1993 Fiscal Year Final Research Report Summary
Identification of endogenous calcium channel activator and develpment of clinical analysis method by spedific antibodies.
| Project/Area Number |
03557108
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
医学一般
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
MAKI Masatoshi Kyoto University, Institute for Virus Research, Associate Professor, ウイルス研究所, 助教授 (40183610)
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| Co-Investigator(Kenkyū-buntansha) |
OKADA Hiromu National Kyoto Hospital, Clinical Investigation Chief, 臨床検査科, 科長
KAMEYAMA Masaki Kagoshima University School of Medicine Professor, 医学部, 教授 (60150059)
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| Project Period (FY) |
1991 – 1993
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| Keywords | Calcium / Calcium-channel / Calpastatin / Calpain / Calmodulin |
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| Research Abstract |
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.
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