Study on brain calcium channel molecules with omega-conotoxin

omega-芋螺毒素脑钙通道分子的研究

基本信息

批准号：
63570052
负责人：
ABE Teruo
金额：
$ 1.34万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

[^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.

合成了omega-芋螺毒素GVIA的[^3H]丙酰衍生物以检查毒素与脑膜部分的结合。该毒素特异性结合两个位点，解离常数 (K_d) 分别为 3 pM 和 3.5 nM。结合不受二氢吡啶或维拉帕米的影响。地尔硫卓仅特异性阻断与高亲和力位点的结合。 omega-芋螺毒素 GVIA (GVIA) 受体可以用各种去污剂溶解。洋地黄皂苷产量最高。根据地尔硫卓对毒素与可溶性受体结合的抑制判断，可能仅恢复了高亲和力位点。为了研究脑中的受体分子，使用了单克隆抗体。从兔骨骼肌中纯化出的针对 L 型钙通道分子（二氢吡啶受体）的单克隆抗体，可沉淀来自牛和兔脑以及牛心肌的 DHP 结合活性超过 80%。然而，该抗体并未显着沉淀 GVIA 结合活性。这些结果强烈表明哺乳动物大脑中的 GVIA 受体分子与 DHP 受体分子大部分不同。据称，GVIA 可抑制神经系统中的 L 型和 N 型钙通道。我们的结果表明 GVIA 不会抑制哺乳动物大脑中的 L 通道。为了获得沉淀溶解的 GVIA 受体的单克隆抗体，我们用牛和大鼠前脑的突触膜片段对小鼠进行免疫。在获得的抗体中，有两种可以沉淀约1/3的总溶解GVIA受体。然而，同时添加这两种抗体仅稍微增加了沉淀，表明它们识别的 GVIA 受体的群体基本相同。这些抗体与免疫印迹上的 M_r、36000 或 28000 蛋白发生反应。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。