Interaction of cysteine string proteins with the alpha1A subunit of the P/Q-type calcium channel.
Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with 125I-omega-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparent KD = 700 nM at pH 7.4 and 4 degreesC) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the alpha1A calcium channel subunit (BI isoform, residues 780-969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.
Leveque
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C
Institut National de la Santé et de la Recherche Médicale, Unité 464, Institut Jean Roche, Faculté de Médecine Secteur Nord, Bd. Pierre Dramard, 13916 Marseille Cedex 20, France.
Pupier
S
S
Marqueze
B
B
Geslin
L
L
Kataoka
M
M
Takahashi
M
M
De Waard
M
M
Seagar
M
M
D000818:Animals; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D015220:Calcium Channels; D050956:HSP40 Heat-Shock Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; C088460:cysteine string protein