like activity.

1190- Pos 1191 - Pos 0-ADRENERGIC RECEPTOR REGULATION OF L-TYPE Ca5+ CURRENT DURING A POINT MUTATION IN THE CARBOXYL TAIL MODIFIES P/Q TYPE Ca2+ HYPOXIA IN GUINEA-PIG VENTRICULAR MYOCYTES CHANNEL BIOPHYSICAL PROPERTIES. Livia C Hool, The University of Western Australia, Hackett Drive, Nedlands, Westem Australia Eleanor A Mathews, E. Garcia, C. M. Santi, T. P. Snutch, University of Bristsh Columbia, 6174 6907 Australia University Blvd., Vancouver, British Columbia V6T IZ3 Canada The effects of hypoxia on the L-type Ca2+ current (Ic.) in the absence and presence of the 3- The Caenorhabditis elegans unc-2 gene encodes a voltage-gated Ca2+ channel a, subunit closely adrenergic receptor agonist isoproterenol (Iso) were examined. Consistent with effects in related to the a,c and CzIA Ca2+ channels expressed in the mammalian nervous system. Using a recombinant human L-type channel aic subunits, exposing guinea-pig ventricular myocytes to mutant screening strategy we isolated a novel allele of unc-2 (ra612) and identified a glycine to hypoxia resulted in a reversible inhibition (25.2 ± 2.5%, n=28) of basal Ic,, Hypoxia also arginine substitution in the carboxyl tail region. The corresponding mutation was introduced into significantly decreased the K3.5 for activation of Ic. by Iso from 5.3 ± 0.7 to 1.6 ± 0.08 nM. To the rat brain tIA P/Q-type channel (QIA-612)- mutant determine if the effects were membrane-delimited, cells were exposed to the membrane- Whole cell currents were recorded in 5 mM BaCl2 impermeant thiol-specific oxidizing compound DTNB. In the presence of 200pM DTNB, the external solution using standard patch clamp inhibition of basal Ic. by hypoxia was attenuated 81.3 ± 9.4% (n=5) while the increase in technique. During a 200 ms test pulse, inactivation sensitivity of Ic, to Iso was unchanged. Hypoxia also increased the sensitivity for activation of IC. was significantly more pronounced in C,A.612 than by the H2-receptor agonist histamine. Since hypoxia may alter nitric oxide production, a possible in the wild type (wt) channel, showing a decay of role for cGMP was investigated. Neither methylene blue (25pM) nor LY-83583 (20sM), 94.22 ± 1.16 % (n = 13) of the peak current, as inhibitors of guanylate cyclase, applied intracellularly had any effect on the basal inhibition of IC. comparedto42.92± 4.43 % (n = 5) for the wt. In 50 m or the decrease in K5.5 for activation of Ic, by Iso during hypoxia. These results suggest that addition, the inactivation time course of the wt hypoxia regulates Ic, through two distinct mechanisms: direct inhibition of basal Ic and an current was fitted with a single exponential (i,.,= 174.65 ± 25.65), while a(A.612 showed double indirect effect in the presence of ,B-adrenergic receptor stimulation that does not appear to involve exponential decay (r, = 9.16 ±0.77, 2 560.71 ± 7.85 at 0 mV). A negative shift in the steady state cGMP. inactivation (V%,,,g = -73.6 and VS,, =-52.5 mV) and a 10 mV positive shift in the I-V relation were also observed. We are investigating whether these changes can be explained by a direct effect of the mutation on channel gating properties or by differential modulation. Supported by MRC of Canada, Human Frontiers Science Program and Heart & Stroke Foundation of BC and Yukon.

1192 - Pos 1193 - Pos CYTOSKELETAL MODULATION OF CARDIAC L-TYPE CALCIUM CHANNEL MODULATION OF T-TYPE CALCIUM CURRENT IN HYPERTROPHIED RAT ACTIVITY IN L6CELLS VENTRICULAR MYOCYTES. Basal M. Hantashl, Nevin Boparai2, John P. Reeves', 'UMDNJ-Grad. Sch. Biomed. Sci., Dept. Carmea Delpdo, MP Heredia, ML Martinez, Institute of Pharmacology and Toxicology (CSIC- Pharmacology & Physiology, 185 S. Orange Ave., Newark, NJ 07103, 2Rutgers University, New UCM), Madrid, 28040 Spain NJ Brunswick, 08901 Recently, T-type calcium current (IC.T) was described in ventricular myocytes isolated from Rat skeletal myoblasts express the cardiac L-type Ca channel, as determined by functional assays rat hearts. In this we and hypertrophied study examined the modulation of this current by a p- sequence analysis by RT-PCR. Channel activity was measured in suspensions of fura-2 adrenoceptor an agonist, Methoxaniine (Mtx), and loaded L6 cells as agonist, Isoproterenol (Iso), a,-adrenoceptor a nifedipine-sensitive Ba (5 mM) influx in a depolarizing salt solution containing specific blocker of Ic.T, Mibefradil (Mib). Left ventricular hypertrophy was induced by abdominal 140 mM K. The initial rate of Ba uptake was inhibited by 52% and 60% upon treating the cells with aortic stenosis. The whole-cell patch-clamp technique was used to record Ca currents in agents that stabilized either microtubules (taxol, 50 sM) or F-actin filaments (iasplakinolide, enzymatically dissociated ventricular cells. T-and L- calcium currents( IC.T, IC.L) were separated 10 FiM). Preincubation with nocodazole, which disrupts microtubules, had no effect on Ba influx, by to different test from a while applying voltage steps potentials holding of-90 mV and -50 mV. "C.T depolymerization of the actin cytoskeleton with cytochalasin D (10 1M) stimulated Ba was defined as the difference current elicited at the two holding potentials. Iso 1pM induced a 60%. Nocodazole did not uptake by protect against inhibition by taxol. Finally, calyculin (100 significant increased in IC.T (at -4OmV: -0.378 ± 0.086 pA/pF vs. -0.626 ± 0.059 pA/pF, n=6). nM), a inhibitor that causes extensive in protein phosphatase alterations intermediate filaments, Mtx 10pM significantly reduced IC.T in these cells (at -4OmV: -1.426 ± 0.302 pA/pF, vs. -0.482 ± microtubules and F-actin filaments, inhibited Ba influx by 75 %. Okadaic acid (I 1kM) and 0.176 Mib also pA/pF, n=7). 1pM produced a substantial blockade of IC.T (at -4OmV: -0.751 ± 0.150 tautomycin (I pM), phosphatase inhibitors, reduced Ba uptake by 86% and 66% respectively. pA/pF vs. -0.065 ± 0.008 pA/pF, n=9). These results suggest that can be modulated by a-and- We conclude that cardiac Ca channel can "C.T L-type activity be modulated both positively and P-adrenergic stimulation in rat ventricular negatively by alterations in cytoskeletal filament systems. The results suggest that cytoskeletal hypertrophied myocytes. dynamics may be an important factor in regulating cardiac contractility.

202A VOLTAGE-GATED CA CHANNELS I MONDAY CA CHANNELS I 1194-1196

1194 - Pos 1195 - Poe PROTEOLYTIC PROCESSING OF THE C-TERMINUS OF THE alc SUBUNIT OF L- DUAL EFFECT OF PROTEIN KINASE C ON L-TYPE CALCIUM CHANNEL TYPE CALCIUM CHANNELS REVEALS NOVEL MECHANISMS OF CHANNEL ACTIVITY IN L6 CELL& REGULATION INVOLVING A PROLINE-RICH DOMAIN. Basil M Hastashl, Andrew P Thomas', Nevin Boparai2, John P. Reeves', 'UMDNJ-Grad. Sch. Brian L Gerhardsteis, Tianyan Gao, Moritz Bllnemans, Tipu S Puri, Adam Adair, Hong Ma, M Biomed. Sci., Dept. Pharmacology & Physiology, 185 South Orange Avenue, Newark, NJ 07103, Marlene Hosey, Northwestern University Medical School 2Rutgers University, New Brunswick, NJ 08901 Although most L-type calcium channel a,c subunits isolated from heart or brain are -190 kDa Rat skeletal myoblasts express the cardiac L-type Ca channel, as determined by functional assays proteins that lack -50 kDa of the C-terminus, the C-terminal domain is present and co-localized and sequence analysis by RT-PCR. Channel activity was measured as nifedipine-sensitive Ba (5 with channel subunits in intact cardiac myocytes. To test the hypothesis that the C-terminus is mM) influx in a depolarizing salt solution containing 140 mM K. Activation of protein kinase C processed but remains functionally associated with the channels, expressed, full-length a,c (PKC) with phorbol-12-myristate-13-acetate (PMA) inhibited the rate of Ba influx by 41%; an subunits were cleaved in vitro by chymotrypsin to generate C-terminal fragments of 30-56 kDa as inactive PMA analogue had no effect. The effects of S 100 nM PMA were completely blocked by well as a 190 kDa, C-terminally truncated protein that was similar in mass to the native C- bisindolylmaleide (Bit) or G(6983, specific inhibitors of PKC; at PMA concentrations >100 nM, terminally truncated protein. Interestingly, although the C-terminal fragments were very these agents were only partially effective. In the absence of PMA, Bis and G66983 alone inhibited hydrophilic, they remained associated with the membrane. A proline-rich domain (PRD) in the C- Ba uptake, effects that were enhanced following depletion of Ca stores by preincubation with terminal fragments was identified and found to be an important mediator of membrane association. ionomycin and thapsigargin. Downregulation of PKC by overnight incubation with PMA Deletion of the PRD resulted in the loss of membrane association of the C-terminal fragments. eliminated the acute effects of 100 nM PMA as well as the inhibition by Bis and Go6983. We The a,c PRD has strong homology to a PRD in dynamin that binds to SH3 domains. Indeed, the conclude that in the basal state, the activity of the cardiac L-type Ca channel is partly maintained aic PRD bound to SH3-domains in src, lyn, hck and the channel 32 subunit. When expressed by a stimulatory PKC isozyme. However, upon activation of PKC with PMA, the predominant together with P2 subunits, aic subunits lacking either the PRD or -50 kDa of the C-terminus effect is a net attenuation of Ca channel activity. The results suggest that different PKC isozymes, generated significantly increased barium currents, demonstrating that these are functionally or different degrees of PKC activation, exert opposing effects on L-type Ca channel activity in L6 inhibitory domains. cells.

1196- Pos EFFECTS OF NI'2 ON Cav3.2 (alK), A LOW VOLTAGE-ACTIVATED Ca2+ CHANNEL CLONED FROM HUMAN HEART Eric A. Ertel, Loic Perchenet, Hoffmann-LaRoche, CH-4070 Basel Switzerland Ni2+ blocks low-voltage-activated (LVA) Ca2+ 100- 25 ochannels preferentially over high-voltage-activated 80i / 20 (HVA) channels. However, Ni+ blocks HVA Block V,a channelsCa2'in a complex fashion, which cannot be described by an action at a single site. This 60 \15 complexity suggests that it is difficult to use Ni2+ block as an unequivocal criterion to clmasify Ca2+ 40 10 channels as LVA (T-type) or HVA (L, N, P, Q-type). Here we examnine the effects of external Ni2+ on the 20 5~~~~~~cloned T-type Ca2' channel Ca,3.2. We find that Ni2+ dose-dependently blocks Cav3.2 in a complex 0 ,; 0 manner. Ni2+ produces: 1) a decrease in current 0 1 PM 1 mM amplitude, 2) a positive shift in voltage-dependence [NFI" of activation (Vm0), 3) an acceleration of deactivation, and 4) no effect on inactivation. Furthermore, the dose-response curves for Ni2+ block in external Ca2+ are markedly biphasic. The shift in V10 and the plateau of the dose-response curve occur at similar Ni2+ concentrations, suggesting that they are related. Yet, one expects that a positive shift in V,2 would increase the apparent block at a fixed potential, rather than weaken it as it is observed. Furthermore, if Ba2+ replaces Ca2+ as the permeant ion, the shift in V,0 remains whereas the plateau of the dose-response curve disappears. Thus, the relationship between shift and plateau remains unclear. The shift in V,0 is well described by a simple model where binding to a saturable site results in altered gating. This suggests the existence of at least two Ni2+ binding sites on Cav3.2: one blocking perneation (high-affinity) and one modulating voltage-dependent gating (low-affinity).

MONDAY KCNQ/MINK & OTHER K+ CHANNELS 1197-1198

1197- Pos 1198 - Pot DISPARATE EFFECTS OF RUBIDIUM ON HOMOMERIC KCNQ1 AND A NOVEL PROTEIN PARTNER FOR THE MinK K+ CHANNEL SUBUNIT. HETEROMERIC KCNQI/MINK K CHANNELS. Sabina Kupershmidt, Margaret Sutherland, Nancy A. Sugg, Dan M. Roden, Vanderbilt Michael Pusch, Lara Bertorello, Franco Conti, CNR, Via de Marini 6, Genova, 16149 University, Nashville, TN 37232 The cardiac K channel, IKs, is formed by an association of the Shaker-like KCNQI protein and The delayed rectifier I y, plays an important role in the repolarization phase of the cardiac action the small protein minK. Heteromeric KCNQI/minK and homomeric KCNQI channels have potential. Two protein subunits, KvLQTI and minK, are required for the recapitulation of this different properties. We studied them in oocytes using two-electrode and patch-clamp in high K current in heterologous systema. and Rb. Inward tail currents (I.,j) of homomeric KCNQI are increased about 3-fold upon We have used the intracellular 63 substitution of K with Rb whereas amino acids of the minK protein as a bait in a yeast-two-hybrid kinetics of I,^ and steady-state activation are only slightly assay. One of the proteins we identified as a partner is the cardiac-specific, four-and-a-half LIM changed. Single channel conductance at negative voltages (y) was estimated by noise analysis. Rb domain containing protein.fhI2. Although the function of fhl2 is unknown, other members of the has only a small effect on homomers (1.2 fold increase) at a 5 kHz bandwidth. Apparent y was LIM domain family of proteins function as protein interaction modules and some have been decreased after filtering at lower frequencies (f) indicative of a fast "flickery" process. The ratio implicated in the development of muscle and heart. We demonstrated physiological relevance of yRb/yK increased at lower f (about 2 fold at 10 Hz), suggesting that the main effect of Rb is to this interaction, by showing that the two proteins can be co- immunoprecipitated from mouse heart decrease the probability of channel "blockage". I,. of heteromers are reduced 2-fold in Rb and and that they interact in a GST-pulldown assay. show a pronounced sigmoidal a time-course. y of heteromers is 2-fold smaller in Rb at 5 kHz. Using our minK knock-out/ lacZ knock-in mouse model to localize minK expression in the mouse Filtering more reduces yRb and YK, while yRb/Ye iS independent of f. Our results suggest the heart, and in situ hybridization for ld2, we demonstrated that the two proteins are expressed in presence of a rapid process that occurs independendy of the gating state. Rb favors the "flicker- overlapping regions of the mouse heart. This interaction links a voltage gated K' channel to a open" state and slows flickering in homomers, whereas Rb does not affect flickering in protein which is probably not directly involved in ion flow, but has the potential to interact with heteromers, indicating that minK interacts with KCNQ1 in the outer vestibule of the channel. other proteins, via its LIM domains. Studies investigating the functional significance of this (Supported by Telethon Italy, grant 1079.) interaction are currently underway.

203A 1199-1204 KCNQ/MINK & OTHER K+ CHANNELS MONDAY

1199 - Pos 1200- Pos MAPPING THE BINDING SITE OF THE CHROMANOL 293B ON THE IKrCHANNEL MOLECULAR BASIS FOR DIFFERENTIAL SENSITIVITY OF KCNQ1 AND IKs Christian Lerchel, G. Seebohm2, C. I. Wagner2, U. Gerlach2, A. Brfiggemann2, B. Attali3, A. E. CHANNELS TO THE COGNITIVE ENHANCER XE991. Busch2, 'University of Ttibingen, Ttlbingen, 72076 Germany, 'HMR Deutschland GmbH, Hong-Sheng Wang', B S Brown', D McKinnon', I S Cohen', 'Dept Physiology and Biophysics, 3Weizmann Instute of Science, Rehovot, Israel SUNY at Stony Brook, Stony Brook, NY 11794-8661, 'General Pharmacology, DuPont The KvLQTI channel, encoded by the KCNQI gene, coassembles with the transmembrane protein Pharmaceuticals Company, Wilmington, DE 19880-0400, 'Dept Neurobiology and Behavior, IsK to form the cardiac IKo-conductance, which contributes to the repolarization of the cardiac SUNY at Stony Brook, NY 11794-5230 action potential. As previously reported (Lerche et al. (1999), Biophys J 76, A89) the chromanol XE991, 10,10-bis(4-pyridinylmethyl)-9(lOH)-anthracnone, has been shown to be an equally 293B specifically blocks IrK-channels, heterologously expressed in Xenopus-Oocytes with an ICs5 potent inhibitor of potassium currents resulting from the expression of either KCNQ2/KCNQ3, of 6 gsM, while KvLQTI alone is only blocked by a significantly higher concentration of 30 FM. which form heteromeric channels that underlie the M-current in the nervous system, or KCNQI Furthermore 293B has almost no affinity to KCNQ2, another potassium channel of the same (KvLQTI), which, along with KCNEI (minK), underlies Ir, in cardiac myocytes. Although family. By building chimeric proteins of these two chamels we identified the S5/H5/S6 region to XE991 enhances neurotransrnitter release in wtro and in wvo, an effect attributed to M-current be involved in binding the chromanol. We could transfer the 293B binding site to KCNQ2 by inhibition, it exerts no known cardiac effects. To explore the possibility that this apparent tissue substituting the region S5 to S6 in KCNQ2 with the equivalent of KvLQTI. Moreover, we specific action of XE991 is due to a differential block of KCNQI alone and KCNQl+minK, these strikingly reduced the block of 293B on KvLQTI by mutating both S6 and S5/H5 to the sequence channels were heterologously expressed in Xenopus oocytes, and their blockade by XE991 was of KCNQ2. Finally single aminoacids located in H5 and S6 could be identified to be crucial for compared. Coexpression with minK markedly decreased the sensitivity of KCNQI to blockade by the drug to block. Taking together these results and the findings of Seebohm et al. (1999, Biophys XE991. When measured at the end of a 500 ms step, XE991 blockade of the KCNQl+minK J 76, A187) we can postulate 293B to be an open channel blocker of the Ilc, channel acting within current had a KD of 11.1 i± 1.8 pM (n=7), approximnately 14 fold less sensitive than the block of the the pore region most likely from the inside. KCNQI current (KD"0.78 ± 0.05 pM, n=6). In addition, XE991 reduced activation and deactivation time constants and caused a rightward shift in the GN activation curve of the KCNQl+minK channel, but affected none of these parameters of KCNQI alone. Also, XE991 block of KCNQl+minK, but not of KCNQI, was time and voltage-dependent with greater block observed with longer depolarizations and slightly lower affinity at more positive potentials. Thus, the presence of the minK subunit in native It, channels is likely to underlie the tissue specific actions of XE991.

1201 - Pos 1202 - Pos CONFORMATION OF EXTRACELLULAR, TRANSMEMBRANE AND OUTER PORE MUTATIONS OF KVLQT1 CHANNELS: INFLUENCE OF MINK CO- INTRACELLULAR DOMAINS OF THE MIRPl POTASSIUM CHANNEL SUBUNIT IN ASSEMBLY ON TEA+-SENSITIVITY AND CHANNEL GATING. AQUEOUS AND LIPID ENVIRONMENTS. Junko Kurolcaws, H Motoike, A Morales, A Dadzie, Robert S. Kass, Columbia University, Geoffrey W. Abbott', Bala Ramesh2, Surjit K.S. Srai2, 'Yale University School of Medicine, 630W 168th Street, PH7W-318, New York, New York 10032 Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, Common residues in the extracellular loop of the Shaker K+ channel outer pore region coordinate 2Royal Free Hospital School of Medicine, University of London, U.K. TEA+-binding and C-type inactivation. To determine whether this region affects the characteristic MiRPI (minK-related peptide 1), the second member of the KCNE superfamily of single slow gating of Ir,. channels, we mutated two residues (K318; V319) at equivalent positions of the transmembrane (TM) domain ion channel subunits, co-assembles with HERG subunits to form the KvLQTl. Effects of the mutations on gating and TEA+ sensitivity were investigated in transiently- IKr cardiac potassium channel complex. To investigate the conformation and surface contact transfected CHO cells. KvLQTl constructs were expressed with and without minK. In the requirements of MiRPI we synthesised the three predicted domains of human MiRPl and studied absence of minK, both the double mutant (K318I + V319Y) and the single mutant V319Y channel them using FTIR spectroscopy. The extracellular (N-terminal) domain was predominantly were reversibly and completely blocked by extracellular TEA+ (5 mM). This contrasts with wild unordered in aqueous media, but a-helical in lysophosphatidylcholine (LPC) micelles. The TM type (WT) KvLQTI channels which were not completely blocked by > 50 mM TEA+. domain was insoluble in aqueous solution, but mainly a-helical with a minor contribution from Surprisingly, both outer pore mutants altered channel kinetics dramatically, promoting intramolecular n-strand in LPC micelles; previous functional studies suggest this extended portion inactivation. When these KvLQTI constructs were co-expressed with minK, the heteromultimeric lies centrally in the conserved FXF motif of the TM domain. The intracellular C-terminal (CT) channels retained the TEA' sensitivity of the homomultimeric KvLQTI channels. This result domain was soluble only in non-aqueous environments, helicity increasing with the solvent order provides evidence that minK co-assembly does not markedly alter the structure of the outer pore SDS, dimyristoylphosphatidylcholine (DMPC), LPC, trifluoroethanol. CT domain conformation of the KvLQTI channel. However, when co-expressed with minK, neither KvLQTI mutant was affected by the DMPC gel-fluid phase transition, indicating lipid insertion. This suggests that significantly affected the kinetics of Ito. currents. These results strongly suggest that minK may in vivo the CT domain may loop back into the plasma membrane, or associate with hydrophobic affect the gating of Irc, channels via either direct or allosteric interactions distinct from the outer patches on intracellular HERG domains. Thus, the three MiRPI domains depend upon pore region of the KvLQTI subunit. hydrophobic contacts for solubility and adoption of ordered structure, consistent with their functional roles in cardiac IKr channel complexes based on mutagenesis studies. Supported by NIH-ROlGM51851 to S. Goldstein.

1203 - Pos 1204 - Pos KVLQT1 GATING MODULATION AND SUBUNIT ASSOCIATION ARE MEDIATED MiRPI MUTANT ASSOCIATED WITH QUINIDINE-INDUCED CARDIAC BY SEPARATE MINK DOMAINS. ARRHYTHMIA SUPPORTS A GENETIC CONTRIBUTION TO "ACQUIRED" Andrew R. Tapper, Alfred L. George, Jr., Department of Pharmacology, Vanderbilt University DISEASE School of Medicine, Nashville, TN 37232 Federico Sestil, Geoff W Abbott', J. Wei', D. M. Roden', A. L. George2, Steve A. goldstein', When expressed alone in heterelogous expression systems KvLQTl channels elicit a rapidly 'Yale Medical School dept. of Pediatrics and Cellular and Molecular Physiology, 2Vanderbilt activating K+ current that slowly deactivates. MinK modulates KvLQTI, greatly slowing University Medical Center dept. of Pharmacology and Medicine activation, increasing current amplitude, and removing inactivation. Using deletion and chimeric MiRPI (minK related peptide 1), a member of an emerging family of single analysis we have determined that gating modulation and subunit association are mediated by transmembrane subunits, assembles with HERG to form cardiac Ic, channels. MiRPl has been separate MinK domains. Coexpression of KvLQTI with a MinK carboxy terminus deletion associated with long QT syndrome, torsades de pointes (TdP) and ventricular fibrillation (VF) mutant (MinKCTD) in Xenopus oocytes resulted in a rapidly activated K+ current identical to (Abbott et al., Cell 97:175-187, 1999). Here, we compare wild type MiRPI/HERG complexes and current recorded from oocytes expressing KvLQTI alone. To determine if MinKCTD was channels formed with a newly-recognized mutant. Al 16V-MiRPI was identified in a 44 year-old associated with KvLQTl, a MinK fimctional tag (G55C) that makes current susceptible to partial female with previous VF and syncope and TdP while on quinidine therapy. block by extemal Cd'+(Tai and Goldstein. Nature 391: 605-8, 1998) was engineered into the transmembrane Channel subunits expressed in Chinese Hamster Ovary (CHO) cells were studied by domain of MinKCTD. Currents resulting from coexpression of KvLQTl with patch-clamp technique. The mutation shifted half-maximal MinKCTD were Cd2+ sensitive suggesting that MinKCTD does associate with KvLQTI but does activation voltage from -21 to -27 mV not modulate gating. To determine which MinK regions and speeded deactivation (Tf = 80 to 51 ms). CHO cells expressing the mutant revealed peak are sufficient for KvLQTI association current densities at -40 mV and modulation, chimeras were generated between MinK and either MinK Related Protein 1 that were decreased from 7 to 4 pA/pF. While single-channel analysis revealed no significant differences, mutant subunits had a shorter mean lifetime at the cell surface. (MiRPl) or the Nae channel beta-I subunit. A chimera consisting of the MinK carboxy terminus These in a MiRPI background could modulate KvLQTI gating, whereas chimeras between MinK and changes produced a 3-fold decrease in potassium flux during a simulated cardiac action beta-l could only modulate KvLQTI if they contained both the MinK transmembrane domain and potential. Conversely, wild type and mutant channels exhibited the same sensitivity to quinidine carboxy terminus. Together these results suggest that the MinK carboxy terminus is necessary but (K1 = 0.8 AM). The findings further support our theory that "acquired" arrhythmia may result from not sufficient for gating modulation. Subunit association via an interaction between the MinK an inherited predisposition combined with additional stressors that diminish capacity of the transmembrane domain and KvLQTl is required for gating modulation. myocardium to repolarize normally (repolarization reserve).

204A MONDAY KCNQ/MINK & OTHER K+ CHANNELS 1205-1210

1205 - Pos 1206- Pos IDENTIFICATION OF A NOVEL REGION INVOLVED IN KVLQT1 INACTIVATION A NOVEL GENE, RKCNQ2, ENCODES THE VOLTAGE-DEPENDENT POTASSIUM Guiscard Seebohm', Christian Lerche2, Andrea Bruggemann', Andreas E Busch', 'Hoechst CHANNEL IN RAT BRAIN. Marion Roussel, DG Cardiovascular Disease, Building H821, Frankfurt am Main, 65926 Flora Jow, KeWei Wang, Wyeth-Ayerst Research Germany, 2University of Tiibingen, Tuibingen, 72076 Germany Human KCNQ2 is a recently cloned novel K+ channel gene responsible for benign familial The voltage gated potassium channel KvLQTI shows a delayed inactivation at pulses more neonatal convulsions (BFNC), a form of epilepsy. By homologue cloning we have isolated from a positive than -30 mV. This inactivation differs from classical C-type inactivation and inactivation brain cDNA library, rKCNQ2, a rat version of KCNQ2. The open reading frame of the translated in Herg channels. On the other hand KvLQTl inactivation seems to depend strongly on the protein comprises 852 amino acids with 6 transmembrane segments and a pore motif between S5 activation of channels like the classical N-type inactivation of Shaker potassium channels (tethered and S6. rKCNQ2 shares 96% similarity with human KCNQ2 at the amino acid level. Multiple ball and chain mechanism). To identify regions involved in the inactivation process we tissue Northerm blot analysis shows an exclusive expression and distribution in CNS and reveals a constructed functional chimeras of KvLQT I and KCNQ 2 potassium channel of the same family. transcript size of 8.6-kb. Functional expression of rKCNQ2 in a HEK 293 cell line by whole-cell In the Xenopus expression system KCNQ 2 potassium channels don't show an inactivation. current recording and in Xenopus oocytes by two-electrode voltage clamp shows outward K+ Introducing the sequence of KvLQTI from S5 to S6 into a KCNQ2 backbone gave inactivating selective currents that display delayed rectifier-type kinetics, showing no inactivation during 2.5- currents. The same KCNQ 2 region introduced into the KvLQT I backbone produced currents second pulses. The G-V curve, fitted with Boltzmann function, shows voltage dependence of with no detectable inactivation. We could map the borders for inactivation more detailed by two activation with a threshold of activation approximately -50 mV. The channels are fully open at 0 other chimeras to the S5, H5 region. Furthermore we performed point mutations in this important mV with half-maximal activation occurring at -30 mV, and the apparent gating charge is 2.6. region. Tetraethylammonium (TEA) and Barium (Ba2+) are found to effectively block the rKCNQ2 currents half-maximally at less than 1.0 mM. In addition, cAMP-dependent phosphorylation up- regulates rKCNQ2 current by -40% in responding cells.

1207- Pos 1208 - Pos MODULATION OF THE POTASSIUM CHANNEL KCNQ4 BY CYTOPLASMIC EFFECTS OF LINOPIRDINE ON K+ CURRENTS IN VESTIBULAR HAIR CELLS. FACTORS. Katherine J Rennie, T X Weng, B Pirtle, M J Correia, University of Texas Medical Branch, Dominik Oliver', Jost Ludwig', Bjom C. Schroeder2, Thomas J. Jentsch2, Bermd Fakler', 'Dept. Otolaryngology, 7.102 MRB, Galveston, TX 77555-1063 of Physiology, Tuebingen University, Ob dem Himmelreich 7, Tuebingen, D-72074 Germany, Linopirdine is a potent blocker of M type channels and a less potent blocker of other members of 2ZMNH, Hamburg University the KCNQ family of K+ channels such as KCNQ4, recently reported to be expressed in outer hair Recently, a new member of the KCNQ potassium channel family, KCNQ4, was cloned. It is cells of the mammalian cochlea (Kubisch et al. Cell, 96:437-446, 1999). We have tested the specifically expressed in outer hair cells (OHCs) of the mammalian cochlea and therefore a good effects of this drug on hair cells from the amniote vestibular system. Conventional patch-clamp candidate for the OHC potassium current, IK,n, whose molecular identity is still uncertain. We techniques were used to record whole cell currents from hair cells in thin slices of pigeon investigated KCNQ4 properties in giant patches from Xenopus oocytes heterologously expressing vestibular neuroepithelia and from hair cells non-enzymatically dissociated from gerbil this channel. In cell-attached patches from oocytes with several AA whole-cell current only very semicircular canals. In type I hair cells, 200 pM linopirdine reduced peak inward currents through small currents could be detected. After excision, current increased up to several nA. When patches the low-voltage activated K+ current (IKI) by up to 66%. In type II hair cells linopirdine blocked were crammed back into the oocyte, currents decreased again to initial levels, indicating that 33-96 % of the steady state outward current (n = 8, Kd < I pM) and unmasked a rapidly activating KCNQ4 is blocked by a factor present in the oocyte cytoplasm in a non-voltage-dependent and rapidly inactivating component (A current). The linopirdine-sensitive component showed manner. The current increase after patch excision was followed by run-down of current. Thus, steady-state inactivation suggesting it was a previously described delayed rectifier (IK10) some intracellular factor is also necessary to maintain channel activity. To test for channel Vestibular hair cells may therefore express K+ channels pertaining to the KCNQ family. phosphorylation, protein kinase A together with 1 mM Mg-ATP was applied to the intracellular Supported by NIDCD DC03287 (KJR) and DC01273 (MJC). face of excised patches, yielding a prominent increase of current. In addition to modulation of the current amplitude, patch excision exerted a surprising effect on the voltage-dependence of KCNQ4. While the half-activating voltage, Vh, of KCNQ4 of whole-cell currents was -17.8 mV, Vh was -50.9 mV in excised patches. Thus, channel activity as well as voltage-dependence are modulated by intracellular factors.

1209 - Pos 1210 - Pos STRUCTURAL DETERMINANTS OF HCN2 CHANNEL GATING: MINIMAL VIRAL KILLER TOXIN ACTS VIA ACTIVATION OF TOK1 POTASSIUM CHANNELS. FUNCTIONAL UNIT AND MUTATIONAL ANALYSIS OF THE S4 DOMAIN Theodore M. Shilh', Aamir Ahmed', Federico Sesti', Nitza Ilan', Stephen L. Sturley2, Steve A. N. Jun Chen, John S. Mitcheson, Michael C. Sanguinetti, University of Utah, 15 North 2030 East, Goldstein', 'Pediatrics and Molecular and Cellular Physiology, Yale University, 295 Congress Salt lake City, UT 84112 Ave, New Haven, CT 06536, 2Physiology and Pediatrics, Columbia University, New York, NY The hyperpolarization-activated, cyclic nucleotide-gated channel gene (HCN) encodes a subunit of 10032 the pacemaker channel in heart and neurons. Although structurally similar to Kv channels, HCN Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein channels only open upon membrane hyperpolarization. To explore the gating mechanism of HCN killer toxins that kill sensitive, virus-free cells. Killer yeasts are used to intentionally suppress channels, we systematically deleted N- and C- terminal regions of HCN2 and defined a minimal competing strains in commercial food and agriculture applications and may play a role in fungal functional unit (a.a. 131-619). Further deletions of the N- or C- termini resulted in loss of function. infections in immunocompromised patients. The toxins are species specific and have potential as Despite deletion of a large portion of the N- and C-terminal regions (including the alpha C-helix of anti-mycotic therapeutics. Mature KI toxin is an 178 residue a-P heterodimer that binds to cell the CNB domain), the channel functioned normally except for a small leftward shift in the voltage- wall ,-glucan receptors before acting at the membrane to produce leakage of potassium, protons dependence of activation. These results suggest that in contrast to the plant hyperpolarization- and small organic molecules leading to cell death. It had been thought that Kl killer toxin formed activated channel KATI, the N- and C-terminal regions of HCN2 do not contribute to gating. holes directly in yeast cell plasma membranes. Moreover, unlike CNG channels, the function of HCN2 is not dependent on the integrity of the We find that K1 toxin activates the plasma membrane potassium channel of yeast cells, CNB domain. The 9 positive charges in the S4 domain of HCN2 were individually mutated to TOKI (Ahmed et al. Cell. 1999. V99: in press). Whereas genetic deletion of TOKI confers Gln, and Ala or Cys. Mutations of K29 1, R294, R297, R300, R312 and R3 18 produced functional resistance to KI toxin, overexpression increases susceptibility. Cells expressing TOKI exhibit channels with altered gating properties, but mutations of K303, R309 and R3 15 resulted in loss of toxin-induced potassium flux while those lacking the gene do not. Moreover, Kl toxin acts in the function. These findings indicate that K303, R309 and R315 have important structural and/or absence of other viral or yeast cell products to increase the open probability of single TOKI functional roles in the HCN2 channel. channels. The effects of KI toxin on the biophysical attributes of TOK1 channels and mechanisms underlying cytolysis will be considered.

205A 1211-1216------KCNQ/MINK & OTHER K+ CHANNELS MONDAY

1211 - Pos 1212 - Pos ROLE OF THE CARBOXYL TAIL IN GATING OF THE OF THE YEAST K+ GYGD PORE MOTIFS IN NEIGHBORING SUBUNITS INTERACT TO DETERMINE K CHANNEL, TOKI. CHANNEL SELECTIVITY Stephen H. Loukin, Xin-Liang Zhou, Chris P. Palmer, Jeremiah Placido, Umair Alathar, Ching Mark L Chapman', Howard S Krovetz2, Antonius M. VanDongen3, 'Duke University Medical Kung, Yoshiro Saimi, University of Wisconsin, Lab of Molecular Biology, 1525 Linden Dr., Center, Dept of Pharmacology, C141 LSRC, Durham, NC 27707, 2NCSU College of Vet. Madison, WI 53706 Medicine, 3Duke University, Pharmacology, C138A LSRC Bldg, La Salle str, Durham, NC 27708 TokI conducts the prominent outward K+ current of the yeast plasma membrane. Although A new class of voltage-independent K channels has been identified with the unique feature that steady-state inward current is primarily prevented by a rapidly transiting "R" state which is most they contain two homologous, subunit-like domains. Each domain contains a pore-forming region likely an intrinsic property of the KI filter region (Loukin and Saimi, 1999, Biophys. J. in press), which is conserved with other K selective channels. A defining feature of these channels is a Tokl also transits more slowly into a set of closed "C" states. Mutations in the cytoplasmic end of characteristic sequence variation seen in the GYGD pore motifs. This variation, GxGD in one the transmembrane helices following either P-region (M6 or M8) disrupt the C states, suggesting domain and GYGx in the other, suggested an interaction between neighboring domains. Using that C may be structurally related to the deactivated state of other cation channels (Loukin et al., constructs of the drkl K channel, we found that mutations at the tyrosine position of the GYGD 1997, EMBO, 16:4817). From a non-directed mutagenesis study, we found that deletion of the motif significandy decreased both selectivity and conductance. Mutations at the aspartate position carboxyl tail restores the C state of one of the M6 mutants, T3221. Deletion of the carboxyl tail had no significant effect on either endpoint. Tandem constructs, engineered to mimic the sequence from the wild-type channel causes a large positive shift in the voltage at which Tokl remains in variation of two-domain channels, delineated a nonadditive intersubunit interaction between these the C states. Normal C-state voltage dependence can be restored by co-expression of a tail positions which was important in determining selectivity and conductance. This inter-subunit extending from S517 to E673. Preliminary evidence indicates that the wild-type cytoplasmic tail interaction between the Y and D positions explains the sequence variation seen in two-domain K of Tokl locks the channel in the O/R states, possibly by interacting with the internal mouth of the channels. permeation pathway in the region of C-state gating.

1213 - Pos 1214 - Pos IN VIVO ANALYSIS OF TWO-PORE POTASSIUM CHANNELS IN CAENORJIBDITIS PORE STRUCTURE OF THE SMALL Ca5+-ACTIVATED K+ CHANNEL, rSK2 - A ELEGANS. COMPARISON WITH THE KcsA PORE. James H. Thomas, Duncan Johnstone, Monika Tzoneva, David Reiner, Dept of Genetics, Heike Jaeger, S. Grissmer, University of Ulm, Albert-Einstein-Allee 1 1, Ulm, Germany D-89069 University of Washington, Seattle, WA 98195-7360 Germany The genome of the nematode C. elegans encodes about 50 members of the recently described family of two-pore domain (twk) potassium channels. From genetic screens, many dominant We addressed the question whether the crystal structure of the KcsA channel from Streptomyces mutations have been isolated that confer behavioral defects in C. elegans. We have found that lividans is a suitable model for the outer vestibule of small Ca2+ activated K+ channels. For this three of the affected genes, unc-11O, egl-23, and unc-S8, encode twk channels. In each, dominant appoach used different peptide toxins, ScTX, CTX and KTX, to block the outer pore of wt and gain-of-function (gi) alleles confer neuronal or muscle excitability defects, whereas null alleles mutant rSK2 channels. cause no obvious phenotype. In collaboration with us, M. Kunkel and L. Salkoff have shown that wild-type unc-110 expresses a potassium-selective channel, and that mutations unc-IIO(go The amino acid substitutions D341 E and N368H of rSK2 decrease the affinity for ScTX. Similar dramatically increase current magnitude. The gfmutations in all three genes result in single amino results were obtained in studies and on et al., acid changes in M2 or M4 (TM domains following each P domain), which probably line part of using apamin d-tubocurare SKI (Ishii J. Biol. Chem. 272:23195-23200, we the channel pore. We hypothesize that these changes destabilize the closed state of the channel. In 1997). Therefore conclude that ScTX and apamin interact with similar sites on the channel. Guided by the receptor on vivo, this would result in elevated potassium efflux from cells expressing the channels, reducing knowledge of the toxin Shaker and KcsA K+ channel we introduced point mutations into the rSK2 channel that should render it sensitive to the excitability. Expression patterns of GFP fusions to the unc-110 and unc-58 promoters are scorpion toxin charydotoxin (CTX). The rSK2 V342G mutant channel is blocked by KTX no consistent with this interpretation. We have also identified extragenic suppressors of unc-58(gJ) CTX and and mutations, potentially identifying other proteins required for twk function. Genetic analysis of twk- longer by ScTX. Like for the KcsA (Q58A) and Shaker (F425G) channel mutants a small encoded channels in C. elegans provides the first in vivo model for this large and poorly uncharged amino acid at this position is necessary for CTX binding. We conclude that the overall understood family of potassium channels. structure of the CTX receptor site of all three channels is very similar.

Supported by a grant from the DFG (Gr 848/4-2).

1215- Pos 1216 - Pos CLONING OF TWO NOVEL HUMAN TWO-PORE I+ CHANNELS CLOSELY IMPROVING THE PORE OF A HUMAN 2 P DOMAIN POTASSIUM CHANNEL RELATED TO TASK1. (hTPKC1) BY RANDOM MUTAGENESIS AND SELECTIVE PRESSURE IN Eleazar C Vega-Saenz de Miera, David Pountney, Wiiliam Coetzee, Bernardo Rudy, New York CEREVISIAE University School of Medicine, Physiology and Neuroscience, 550 First Avenue, New York, NY Detlef Boekenhaner', Steve A. N. Goldstein2, Mark H Pausch3, 'Yale University School of 10016 Medicine, Pediatrics, Boyer Center for Molecular Medicine, Room #436, 295 Congress Ave, New We have cloned two human members of the emerging 2-pore K+ channel (KT; K=K+, T=2-pore) Haven, CT 06536-0812, 2Yale University, 3Wyeth-Ayerst Research, Cyanamid Agricultural family, structurally related to TASK (KT3.1) (>64% identity). Based on sequence similarity and Research Center, Box 400, Princeton, NJ 08543-0400 phylogenetic analyses of all KT subunits cloned to date, we determined that together with TASK, Two P domain potassium are characterized by the presence of two P loops per channel subunit. the new sequences constitute a KT subfamily. Therefore, they have been named KT3.2 and KT3.3. hTPKCI was cloned fiom human brain using a degenerate PCR approach, as previously described Xenopus oocytes injected with KT3.2 cRNA (in ND96) had an E. of -89±2 mV (close to EK and (J. Molec. Med. 1998; 76: 13-20). The predicted protein has 83% identity with mTREK, which 45 mV more negative than in control oocytes). Two-electrode voltage clamping showed the appears to be its murine homologue. Its expression in Xenopus oocytes induces an outwardly- expression of an outward current in ND96. In KD98 (98mM KCI), both inward and outward rectifying current that is potassium selective, largely instantaneous and is non-inactivating. currents were 10 observed. Measurement of E,,, at different [K], reveals a slope of 48 mV per fold Yeast cells were rendered unable to grow without high levels of extemal potassium or in change K+ concentration. Unlike TASKI, KT3.2 currents were insensitive to changes in heterologous expression of a potassium channel gene by deletion of their transport genes trkl, 2. intracellular or extracellular pH in the physiological range. KT3.2 currents were inhibited by Expression of hTPKC1 conferred survival in low potassium medium at pH 6.8, but not at pH < activation of PKC by PMA (1OnM; block in 20 Whereas TASK expresses mainly 50"/% min). in the 4.7. To investigate the basis of this effect, hTPKCI was altered randomly by passage of a plasmid heart, KT3.2 expression was most prevalent in the cerebellum. The emergence new of members of bearing the hTPKCI gene through the mutation-prone bacterial strain XLI-Red (mutS, mutD, the KT will our family facilitate understanding of the structure-function of these channels. mutT). Three point mutations, all within the second P domain, were identified based on their Supported by American Heart Association grant 9951070T ability to confer growth to transport-deficient cells in low potassium medium at pH 4.7. The attributes of wild type and mutant channels in yeast cells and Xenopus oocytes will be considered.

206A MONDAY KCNQ/MINK & OTHER K+ CHANNELS 1217

1217 - Pos TASK-3, A NEW MEMBER OF THE TANDEM PORE K+ CHANNEL FAMILY Yangmi Kim, Hyoweon Bang, Donghee Kim, Chicago Medical School, 3333 Green Bay Road, North Chicago, IL Most recently described family of K+ channels has four transmembrane (4TM) segments and two pore-forming (2P) domains. We have isolated a complementary DNA encoding a new member of this tandem pore K+ channel family from rat cerebellum cDNA library. Its amino acid sequence shares 52% homology with that of TASK-I, but less than 30%/o with those of TASK-2 and other tandem pore K+ channels (TWIK, TREK, TRAAK). Therefore, the new clone was named TASK- 3. RT-PCR analysis showed that TASK-3 mRNA is expressed in many rat tissues including brain, ventricle, kidney, liver, lung, colon, stomach, spleen, testis, and skeletal muscle. When expressed in COS-7 cells, TASK-3 exhibited a time-independent, non-inactivating K+-selective current. Single-channel conductance was 27 pS at -60 mV and 17 pS at +60 mV, and the mean open time was 1.1 ms when measured in symmetrical 140 mM KCI. TASK-3 current was highly sensitive to changes in extracellular pH (pH.), a halhnark of the TASK family of K+ channels. Thus, a change in pH. from 7.2 to 6.4 and 6.0 decreased TASK-3 current by -50%/o and -90%, respectively. K+ channel blockers such as tetraethylammonium (I mM), quinidine (100 FM) and Ba2+ (3 mM) reduced the TASK-3 current by 0%, 35% and 60%, respectively. Lidocaine (I mM) reduced TASK-3 current by 42%. Thus, TASK-3 is a new member of the acid-sensing K+ channel subfamily (TASK) and may be involved in pH-sensitive regulation of K+ conductance in many types of cells.

MONDAY K+ CHANNEL REGULATION 1219-1222

1219 - Pos 1220 - Pos ACCELERATION OF P/C-TYPE INACTIVATION IN VOLTAGE-GATED K* H202 INCREASES A HUMAN VOLTAGE-GATED K+ CHANNEL hKvl.5 CURRENT BY CHANNELS BY METHIONINE OXIDATION. SHIFFING THE VOLTAGE DEPENDENCE OF ACTIVATION. Jianguo Chen', V. B. Avdonin', M. Ciorba', S. H. Heinemann2, 'The University of Iowa, Bowen Yong-Geun Kwak, SW Chae, Chonbuk National University Medical School, Pharmacology, 5660, Iowa City, IA, 2Universitat Jena Keum-Am Dong San 2-20, Chonju, Chonbuk 560-182 Korea, Republic of We showed previously that methionine oxidation and reduction mediated by the enzyme H202 has several adverse effects on the myocardium including induction of cardiac arrhythmias methionine sulfoxide reductase regulate N-type inactivation in the ShC/B potassium channel during myocardial ischemia and subsequent reperfusion. The underlying ionic mechanisms of mediated by the ball and chain mechanism. We investigated how methionine oxidation affects H202 effects on myocardium remain unclear. In addition, voltage-gated K+ channels represent the P/C-type inactivation in the ShB 6-46:T449S channel without N-type inactivation. We found that most complex group of ion channel genes expressed in cardiovascular system. The human Kvl.5 the time course of P/C-type inactivation of this channel expressed in Xenopus oocytes is variable channel (hKvl.5) represents the IK., repolarizing current in atrial myocytes. The hKvl.5 channel is and that it was irreversibly accelerated by patch excision. The single-channel recordings showed functionally modulated by the Kvbeta subunits. In present study, we examined the effects of H202 that patch excision decreased the mean open and burst durations. The effect of patch excision was following coexpression of hKvl.5 with the Kvbetal.3 subunit in Ltk- cells and HEK-293 cells. mimicked by application of H202 or 02- Substitution of a methionine residue located in the P- H202 (1 mM) reversibly increased the K+ currents in HEK-293 cells transected with hKvl.5 alone segment of the channel with a leucine largely eliminated the channel's sensitivity to patch (315% increase at +50 mV, n=6) as well as in the cells cotransfected with Kvbetal.3 (42±5%, excision, H202, and high 02- The results suggcst that oxidation of methionine is an important n=5) or Kvbeta2.1 subunits (36±6%, n=7). This increase in K' current was associated with marked regulator of P/C-type inactivation and that it may play a role in mediating the cellular responses to slowing of macroscopic inactivation and hyperpolarizing shift of activation curve by about 20 mV. hypoxia/hyperoxia. Supported by NIH GM57654. Additionally, H202 increased the K+ currents in human atrial myocytes (68±7%, n=4). These results clarified the biophysical mechanism of H202 effects on hKv1.5 current, thereby being able to explain the potential roles ofH202 during myocardial reperfusion injury.

1221 - Pos 1222 - Pos MODULATION OF hKv1.1 AND hKvl.2 VOLTAGE GATING AND C-TYPE TGF-0 UPREGULATES Kvl3 CHANNELS IN MICROGLIA. INACTIVATION BY 5-HT2C RECEPTORS Tom SchillNg', Vladimir V. Cherny, Wei Zhou2, Fred N. Quande, Uwe Heinemann', Thomas E. P. Imbrici', S. J. Tucker2, M.C. DAdamo', F. Sponcichetti', Mauro Pessia', 'Istituto di Ricerche DeCoursey9, Claudia Eder', 'Humboldt University Berlin, Tucholskystr. 2, Berlin, 10117 Farmacologiche "Mario Negri" Italy, 2University, Laboratory of Physiology, Oxford, OXI 3PT, Germany, 2Rush University, Chicago UK. Effects of transforming growth factor-P (TGF-P) on K+ channel expression were studied in cultured The activity of voltage-gated potassium channels is dynamically modulated by several events murine microglial oells. In TGF-,-treated microglia, delayed rectifier (DR) cunrent density was >6- including neurotransmitters stimulated biochemical cascades mediated by G protein-coupled fold large than in untreated microglia. The TGF-p-induced up-regulation of DR channels could be receptors. The superfusion of oocytes expressing 5-HT2 receptors and either hKvl.l or hKvl.2 inhibited by the protein kinase inhibitor H7. subunits with solutions containing 5-HT inhibited channel activity in a concentration-dependent DR currents of TGF-,B-treated cells exhibited the following properties: activation at fashion (Kd= 30-il0 pM, n=15). Moreover, the voltage-dependent gating (n=41) and the C-type than inactivation properties (n=52) of both channels were potentials more positive -40 mV, half-maximal activation at -27 mV, half-maximal significantly affected. The voltage-dependent inactivation at -38 mV, time-and use-dependent inactivation. The average single DR parameters of activation V1,2 and the slope factor k, computed from the Boltzmann fit to the channel relevant hKvl.l tail conductance was 13 pS in Ringer's solution. DR channels were highly sensitive to charybdotoxin currents, were respectively -27.0i+0.9 mV and 7.9t0.8 mV (n=6) in control and kaliotoxin, but not conditions and, -29.8g1.6 mV and 4.3*0.7 mV (n=6) after the application of 5HT O.1nM. The a-dendrotoxin. hKvl.l C-type inactivation time constant at +2OmV was 3.2±0.3 s (n=32) in control conditions Using RT-PCR, mRNA for Kvl.3 was detected in microglia. In parallel with the observed and, 0.9440.2 s (n=6) after the application of 5HT InM. Orthovanadate, a tyrosine phosphatase changes in DR current density, the mRNA level for Kvl.3 increased 4.7-fold after treatment of inhibitor, mimicked all the effects mediated by 5-HT2c receptors stimulation on both hKvl.l and microglia with TGF-P. hKvl.2 channels. In contrast, genistein (5-50 gM), a tyrosine kinase inhibitor, blocked these effects. The results suggest that the stimulation of 5-HT2c receptors inhibit hKvl.l and hKvl.2 Supported by DFG grant SFB 507/C3, NIH grant HL52671 and the Michael Reese Foundation. channel activity and modulates the voltage-dependent gating and the C-type inactivation kinetics of both channels by affecting the phosphorylated/dephosphorylated state of the channel. The molecular mechanisms underlying these phenomena are under investigation.

207A