Study on brain calcium channel molecules with omega-conotoxin

合成ω-芋螺毒素GVIA的[^3H]丙酰基衍生物以检测毒素与脑膜组分的结合。该毒素与两个位点特异性结合，解离常数（Kd）分别为3 pM和3.5nM。结合不受二氢吡啶类或维拉帕米的影响。地尔硫卓特异性地阻断仅与高亲和力位点的结合。ω-芋螺毒素GVIA（GVIA）受体可以用各种去污剂溶解。毛地黄皂苷产量最高。可能只有高亲和力的网站被回收，从抑制毒素结合到溶解受体的地尔硫卓判断。从兔骨骼肌中纯化的抗L-型钙通道分子（二氢吡啶受体）的单克隆抗体沉淀了从牛和兔脑和牛心肌中溶解的DHP结合活性，超过80%。然而，抗体没有显著沉淀GVIA结合活性。这些结果有力地表明，GVIA受体分子大多不同于哺乳动物脑中的DHP受体分子。已经声称GVIA抑制神经系统中的L型和N型钙通道。我们的研究结果表明，GVIA不抑制哺乳动物大脑中的L通道。为了获得沉淀溶解的GVIA受体的单克隆抗体，我们用来自牛和大鼠前脑的突触膜组分免疫小鼠。在所获得的抗体中，有两种可沉淀约1/3的总溶解的GVIA受体。然而，同时添加这两种抗体仅略微增加沉淀，表明它们识别大多数相同的GVIA受体群体。这些抗体与M_r、36000或28000蛋白质发生免疫印迹反应。这些蛋白质高度集中在从小鸡到人类的各种动物的大脑中。

[^3H] Propionyl derivative of omega-conotoxin GVIA was synthesized to examine the binding of the toxin to brain membrane fractions. The toxin specifically bound to two sites with dissociation constants (K_d) of 3 pM and 3.5 nM. Binding was not affected by dihydropyridines or verapamil. Diltiazem specifically blocked the binding to only the high-affinity sites. The omega-conotoxin GVIA (GVIA) receptor could be solubilized with various detergents. Digitonin gave best yield. Probably only the high-affinity site was recovered, as judged from the inhibition of toxin binding to the solubilized receptor by diltiazem.To investigate the receptor molecule in the brain, monoclonal antibodies were used. A monoclonal antibody raised against the L-type calcium channel molecule (dihydropyridine receptor) purified,from rabbit skeletal muscle precipitated the DHP-binding activity solubilized from bovine and rabbit brains and bovine cardiac muscle by more than 80%. However, the antibody did not significantly precipitate the GVIA-binding activity. These results strongly suggest that the GVIA receptor molecules are mostly different from DHP receptor molecules in mammalian brains. It has been claimed that GVIA inhibits both L and N types of calcium channels in the nervous system. Our results indicate that GVIA does not inhibit L channels in the mammalian brains. To obtain monoclonal antibodies that precipitate the solubilized GVIA receptor, we immunized mice with synaptic membrane fractions from bovine and rat forebrains. Among the antibodies obtained two could precipitate some 1/3 of total solubilized GVIA receptor. However, simultaneous addition of these two antibodies increased the precipitation only a little, indicating that they recognize mostly the identical population of the GVIA receptor. These antibodies reacted with proteins of M_r, 36000 or 28000 on immunoblots. These proteins were highly concentrated in brains of various animals from chick to human.