Study on brain calcium channel molecules with omega-conotoxin

[^3H]合成了ω - concontoxin GVIA的丙酰衍生物，以检测该毒素与脑膜组分的结合。毒素特异性结合到两个位点，解离常数（K_d）分别为3pm和3.5 nM。二氢吡啶或维拉帕米不影响结合。地尔硫卓特异性地阻断了仅与高亲和力位点的结合。omega- concontoxin GVIA （GVIA）受体可被多种洗涤剂溶解。洋地黄苷产量最高。从地尔硫卓抑制毒素与溶化受体的结合来看，可能只有高亲和位点被恢复。为了研究大脑中的受体分子，我们使用了单克隆抗体。从兔骨骼肌中纯化出一种针对l型钙通道分子（二氢吡啶受体）的单克隆抗体，可使从牛、兔脑和牛心肌中溶解的dhp结合活性降低80%以上。然而，该抗体对gvia结合活性无明显影响。这些结果有力地表明，在哺乳动物大脑中，GVIA受体分子与DHP受体分子有很大的不同。据称，GVIA抑制了神经系统中L型和N型钙通道。我们的研究结果表明，GVIA不会抑制哺乳动物大脑中的L通道。为了获得沉淀溶解GVIA受体的单克隆抗体，我们用牛和大鼠前脑的突触膜部分免疫小鼠。所得抗体中有两种抗体可沉淀约1/3的可溶性GVIA受体。然而，同时加入这两种抗体只增加了一点点沉淀，这表明它们识别的大多是相同的GVIA受体群体。这些抗体与免疫印迹上的M_r、36000或28000蛋白反应。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.