2001 Fiscal Year Final Research Report Summary
Searching responsible sites for the regulation of the L-type Ca channel through phosphorylation
| Project/Area Number |
11470011
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | HIROSHIMA UNIVERSITY |
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Principal Investigator |
YAMAOKA Kaoru Faculty of Medicine, HIROSHIMA UNIVERSITY, Associate Professor, 医学部, 助教授 (10200586)
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| Co-Investigator(Kenkyū-buntansha) |
YUKI Tsunetsagu Faculty of Medicine, HIROSHIMA UNIVERSITY, Research Associate, 医学部, 助手 (90294561)
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| Project Period (FY) |
1999 – 2001
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| Keywords | L-type Ca channel / Magnesium / phosphorylation / Frog / Gunea-pig / Temperature-sensitive |
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| Research Abstract |
[Overview]
The ultimate aim of this study is to identify responsible sites for the regulations of L-type Ca channels in the heart, such as intracellular Mg^<2+> block, or enhancement of channel activity accelerated by phsphorylation. As a result, we found that something is lacking to fully reconstruct channel activity only using L-type Ca channels with non-cardiac cells. Especcially, Mg^<2+> response was totally different from the behavior found in native cardiac cells. Depletion of intracellular Mg^<2+> led to the rapid wash out of channel activity in reconstructed systems, while in native cardiac cells, depletion of intracellular Mg^<2+> normally causes prominent increase in Ca channel current (I_<ca>). This indicates the requirement of extensive improvement of reconstruction systems regarding host cell conditions
[Results]
1. Increase in I_<ca> responding to reduction in intracellular Mg^<2+> was disturbed in guinea-pig cardiac cells, when temperature was below 28℃. This temperature dependency was not explained by simple thermodynamic activities.
2. We identified whole length of cDNAs encoding α_1, β_<2C> and α_<2/δ> subunits of the L-type Ca channel in frog ventricular cells. They exhibited more than 80 % homology of those reported in rat or mouse. Co-expression of all subunits of α_1, β_<2C> and α_<2/δ> in BHK cells exhibited functional L-type Ca channel activity.
3. Reconstructed Ca channels in BHK cells rapidly ran down, when cells were dialysed with log Mg^<2+> (below 10^<-5>M) patch solutions below 10^<-5>M. This is in contrast to data found in native cardiac cells. We need to search unkown proteins to accommodate the lacking of important regulations of the channel.
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