Abstract
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.
MeSH terms
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Animals
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Calcium / metabolism
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Calcium Channels / immunology
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Calcium Channels / isolation & purification
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Calcium Channels / metabolism*
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Calcium Channels, N-Type*
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Calcium Signaling
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Calcium-Binding Proteins*
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Chickens
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Exocytosis
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Macromolecular Substances
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Membrane Glycoproteins / immunology
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Membrane Glycoproteins / isolation & purification
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Membrane Glycoproteins / metabolism
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Membrane Proteins / immunology
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Membrane Proteins / isolation & purification
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Membrane Proteins / metabolism
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Mice
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Models, Biological
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Nerve Tissue Proteins / immunology
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Nerve Tissue Proteins / isolation & purification
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Nerve Tissue Proteins / metabolism*
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Peptides / pharmacology*
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Presynaptic Terminals / metabolism*
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Protein Binding
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Qa-SNARE Proteins
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R-SNARE Proteins
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Rats
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Sodium-Potassium-Exchanging ATPase / metabolism
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Synaptic Vesicles / metabolism*
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Synaptosomal-Associated Protein 25
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Synaptotagmins
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Syntaxin 1
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omega-Conotoxin GVIA
Substances
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Calcium Channels
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Calcium Channels, N-Type
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Calcium-Binding Proteins
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Macromolecular Substances
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Membrane Glycoproteins
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Membrane Proteins
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Nerve Tissue Proteins
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Peptides
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Qa-SNARE Proteins
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R-SNARE Proteins
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Snap25 protein, mouse
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Snap25 protein, rat
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Stx1a protein, mouse
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Stx1a protein, rat
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Synaptosomal-Associated Protein 25
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Syntaxin 1
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omega-conotoxin receptor
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voltage-dependent calcium channel (P-Q type)
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Synaptotagmins
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omega-Conotoxin GVIA
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Sodium-Potassium-Exchanging ATPase
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Calcium