| Project/Area Number |
01460269
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Kyushu University |
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Principal Investigator |
KIRINO Yutaka Kyushu University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (10012668)
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| Co-Investigator(Kenkyū-buntansha) |
HIRASHIMA Naohide Kyushu University Faculty of Pharmaceutical Sciences, Assistant Professor, 薬学部, 助手 (10192296)
ANZAI Kazunori Kyushu University Faculty of Pharmaceutical Sciences, Assistant Professor, 薬学部, 助手 (70128643)
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| Project Period (FY) |
1989 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1991: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1990: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1989: ¥4,300,000 (Direct Cost: ¥4,300,000)
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| Keywords | ion channels / reconstitution / planar bilayer / cardiac sarcolemmal membranes / 心筋細胞膜ベシクル / 脂質平面膜 / 単一チャネル電流 / 細胞質因子 / B型Ca^<2+>チャネル / 脂質平面膜法 / L型Caチャネル / ジヒドロピリジン化合物 / 活性酸素 / Ca取り込み |
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| Research Abstract |
Preparation method for sarcolemmal membrane vesicles from porcine cardiac myocytes in high purity was established. In order to evaluate the purity of the sarcolemmal membranes, it is useful to measure marker enzyme activity such as Na^+, K^+-ATPase activity as a whole by unmasking the latent activities. For the unmasking reagent, saponin was found to be very effective and convenient.
Dihydropyridine receptor complex was purified in about 2000-fold by the use of immune affinity chromatography with a gel attaching a monoclonal antibody against alpha_2delta subunit complex of L-PM Ca^<2+> channel from rabbit skeletal muscles. alpha_1, alpha_2, and delta subunit were detected in SDS-PAGE analysis, while other subunit such as beta and gamma were not detected. The procedure established in this research takes shorter time than the procedures reported before. It may thus make us possible to reconstitute the purified subunit into proteoliposomes or planar bilayer membranes with keeping their activities in natural.
The sarcolemmal vesicles were fused to planar lipid bilayers and ion channel activities were measured. In the solution containing 100 mM Ba^<2+>, lion-inactivating channels were detected at negative membrane potentials. One of the channel was identified as B-QW Ca^<2+> channel, which had been reported in the patch clamp experiment. In the solution containing 600 mM Na^+, a cation channel with a conductance of 10 pS between -80 to 0 mV was detected. The charmer also allows K^+ to pass through, and the permeability ratio between Na^+ and K^+ was unity. The channel therefore is a new non-selective cation channel, which has not been reported before.
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