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Study on the new regulatory mechanisms of L-type calcium channels

L型钙通道调控新机制研究

基本信息

批准号：
11670048
负责人：
KAMEYAMA Masaki
金额：
$ 2.18万
依托单位：
Kagoshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670048/
关键词：
Ca channel run-down calpastatin patch-clamp cardiac myocyte Caチャネル run-down

项目摘要

L-type Ca channel plays important roles in nerve and muscle tissues. Althrough Ca-mediated inactivation, phosphorylation by A kinas and/or C kinase and direct inhitory/stimulatory action of G proteins are reported for its regulatory mechanisms, much work remains to be done. We have previously found that run-down of cardiac Ca channel in cell-free systems is reversed by cytoplasmic factor(s) and ATP.This study is carried out to evaluate our hypothesis that Ca channel is regulated by unindentified cytoplasmic factors. By gel filtration, cytoplasm seems to contain at least two factors (P factor and H factor). The P factor is identified as calpastatin (CS), the endogenous calpain inhibitor, based on the experiments of eletrophoresis, immunology and electro-physiology. Althrough we have clarified properties of H factor in ion-exchange chromatography and trypsin and phospholipase sensitivity, its idenfication remains to be done. We have also examined the region of CS responsible for Ca channel regulation. The CS consists of N-terminal L domain (function unknown) and flanking 4 homologous calpain inhibitory domains (D1-D4). The L domain, but not D1-D4 domain, partially reversed run-down of Ca channel, suggesting L domain to be responsible for the channel regulation. In other experiments, it is also found that the action of the cytoplasmic factors does not seem to involve channel phosphorylation mediated by A kinase.These results support our hypothesis that Ca channel is regulated by cytoplasmic regulatory proteins.

L型钙通道在神经和肌肉组织中起着重要作用。尽管钙离子介导的失活、A激酶和/或C激酶的磷酸化以及G蛋白的直接抑制/刺激作用已被报道，但其调节机制仍有许多工作要做。我们以前发现，在无细胞系统中，心肌钙通道的下降可被胞质因子和ATP逆转，本研究旨在验证我们的假说，即钙通道受未鉴定的胞质因子的调节。通过凝胶过滤，细胞质似乎含有至少两种因子（P因子和H因子）。通过电泳、免疫学和电生理学实验，鉴定P因子为钙蛋白酶抑制剂（calpastatin，CS）。虽然我们已经阐明了H因子在离子交换层析中的性质以及对胰蛋白酶和磷脂酶的敏感性，但它的纯化仍有待进一步研究。我们还研究了负责Ca通道调节的CS区域。CS由N端L结构域（功能未知）和侧翼4个同源的钙蛋白酶抑制结构域（D1-D4）组成。L结构域部分逆转Ca通道的下降，而D1-D4结构域不起作用，提示L结构域可能参与Ca通道的调节。在其他实验中也发现胞质因子的作用似乎不涉及A激酶介导的通道磷酸化，这些结果支持了我们的假说，即Ca通道受胞质调节蛋白的调节。