Purification of the N-type calcium channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis.
omega-Conotoxin-sensitive N-type calcium channels control neurotransmitter release at the nerve terminal and interact with proteins implicated in secretion. Solubilized omega-conotoxin receptors from rat brain synaptic membrane were immunoprecipitated by antibodies against calcium channel alpha 1 subunits, syntaxin, and a 105-kDa plasma membrane protein. A multimeric complex, composed of calcium channel subunits, and synaptic proteins that showed varying degrees of association, was purified by a procedure involving anti-syntaxin immunoaffinity chromatography. A 250-kDa N-type alpha 1 subunit, containing cAMP-dependent phosphorylation site(s), was identified by photoaffinity labeling with 125I-azidonitrobenzoyl omega-conotoxin and immunoblotting with sequence-directed antibodies. An immunologically related 210-kDa form of the alpha 1 subunit was detected that displayed different pharmacological and regulatory properties. Protein bands of 140, 70, 58, and 35 kDa comigrated with purified alpha 1 subunits upon sucrose gradient centrifugation, whereas the 105-kDa protein was removed. The 58- and 35-kDa bands contained, respectively, the synaptic vesicle protein synaptotagmin and syntaxin, a plasma membrane protein that binds synaptic vesicle proteins. Purified omega-contoxin receptors were quantitatively immunoprecipitated by anti-syntaxin antibodies. These proteins may constitute an isolated exocytotic complex in which the N-type calcium channel tightly interacts with a synaptic vesicle docking site.
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Institut National de la Santé et de la Recherche Médicale U374, Institut Jean Roche, Faculté de Medécine, Secteur Nord, Moarseille, France.
el Far
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O
Martin-Moutot
N
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Sato
K
K
Kato
R
R
Takahashi
M
M
Seagar
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013570:Synaptic Membranes / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*; D050857:Synaptotagmins; D020960:omega-Conotoxins
D000911:Antibodies, Monoclonal; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D020960:omega-Conotoxins; C053423:Conus magus toxin; D050857:Synaptotagmins