1997 Fiscal Year Final Research Report Summary
Molecular Mechanism of Function of Cation Antiporters.
| Project/Area Number |
07457015
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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 | National Cardiovascular Center Research Institute |
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Principal Investigator |
SHIGEKAWA Munekazu National Cardiovascular Center Research Institute, Molecular Physiology, Department Head, 循環分子生理部, 部長 (00113738)
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| Co-Investigator(Kenkyū-buntansha) |
IWAMOTO Takahiro National Cardiovascular Center Research Institute, Molecular Phsiology, Research, 循環分子生理部, 室員 (20300973)
若林 繁夫 国立循環器病センター研究所, バイオサイエンス部, 室長 (70158583)
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| Project Period (FY) |
1995 – 1997
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| Keywords | Cation exchanger / Na^+ / H^+ exchanger / Na^+ / Ca^<2+> exchanger |
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| Research Abstract |
We have investigated the molecular mechanisms of the functions of Na^+/H^+ and Na^+/Ca^<2+> exchangers. We used cells transfected with their mutated cDNAs and obtained the follwoing results : 1) Previously, we showed that the human NHE1 isoform of Na^+/H^+ exchanger is activated by calmodulin (CaM), which binds to the cytoplasmic autoinhibitory domain of NHE1, thus preventing the expression of its inhibitory function, when intracellular Ca^<2+> is increased. We sytematically introduced mutations into the autoinhibitory domain of NHE1 and found that the interaction of this domain with its putative acceptor within NHE1 is highly sequence-specific. We also characterized other portions of the cytoplasmic domain of NHE1 and provided evidence that the most N-terminal subdomain (aa515-595) is essential for the maintenance of high pH_i sensitivity of NHE1 and that specific segments within this subdomain are involved in the NHE1 regulation by growth factors and cell ATP.In addition, we construc
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ted chimera among NHEs 1 to 4 and examined their responses to growth factors and hyperosmotic stress. 2) We have shown that the Na^+/Ca^<2+> exchanger (NCX1) in cardiac and smooth muscle is activated by vasoactive substances such as endothelin by a mechanism involving protein kinase C.We showed that the NCX1 protein is phosphorylated under such conditions and then explored whether phosphorylation of the NCX1 protein is required for the activation of Na^+/Ca^<2+> exchange. In addition, we newly developed an isothiourea derivative (KB-R7943) that specifically inhibits NCX activity. Interestingly, this agent preferentially inhibited the reverse mode ofNa^+/Ca^<2+> exchange. We then found that both this inhibitor and a classic cation inhibitor Ni^i extert differential effects on activities of different isoforms of NCX.We constructed chimera between NCX1 and NCX3 and used them for the determination of the site of action for these inhibitors. Since our kinetical study indicated that Ni^i competes with Ca^<2+> for the external Ca^<2+> transport site, this approach sould give important structural informatin about the ion transport pathway in NCX. Less
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