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。

• 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

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抑制豚鼠心室肌细胞L型钙通道由内向外快速收缩的作用。CCAP存在于心肌细胞的胞浆中，并具有恢复钙通道活动的能力。部分纯化的CCAP的生化性质与钙依赖蛋白(Calain)的内源抑制蛋白(Calastatin)相同。用多克隆抗体抑制CCAP的活性。此外，通过基因工程从猪心中提纯并在大肠杆菌中生产的calastatin具有CCAP活性，这表明calastatin是CCAP。然而，作为CCAP的calastatin活性较低，仅能恢复20%的通道活性。(I)由于CCAP需要ATP或其他核苷酸，蛋白激酶A等辅助因子可能是充分活性所必需的。(Ii)对人calastatin基因及其mRNA的分析表明，通过选择性剪接产生了多种异构体。用钙调蛋白抗体进行Western印迹分析，证实存在不同的钙调蛋白亚型。因此，特定亚型的钙调蛋白有可能作为具有较高活性的CCAP。(Iii)对钙调蛋白的结构-功能分析表明，保守的A和C亚区对于抑制钙蛋白酶是必不可少的，但对于与钙调蛋白样结构域的相互作用是重要的，而B亚区对于这种抑制是必不可少的。由于低相对分子质量的钙蛋白酶抑制剂亮肽素不能抑制通道的活性，而CCAP可以恢复通道的活性，因此可能不涉及Calain的蛋白分解。要了解CCAPS抑制钙通道衰退和恢复的机制，还需要进一步的工作。

项目成果

Ma,H.et al.: "Requirement of different subdomains of calpastatin for calpain inhibition and for binding to the calmodulinlike domains" Journal of Biochemistry.

Ma,H.等人：“钙蛋白酶抑制和与钙调蛋白样结构域结合需要钙蛋白酶抑制素的不同子结构域”《生物化学杂志》。

Maki, M: "Calpastatins : Biochemical and molecular biological studies" Biomed. Biochem. Acta. 50. 509-516 (1991)

Maki，M：“钙蛋白酶抑制剂：生化和分子生物学研究”Biomed。

亀山 正樹: "心筋Caチャンネルとその調節" 実験医学. 10. 605-610 (1992)

Masaki Kameyama：“心肌 Ca 通道及其调节”实验医学 10. 605-610 (1992)。

Kameyama, Masaki: "Cardiac calcium channel and its regulation (in Japanese)" Jikkenn Igaku (Experimental Medicine). 10. 605-610 (1992)

Kameyama, Masaki：“心脏钙通道及其调节（日语）”Jikkenn Igaku（实验医学）。

Emiko Takano: "Molecular diversity of calpastatin in human erythroid cells" Archives of Biochemistry and Biophysics. 303. 349-354 (1993)

Emiko Takano：“人红系细胞中钙蛋白酶抑制素的分子多样性”生物化学和生物物理学档案。