| Project/Area Number |
13142207
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | Osaka University |
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Principal Investigator |
KANAZAWA Hiroshi Osaka University, Graduate School of Science, Professor, 理学研究科, 教授 (50116448)
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| Co-Investigator(Kenkyū-buntansha) |
FUKUYAMA Keiichi Osaka university, Graduate School of Science, Professor, 理学研究科, 教授 (80032283)
MITSUI Keiji Osaka university, Graduate School of Science, Assistant Professor, 理学研究科, 助手 (60379279)
MATSUSHITA Masafumi Osaka university, Graduate school of science, Assistant Professor, 理学研究科, 助手 (50403100)
NAKAMURA Norihiro Osaka university, Graduate School of Science, Assistant Professor, 理学研究科, 助手 (90324748)
INOUE Hiroki Osaka university, Graduate School of Science, Assistant Professor, 理学研究科, 助手 (10294448)
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| Project Period (FY) |
2001 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥84,700,000 (Direct Cost: ¥84,700,000)
Fiscal Year 2005: ¥11,900,000 (Direct Cost: ¥11,900,000)
Fiscal Year 2004: ¥15,200,000 (Direct Cost: ¥15,200,000)
Fiscal Year 2003: ¥20,000,000 (Direct Cost: ¥20,000,000)
Fiscal Year 2002: ¥19,800,000 (Direct Cost: ¥19,800,000)
Fiscal Year 2001: ¥17,800,000 (Direct Cost: ¥17,800,000)
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| Keywords | intracellular ion homeostasis / Na+ / H+ exchangers / pH regulation / salinity resistance / active transporters / molecular structure of membrane proteins / intracellular localization of membrane proteins / molecular biological approach / 細胞内イオン恒常性 / 膜蛋白質のイオン透過機構 / 生物分子多様性 / 生体エネルギー共役 / 分子遺伝学 / 蛋白質工学 / 膜蛋白局在化 / 細胞の塩環境適応 / Na^+ / H^+交換輸送蛋白質 / 遺伝子解析 / イオン輸送の分子機構 / 能動透過系のエネルギー共役 / 遺伝子工学 / FRET解析 / Cys走査変異解析 / Na^+恒常性 / H^+交換輸送担体 / 能動輸送 / 膜蛋白質 / 生物学的統一性 / イオン輸送蛋白 / 機能促進因子 / 分子遺伝学的アプローチ / イオン輸送蛋白質の構想と機能 / 能動輸送の分子機構 / pHセンサー / 新規カルシューム結合性蛋白質 / 膜蛋白質の精製と再構成 / キメラ蛋白質の合成 / 膜タンパクの細胞内局在化 / H^+交換輸送 / イオン輸送分子機構 / ランダムミューテーション / アポトーシスキナ-ゼ / 細胞膜小胚輸送 / カルシューム結合蛋白 |
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| Research Abstract |
Regulation of intracellular pH, Na^+ concentration, and osmolality are the most important factors for cell survival. These regulations are performed mainly by Na^+/H^+ exchangers named NhaA or NHE on the cytoplasmic as well as endocytic membranes. In this project we aimed to elucidate molecular structure-function relationship and functional regulation of these antiporters including intracellular localization of these molecules for these molecules from bacteria, yeast and mammalian cells. As a result, following achievement was performed. (1) For H pylori NhaA membrane integral domains essential for the ion transport were identified. Further the residues important or essential for Na^+ and H^+ binding were estimated and their hydrophobic environment within the membranes were elucidated. A new detection system of conformational change of NhaA during the ion transport was set up. (2) For yeast Nhalp essential or important residues for the ion transport were identified. Function of the hydrophilic C terminal half domain which seems to be similar to the mammalian NHE were analyzed. A membrane jaxta-positional 16 amino acid residues and its flanking 38 amino acid residues were found to be essential for destination of Nhalp to the cytoplasmic membrane, and binding Cos3p, a noble protein capable enhancing the ion exchange, respectively. (3) For mammalian NHE, we identified two new isoforms, NHE8 and 9. The isoforms NHE 6 to 9 were found to be in the membranes of various endocytic vesicles and function as K+/H+ antipoter rather than Na^+/H^+ antiporter, leading to regulate pH within endocytic vesicles. CHP capable binding NHE1-5 found by us was found to bind other proteins, DRAK2 (apoptotic protein kinase) and KIFIB□ (Kinesin isoform). The present findings will contribute to understand the function and structure relation ship and the regulations of these proteins as well as for elucidating diversity and unity of Na+/H+ antoporters among various organs and species.
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