Interaction of Cysteine String Proteins with the α1A Subunit of the P/Q-type Calcium Channel

• DOI: 10.1074/jbc.273.22.13488
• Publication Date: 2002-07-26T15:13:03Z
• Authors: Christian Leveque; Sandrine Pupier; Beatrice Marqueze; Lionel Geslin; Masakazu Kataoka; Masami Takahashi; Michel De Waard; Michael Seagar
• Keywords: N-type calcium channel; R-type calcium channel; STX1A; Syntaxin 3; Synaptobrevin; Synaptotagmin 1

Cysteine string proteins (Csps) are J-domain chaperone anchored at the surface of synaptic vesicles. Csps involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although molecular mechanisms unknown. Interactions between Csps, core (SNARE) complex, P/Q-type channels were therefore explored. Co-immunoprecipitation suggested that occur complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor labeled with125I-ω-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparentK D = 700 nm pH 7.4 4 °C) a fusion protein segment cytoplasmic loop linking homologous domains II-III α1A subunit (BI isoform, residues 780–969). Binding was specific as it displaced by unlabeled Csp1, no interactions detected other domains, VAMP, or 1A. A Csp site on is thus located within 200 residue can also bind I, synaptotagmin I. act to direct assembly disassembly exocytotic channel. 1The abbreviations used are: Csp(s), cysteine protein(s); Hsc70 Hsp70, heat shock proteins; MBP, maltose-binding protein; GST, glutathione S-transferase; vesicle-associated membrane synaptobrevin; TBS, Tris-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; synaptosomal associated 25-kDa BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis. initially discovered Drosophila neuronal antigens predominantly localized regions (1Zinsmaier K.E. Hofbauer A. Heimbeck G. Pflugfelder G.O. Buchner S. E. J. Neurogenet. 1990; 7: 15-29Crossref PubMed Scopus (186) Google Scholar). Subsequently identified inTorpedo (2Gundersen C.B. Umbach J.A. Neuron. 1992; 9: 527-537Abstract Full Text PDF (121) Scholar) mammals (3Mastrogiacomo Gundersen Mol. Brain Res. 1995; 28: 12-18Crossref (42) Scholar, 4Chamberlain L.H. 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Walter N. Benzer 977-980Crossref (319) due impaired excitation-secretion coupling nerve terminals (14Umbach Zinsmaier 13: 899-907Abstract (144) 15Heckmann Adelsberger H. Dudel 228: 167-170Crossref (25) Recent findings suggest default involves either deficit entry ability trigger (16Umbach 7203-7209Crossref These data consistent earlier results indicating positive modulators ω-conotoxin GVIA-sensitive from Torpedo electric organ, heterologously expressed inXenopus oocytes Fusion competent thought be docked active zone close proximity voltage-gated release. It has been proposed necessary for open response depolarization Scholar), biochemical evidence favor this reported. We have examined complex (17Sollner Whiteheart S.W. Brunner Erdjument-Bromage Geromanos Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref (2645) channels, which support major fraction transmitter many fields mammalian brain (18Wheeler D.B. Randall Tsien R.W. 258: 917-924Google (MBP)-Csp1 prepared (10Pupier purified affinity chromatography amylose column (New England Biolabs). GlutathioneS-transferase (GST) rabbit (BI-2 isoform) (19Mori Y. Friedrich Kim M.S. Mikami Nakai Ruth Bosse Homann F. Flockerzi V. Furuichi Mikoshiba Imoto Tanabe Numa 1991; 350: 398-402Crossref (712) 1A, VAMP 2, IV generated reported previously (20De Waard Liu Walker D. Scott V.E. Gurnett C.A. Campbell K.P. 385: 446-450Crossref (375) 21Charvin Lévêque Berton Raymond Shoji-Kasai De M.J. EMBO 16: 4591-4596Crossref (110) GST protein, including sequence III-IV α1A, constructed amplifiying base pairs 4561–4725 subcloning into pGEXKG. production induced cultures protease-deficientEscherichia coli strain BL21, lysates glutathione-agarose. 35S-Csp1 synthesized coupled transcription translation vitro, presence [35S]cysteine (>600 Ci/mmol, DuPont), using TNT™ system (Promega). Polyclonal antibodies against MBP-Csp1 A-Sepharose Fast Flow beads (Amersham Pharmacia Biotech). Immunoprecipitation of125I-ω-conotoxin MVIIC-labeled CHAPS extracts rat cerebellar P2 performed described (22Martin-Moutot Charvin Sato Nishiki Kozaki 6567-6570Abstract (115) incubated 2 h °C 10 μg anti-Csp saline (TBS) 0.3% (w/v) BSA. 35S-Csp·antibody then recovered mixing 1 °C, centrifuging 30 s 10000 × g, washed three times pellet resuspended ml scintillation solvant, 35S-Csp quantified counting. assays incubating vitro-translated35S-Csp 300 μl TBS 0.5% BSA glutathione-agarose Beads centrifugation same buffer, and35S-Csp Rat homogenates solubilized 1% indicated detergent (see figure legends) mm Hepes, 0.15m NaCl, MgCl2, EGTA, adjusted NaOH, 5 bound beads. once SDS-PAGE immunoblotting Spectroscopy BIAcore apparatus (Pharmacia Biosensor), immobilized sensor chip via covalently linked anti-GST antibodies. μm MBP-Csp MBP introduced flow rate μl/min 22 °C. Experiments order explore exocytosis. First we co-immunoprecipitation examine hypothesis associate components trimeric channels. membranes non-immune immunoglobulins, polyclonal raised absence MBP-Csp1, anti-VAMP Immune beads, washed, eluted sample buffer. Western blots probed directed (SNAP-25) VAMP. Anti-Csp found co-immunoprecipitate small total neither SNAP-25 (Fig.1). added excess MBP-Csp1-blocked recovery both Conversely, when antibody, SNARE all among immunoprecipitated (Fig. 1). trap whereas recover Csp/VAMP SNAP-25. While approach demonstrates population does occur, indicate whether these interact directly indirectly unidentified partners. Cross-linking bifunctional reagent disuccinimidyl suberate, failed reveal proteins. Furthermore vitro translated35S-Csp1 GST-VAMP did provide any two below). crude (P2) cerebellum 125I-ω-conotoxin MVIIC conditions allow labeling extracted As prelabeled monoclonal anti-syntaxin antibody 10H5 2) In contrast capture solubilized125I-ω-conotoxin receptors. amount radioactivity significantly different control preincubated large IgG used. detect endogenous recombinant produced [35S]cysteine. autoradiography revealed single radioactive species migrating 28 kDa accordance predicted mass (Fig.3 A, left panel). immunoglobulins 3 right staining predominant forms GST-II-IIIA, B) migrated 70, 43, 48, 27 respectively, apparent masses 21 polypeptide linker corresponding 780–969 BI isoform subunit. range concentrations GST-II-IIIAor GST. Thus assay considered being ligand. Saturable GST-II-IIIA occurred equilibrium dissociation constant C). Interaction identical concentration addition molar inhibited of35S-Csp1 GST-II-III D). No significant I-II D), amino- carboxyl-terminal (not shown) Surface plasmon resonance Biacore confirmed shown). monitored method again domain subunit, fused Preliminary diminished CHAPS. effect series detergents (digitonin, cholate, Lubrol, Triton X-100, Nonidet P-40, CHAPS) examined. All tested (at 1%, w/v) 60–80% required solubilize native membranes, possible Csp/calcium account our inability Fig. 2). possibility studied. Detergent GST-III-IVA, glutathione-Sepharose After washing, centrifugation, treated analyzed blotting A). This specifically despite X-100. Similar Csp. under agitation captured antibodies, quantity trapped alone B). Evidence points essential role neurotransmitters 14Umbach regulating 16Umbach asked composed vesicle protein: (VAMP) plasma membrane: constitute hub protein/protein lead up calcium-dependent (reviewed Refs.23Südhof T.C. 375: 645-653Crossref (1773) Scholar 24Bajjalieh 270: 1971-1974Abstract (185) 25Rettig Sheng Z.H. D.K. Hodson C.D. Snutch T.P. Catterall W.A. Proc. Natl. Acad. Sci. U. 93: 7363-7368Crossref (271) co-expression modulates gating properties (26Bezprozvanny 378: 623-627Crossref (382) regulate function interacting complex. Synaptophysin, interacts (27Edelmann L. Hanson P.I. Chapman E.R. Jahn R. 14: 224-231Crossref (391) Direct detected. translated bacterially palmitoylated, could require acylation. indirect mediated calcium-conducting pore formed contains four each six helical transmembrane segments GST-fusion II III displayed saturable [35S]cysteine-labeled moderate 7.4. binds approximately (residues isoform). Specific spectroscopy, chip. view Csp, important underline specificity binding. Hsp70 DnaJ-like chaperones polypeptides, selectivity. unfolded recognizing short stretches amino acids extended conformation. contrast, such generally exhibiting secondary tertiary structure, exhibit very low polypeptides conformations Ref. 28Cyr D.M. Langer Douglas M.G. Trends Biochem. 19: 176-181Abstract (403) Our demonstrate nonselectively irrespective their sequence, alone, reasonable assume folding provides closer approximation conformation than tag sequences. Although illustrated obtained vitrotranslated verified thatin [35S]methionine-labeled II-IIIA occurs partner protein. Finally selectively strongly implicated coupling. 722–1036, I 780–969, functional relevance supported injection equivalent α1B (29Sheng Z-H. Rettig Cook 379: 451-454Crossref (318) disrupts sympathetic neurons (30Mochida Z.-H. Baker Kobayashi 781-788Abstract (244) amphibian neuromuscular junction (31Rettig Heinemann Ashery Yokoyama C.T. Neher 6647-6656Crossref proteins, immunoprecipitation terminals. masking epitopes alternatively disruption during solubilization. GST-II-IIIAfusion 35S-Csp. prevent MBP-Csp, suggesting limiting parameter accessibility Syntaxin display higher (25Rettig stably (21Charvin 22Martin-Moutot exogenous Csps. If promote opening how intrinsic cooperation involved? Apart translation, contribute multi-protein complexes. (DnaK) (DnaJ) stabilization glucocorticoid aporeceptor 32Bohen S.P. Kralli Yamamoto K.R. 268: 1303-1304Crossref (183) bacteriophage λ DNA replication 33Georgopoulos 295-299Abstract (201) clathrin coated (34Ungewickell Ungewickell Holstein S.E.H. Lindner Prasad Barouch W. Martin Greene L.E. Eisenberg 632-635Crossref (399) Co-expression Q-type decreases availability stabilizing inactivated state principle facilitate activation promoting syntaxin. speculate coordinate sequential partners, consequently interesting note strikingly similar pattern evoked responses deletion motoneurones larvae (15Heckmann Both manipulations reduction synchronous release, while asynchronous paired pulse facilitation increased. regulation ensure transmission. Further studies will 1-SNAP-25-VAMP-synaptotagmin regulated Hsp70.