Purification of the N-type calcium channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis

• Published in: The Journal of biological chemistry 1994-03, Vol.269 (9), p.6306-6312
• Main Authors: Lévêque, C, el Far, O, Martin-Moutot, N, Sato, K, Kato, R, Takahashi, M, Seagar, M J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Biological and medical sciences; Brain - metabolism; Calcium Channel Blockers - pharmacology; Calcium Channels - drug effects; Calcium Channels - isolation & purification; Calcium Channels - metabolism; Calcium-Binding Proteins; Cell membranes. Ionic channels. Membrane pores; Cell structures and functions; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Exocytosis; Fundamental and applied biological sciences. Psychology; Immunoblotting; Membrane Glycoproteins - isolation & purification; Membrane Glycoproteins - metabolism; Molecular and cellular biology; Molecular Sequence Data; Molecular Weight; Nerve Tissue Proteins - isolation & purification; Nerve Tissue Proteins - metabolism; omega-Conotoxins; Peptides - chemical synthesis; Peptides - immunology; Peptides - pharmacology; Rats; Rats, Wistar; Synaptic Membranes - metabolism; Synaptic Vesicles - metabolism; Synaptotagmins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37372-6

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.