Lévêque C, el Far O, Martin-Moutot N, Sato K, Kato R, Takahashi M, Seagar M J
Institut National de la Santé et de la Recherche Médicale U374, Institut Jean Roche, Faculté de Medécine, Secteur Nord, Moarseille, France.
J Biol Chem. 1994 Mar 4;269(9):6306-12.
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.
ω-芋螺毒素敏感的N型钙通道控制神经递质在神经末梢的释放，并与参与分泌的蛋白质相互作用。用针对钙通道α1亚基、 syntaxin和一种105 kDa质膜蛋白的抗体对大鼠脑突触膜中可溶解的ω-芋螺毒素受体进行免疫沉淀。通过涉及抗syntaxin免疫亲和层析的方法纯化了一种由钙通道亚基和显示出不同程度关联的突触蛋白组成的多聚体复合物。通过用125I-叠氮硝基苯甲酰ω-芋螺毒素进行光亲和标记并用序列导向抗体进行免疫印迹，鉴定出一种含有cAMP依赖性磷酸化位点的250 kDa N型α1亚基。检测到一种免疫相关的210 kDa形式的α1亚基，其表现出不同的药理学和调节特性。在蔗糖梯度离心时，140、70、58和35 kDa的蛋白条带与纯化的α1亚基一起迁移，而105 kDa的蛋白被去除。58 kDa和35 kDa的条带分别包含突触囊泡蛋白突触结合蛋白和syntaxin，一种结合突触囊泡蛋白的质膜蛋白。纯化的ω-芋螺毒素受体被抗syntaxin抗体定量免疫沉淀。这些蛋白质可能构成一个孤立的胞吐复合物，其中N型钙通道与突触囊泡对接位点紧密相互作用。
