| Project/Area Number |
10670086
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General pharmacology
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
INOUE Ryuji Dept.Pharmacol.Grad.Sch.Med.Sci., KYUSHU UNIVERSITY Associate professor, 大学院・医学研究院, 助教授 (30232573)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | receptor / cation channel / plasma membrane vesicles / smooth muscle / lipid bilayer reconstitution / 微細再構成形質膜 |
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| Research Abstract |
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.
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