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Investigation on regulatory mechanisms for smooth muscle receptor-operated Ca^<2+> permeable cation channels using lipid bilayer incorporation of plasma membrane vesicles.

使用质膜囊泡的脂质双层掺入研究平滑肌受体操作的Ca^2可渗透阳离子通道的调节机制。

基本信息

批准号：
10670086
负责人：
INOUE Ryuji
金额：
$ 2.11万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

We have established the protocol of reconstituting G-protein-coupled receptor activated cation channels into the lipid bilayer, with plasma membrane vesicles prepared from guinea-pig ileal smooth muscle using the purification technique previously applied to the large conductance Ca^<2+>-dependent and ATP-sensitive K^+ channels (Toro et al., 1990). Ultracentrifugation of crude plasma membrane vesicles across the five discontinuous sucrose gradients provided a plasma membrane fraction (at 25/30% (w/w) sucrose interface) highly enriched with plasmalemmal proteins. Under Na^+-rich conditions, incorporation of these plasma membrane vesicles into the bilayer produced GTPγS (100μM)-activatable channel activities that are inhibited by GDPβS (1mM), sensitive to Ca^<2+> and enhanced by depolarization. The reversal potential and unitary conductance (tens of picosiemens) of these channels varied depending on Na^+ concentration, but not affected by Cl^-. These results strongly indicate that the reconstituted channels activated by GTPγS belong to a class of voltage-dependent, Ca^<2+>-sensitive cation-selective channels that are activated through a G-protein, and correspond most likely to the muscarinic receptor-activated cation channels previously identified in the same preparation. These results also strongly point to the usefulness of bilayer incorporation technique to investigate the receptor-operated cation channels in smooth muscle in the future.

我们已经建立了将G蛋白偶联受体激活的阳离子通道重建到脂质双层中的方案，使用之前应用于大电导Ca ^2+依赖性和ATP敏感性K ^+通道的纯化技术，用豚鼠回肠平滑肌制备质膜囊泡（Toro et al.，1990年）。在五个不连续的蔗糖梯度中，粗质膜囊泡的超离心提供了高度富集质膜蛋白的质膜部分（在25/30%（w/w）蔗糖界面处）。在富含Na ^+的条件下，这些质膜囊泡进入双分子层后，产生了GTP γ S（100 μ M）激活的通道活性，该活性被GDP β S（1 mM）抑制，对Ca ^<2 +>敏感，并被去极化增强。这些通道的逆转电位和单位电导（数十皮西门子）取决于Na ^+浓度，但不受Cl ^-的影响。这些结果强烈表明，由GTP γ S激活的重建通道属于一类通过G蛋白激活的电压依赖性、Ca ^<2 +>敏感性阳离子选择性通道，并且最有可能对应于之前在同一制剂中鉴定的毒蕈碱受体激活阳离子通道。这些结果也有力地指出了有用的双层掺入技术研究受体操纵的阳离子通道在平滑肌的未来。