Identification of endogenous calcium channel activator and develpment of clinical analysis method by spedific antibodies.

内源性钙通道激活剂的鉴定及特异性抗体临床分析方法的开发。

基本信息

批准号：
03557108
负责人：
MAKI Masatoshi
金额：
$ 8.38万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03557108/
关键词：
Calcium Calcium-channel Calpastatin Calpain Calmodulin スプライシング PCR パッチクランプ

项目摘要

We have analyzed the endogenous calcium channel activating protein (CCAP) which suppresses the rapid run-down of the L-type Ca channel in the inside-out patch mode in guineapig ventricular myocytes. CCAP has been found in cytoplasmic fraction of the myocytes and has ability to restore the Ca channel activity after run-down. Biochemical properties of the partially purified CCAP showed those of the endogenous inhibitor protein (calpastatin) of Ca-dependent protease (calpain). A polyclonal antibody against calpastatin inhibited the CCAP activity. Moreover, calpastatin purified from pig heart and produced in E.coli by gene engineering exhibited CCAP activity, suggesting calpastatin is CCAP.The activity of calpastatin as CCAP, however, was low and could restore only 20% of the channel activity.(i) Since the CCAP requires ATP or other nucleotides, co-factors such as protein kinase A might me necessary for full activity.(ii) Analysis of the human calpastatin gene and its mRNA has revealed occurrence of multiple isoforms generated by alternative splicing. Presence of diverse calpastatin isoforms were confirmed by Western blot using calpastatin antibodies. Thus, it may be possible that specific isoform of calpastatin acts as CCAP of higher activity.(iii) Structure-function analysis of calpastatin has suggested that conserved subdomains A and C is dispensable for calpain inhibition but important for interaction with calmodulin-like domain of calpain, while subdomain B is essential for the inhibition.Since the low molecular weight calpain inhibitor leupeptin could not suppress the channel activity and the CCAP could resore the activity, proteolysis by calpain may not be involved in the system. Further works are necessary to understand the mechanism of suppression of run-down and restoration of the Ca-channel by CCAPs.

我们分析了内源性钙通道激活蛋白（CCAP），它在豚鼠心室肌细胞中以由内向外的补片模式抑制 L 型 Ca 通道的快速消耗。 CCAP 存在于肌细胞的细胞质部分中，并且能够在耗尽后恢复 Ca 通道活性。部分纯化的 CCAP 的生化特性显示了 Ca 依赖性蛋白酶（钙蛋白酶）的内源性抑制剂蛋白（钙蛋白酶抑制蛋白）的生化特性。抗 calpastatin 的多克隆抗体抑制 CCAP 活性。此外，从猪心脏中纯化并通过基因工程在大肠杆菌中产生的卡帕他汀显示出CCAP活性，表明卡帕他汀就是CCAP。然而，作为CCAP的卡帕他汀的活性很低，只能恢复20%的通道活性。由于 CCAP 需要 ATP 或其他核苷酸，因此蛋白激酶 A 等辅因子可能是充分发挥活性所必需的。 (ii) 对人类 calpastatin 基因及其 mRNA 的分析表明，存在多个由选择性剪接产生的异构体。使用钙蛋白酶抑制剂抗体通过蛋白质印迹证实了多种钙蛋白酶抑制剂亚型的存在。因此，钙蛋白酶抑制剂的特定亚型可能充当更高活性的CCAP。(iii)钙蛋白酶抑制剂的结构功能分析表明，保守的亚结构域A和C对于钙蛋白酶抑制是可有可无的，但对于与钙调蛋白样结构域的相互作用很重要。钙蛋白酶，而子结构域 B 对于抑制至关重要。由于低分子量钙蛋白酶抑制剂亮肽素不能抑制通道活性，而 CCAP 可以恢复该活性，该系统可能不涉及钙蛋白酶的蛋白水解作用。需要进一步的工作来了解 CCAP 抑制 Ca 通道损耗和恢复的机制。