Wollner D A, Messner D J, Catterall W A
Department of Pharmacology, University of Washington School of Medicine, Seattle 98195.
J Biol Chem. 1987 Oct 25;262(30):14709-15.
The sodium channel purified from rat brain is composed of three subunits: alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000). alpha and beta 2 subunits are linked through disulfide bonds. Procedures are described for preparative isolation of the beta 1 and beta 2 subunits under native conditions. Pure beta 2 subunits obtained by this procedure were used to prepare a specific anti-beta 2 subunit antiserum. Antibodies purified from this serum by antigen affinity chromatography recognize only disulfide-linked alpha beta 2 complexes and beta 2 subunits in immunoblots, and immunoprecipitate 32P-labeled alpha subunits of purified sodium channels having intact disulfide bonds, but not those of sodium channels from which beta 2 subunits have been detached by reduction of disulfide bonds. These antibodies also immunoprecipitate 89% of the high affinity saxitoxin-binding sites from rat brain membranes, indicating that nearly all sodium channels in rat brain have disulfide-linked alpha beta 2 subunits. Approximately 22% of beta 2 subunits in adult rat brain are not disulfide-linked to alpha subunits. Anti-beta 2 subunit antibodies are specific for sodium channels in the central nervous system and will not cross-react with sodium channels in skeletal muscle or sciatic nerve. The brains of a broad range of vertebrate species, including electric eel, are shown to express sodium channels with disulfide-linked alpha beta 2 subunits.
α（分子量260,000）、β1（分子量36,000）和β2（分子量33,000）。α亚基和β2亚基通过二硫键相连。本文描述了在天然条件下制备分离β1和β2亚基的方法。通过该方法获得的纯β2亚基被用于制备特异性抗β2亚基抗血清。通过抗原亲和层析从该血清中纯化得到的抗体在免疫印迹中仅识别二硫键连接的αβ2复合物和β2亚基，并且能免疫沉淀具有完整二硫键的纯化钠通道的32P标记的α亚基，但不能沉淀通过二硫键还原而使β2亚基脱离的钠通道的α亚基。这些抗体还能从大鼠脑膜中免疫沉淀89%的高亲和力石房蛤毒素结合位点，表明大鼠脑中几乎所有的钠通道都具有二硫键连接的αβ2亚基。成年大鼠脑中约22%的β2亚基未与α亚基通过二硫键相连。抗β2亚基抗体对中枢神经系统中的钠通道具有特异性，不会与骨骼肌或坐骨神经中的钠通道发生交叉反应。包括电鳗在内的多种脊椎动物的脑均显示表达具有二硫键连接的αβ2亚基的钠通道。
