Interactions between presynaptic calcium channels and proteins implicated in synaptic vesicle trafficking and exocytosis.
Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized omega conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.
Seagar
M
M
INSERM Unité 464, Institut Jean Roche, Faculté de Médecine, Secteur Nord, Marseille, France.
Takahashi
M
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D020864:Calcium Channels, N-Type; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins; D002645:Chickens; D005089:Exocytosis; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology*; D017729:Presynaptic Terminals / Q000378:metabolism*; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D050827:Syntaxin 1; D020866:omega-Conotoxin GVIA
D002118:Calcium; D015220:Calcium Channels; D020864:Calcium Channels, N-Type; D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D000254:Sodium-Potassium-Exchanging ATPase; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D050827:Syntaxin 1; D020866:omega-Conotoxin GVIA; C494209:Snap25 protein, mouse; C494210:Snap25 protein, rat; C494214:Stx1a protein, mouse; C494213:Stx1a protein, rat; C053157:omega-conotoxin receptor