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

我们已经建立了将G蛋白偶联受体激活的阳离子通道重组到脂质双层的方案，用豚鼠回肠平滑肌制备的质膜囊泡，使用先前应用于大电导钙依赖和ATP敏感的K^+通道的纯化技术(Toro等人，1990)。通过五种不连续的蔗糖梯度超速离心粗质膜小泡，可获得高度富含质膜蛋白的质膜组分(25/30%(w/w)蔗糖界面)。在富钠条件下，将这些质膜微囊掺入双层膜产生GTPγS(100μM)激活的通道活性，该活性可被βS(1 MM)抑制，对Ca~(2+)敏感，并被去极化增强。这些通道的翻转电位和单位电导随Na~(++)浓度的变化而变化，但不受Cl~(2+)的影响。这些结果有力地表明，GTPγS激活的重组通道属于一类电压依赖的、钙敏感的阳离子选择通道，它是通过G蛋白激活的，很可能与先前在同一制剂中发现的M受体激活的阳离子通道相对应。这些结果也有力地表明了双层掺入技术在未来研究平滑肌受体操控的阳离子通道中的作用。

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.