Adaptation of cells to high salinity conditions and basic mechanisms of ion transport in biological membranes

细胞对高盐条件的适应和生物膜中离子传输的基本机制

基本信息

批准号：
15370054
负责人：
KANAZAWA Hiroshi
金额：
$ 7.42万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15370054/
关键词：
Salinity torelance Molecular basis of ion transporters Na+H+ antiporters genetic and biochemical analyses bioenergetics of transporters FRET analyses Cys scanning mutagenesis gene engineering 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

项目摘要

Intracellular ionic conditions are maintained within a certain range for every ions including H^+ and Na^+. Even for the higher salinity conditions of outside of the cells, cells e remove alkaline cations like Na^+ and Li+ from the inside of cells for keeping homeostasis. This exclusion of toxic alkaline cations from inside of cells is performed by Na^+/H^+ antiporters which exists in most of the cells from bacterial to human. To understand the adaptation mechanisms of cells to the high salinity conditions, molecular mechanism of Na^+/H^+ antiporters should be clarified. In this study, we analyzed molecular structure of Na^+/H^+ antipotrer NhaA from E.coli and H pylori mainly by mutagenic and biochemical studies. As the results, we have shown that essential transmembrane segments for ion transport involves TM4,5,10 and 11 and further shown that essential or important residues in these Tms (J.Biol Chem.(2003)). We also established a method by using FRET(fluorescence resonance energy tra … More nsfer) to detect protein conformational change during ion transport (Manuscript submitted). We isolated a series of mutants with substitution to Cys for each residues in the TM 4,5,10 and 11. Analyses of the mutants in terms of antiporter activities and accessibility of N-ethyl maleimide revealed presense of a putative channel like structure within the NhaA surrounded by the Tms (J.Biol Chem.(2004)). We have cloned new isoforms of human Na^+/H^+ antiporters named NHE8 and 9 and revealed their function and intracellular distribution within different intracellular vesicles (J.Biol.Chem.(2005)). These endomembrane type antiporter function as K^+/H^+ antiporter and regulate endosomal pH. We also revealed a new function of CHP we had found a binding partner of NHE1 previously. We have constructed a knockout cells of gene for CHP and found that NHE does not translocate to the cytoplasmic membrane for the knockout cells, suggesting that CHP is required for intracellular destination of NHE to the cytoplasmic membrane which differs from the previous observation by Pang et al. Less

包括H^+和Na^+在内的每种离子的细胞内离子条件都维持在一定范围内，即使在细胞外部的较高盐度条件下，细胞也会从细胞内部去除Na^+和Li+等碱性阳离子。保持稳态。从细胞内部排除有毒碱性阳离子是由 Na^+/H^+ 逆向转运蛋白完成的，这种逆向转运蛋白存在于从细菌到人类的大多数细胞中。盐度条件下，Na^+/H^+反向转运蛋白的分子机制有待阐明。本研究主要通过诱变分析了来自大肠杆菌和幽门螺杆菌的Na^+/H^+反向转运蛋白NhaA的分子结构。和生化研究结果表明，离子转运必需的跨膜片段涉及TM4、5、10和11，并进一步表明这些Tms中必需或重要的残基（J.Biol）。 Chem.(2003))。我们还建立了一种使用 FRET（荧光共振能量转移）检测离子转运过程中蛋白质构象变化的方法（已提交手稿）。在 TM 4、5、10 和 11 中。根据逆向转运蛋白活性和 N-乙基马来酰亚胺的可及性对突变体进行分析，揭示了假定通道的存在，如NhaA 内被 Tms 包围的结构（J.Biol Chem.(2004)）我们克隆了人类 Na^+/H^+ 逆向转运蛋白的新亚型，命名为 NHE8 和 9，并揭示了它们在不同细胞内的功能和细胞内分布。囊泡（J.Biol.Chem.（2005））。这些内膜型逆向转运蛋白发挥 K^+/H^+ 逆向转运蛋白的作用并调节内体 pH。还揭示了CHP的新功能，我们之前发现了NHE1的结合伴侣，我们构建了CHP基因的敲除细胞，发现NHE不会易位到敲除细胞的细胞质膜上，这表明CHP是细胞内所必需的。 NHE 到达细胞质膜的目的地与 Pang 等人之前的观察不同。