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CA通道在神经和肌肉组织中起重要作用。据报道，CA介导的灭活，KINA和/或C激酶的磷酸化以及G蛋白的直接吸入/刺激作用的磷酸化据报道了其调节机制，但仍有许多工作要做。我们以前已经发现，无细胞系统中心脏CA通道的倒数被细胞质因子和ATP逆转。这项研究是为了评估我们的假设，即CA通道受未经注明的细胞质因子调节。通过凝胶过滤，细胞质似乎至少包含两个因素（P因子和H因子）。 P因子被确定为内源性钙蛋白酶抑制剂Calpastatin（CS），基于el骨，免疫学和电生理学的实验。我们已经阐明了H因子在离子 - 交换色谱和胰蛋白酶和磷脂酶敏感性中的特性，其idenfication仍然有待完成。我们还检查了负责CA通道调节的CS区域。 CS由N末端L结构域（功能未知）和侧面4个同源钙蛋白酶抑制域（D1-D4）组成。 L域，而不是D1-D4域，部分反向CA通道的倒数，这表明L域负责通道调节。在其他实验中，还发现细胞质因子的作用似乎不涉及由激酶介导的通道磷酸化。这些结果支持我们的假设：CA通道受细胞质调节蛋白的调节。