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。

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670086/
• 关键词: receptor; cation channel; plasma membrane vesicles; smooth muscle; lipid bilayer reconstitution; 微細再構成形質膜

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.

我们已经建立了重新蛋白偶联的活化脱脂的阳离子通道进入脂质双层，即使用先前应用于大型电导率ca^2+> - 依赖和ATP敏感的k-sentpement和ATP敏感的K ^+通道（Toro等，1990年）。 GDPβS（1mm）thiber的囊泡（100μm） - 对Ca^<2+>的敏感性，并通过去极化增强。 +浓度不受cl^ - 的影响。阳离子通道先前在相同的制剂中鉴定出来。

项目成果

Inoue,Ryuji: "Intracellular ATP slows time-dependent decline of muscarinic cation current in guinea-pig ileal smooth muscle."American Journal of Physiology Cell Physiology. 297. C1307-C1318 (2000)

Inoue, Ryuji：“细胞内 ATP 减缓了豚鼠回肠平滑肌中毒蕈碱阳离子电流随时间的下降。”美国生理学细胞生理学杂志。