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

我们分析了内源性钙通道激活蛋白（CCAP）在豚鼠心室肌细胞内向外贴片模式下抑制l型钙通道的快速衰竭。在肌细胞的细胞质部分中发现了CCAP，并具有恢复Ca通道活性的能力。部分纯化的CCAP具有钙依赖性蛋白酶（calpain）内源性抑制剂（calpastatin）的生化特性。calpastatin多克隆抗体抑制CCAP活性。此外，从猪心脏中纯化并在大肠杆菌中基因工程制备的calpastatin显示出CCAP活性，表明calpastatin就是CCAP。然而，钙pastatin作为CCAP的活性很低，只能恢复20%的通道活性。(i)由于CCAP需要ATP或其他核苷酸，辅助因子如蛋白激酶A可能是充分活性所必需的。（ii）对人类calpastatin基因及其mRNA的分析显示，存在由选择性剪接产生的多种异构体。使用calpastatin抗体，Western blot证实存在多种calpastatin亚型。因此，钙pastatin的特异性异构体可能作为具有较高活性的CCAP。（iii） calpastatin的结构-功能分析表明，保守的亚结构域A和C对于calpain的抑制是必不可少的，但对于与calpain的钙调素样结构域的相互作用是重要的，而亚结构域B对于抑制是必不可少的。由于低分子量的calpain抑制剂lepeptin不能抑制通道活性，而CCAP可以恢复通道活性，因此可能不参与calpain的蛋白水解。进一步的研究需要进一步了解CCAPs对钙通道的抑制和恢复机制。

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.