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 neurotransmi tter release at the nerve terminal and interact with proteins implicat ed in secretion. Solubilized omega-conotoxin receptors from rat brain synaptic membranes were immunoprecipitated by antibodies against calci um channel alpha 1 subunits, syntaxin, and a 105-kDa plasma membrane p rotein. A multimeric complex, composed of calcium channel subunits, an d synaptic proteins that showed varying degrees of association, was pu rified by a procedure involving anti-syntaxin immunoaffinity chromatog raphy. A 250-kDa N-type alpha 1 subunit, containing cAMP-dependent pho sphorylation site(s), was identified by photoaffinity labelling with I -125-azidonitrobenzoyl omega-conotoxin and immunoblotting with sequenc e-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 co migrated with purified alpha 1 subunits upon sucrose gradient centrifu gation, whereas the 105-kDa protein was removed. The 58- and 35-kDa ba nds contained, respectively, the synaptic vesicle protein synaptotagmi n and syntaxin, a plasma membrane protein that binds synaptic vesicle proteins. Purified omega-conotoxin receptors were quantitatively immun oprecipitated by anti-syntaxin antibodies. These proteins may constitu te an isolated exo-cytotic complex in which the N-type calcium channel tightly interacts with a synaptic vesicle docking site.