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

包括H^+和Na^+在内的所有离子的胞内离子条件都保持在一定范围内。即使在细胞外部的高盐度条件下，细胞也会从细胞内部清除Na^+和Li+等碱性阳离子，以保持体内平衡。从细胞内排除有毒碱性阳离子是由Na^+/H^+反转运蛋白完成的，它存在于从细菌到人类的大多数细胞中。为了了解细胞对高盐度环境的适应机制，需要明确Na^+/H^+反转运蛋白的分子机制。本研究主要通过诱变和生化研究分析了大肠杆菌和幽门螺杆菌中Na^+/H^+反转录物NhaA的分子结构。结果表明，离子运输的必要跨膜区段涉及TM4、5、10和11，并进一步表明这些Tms中存在必要或重要的残基（J.Biol Chem.(2003)）。我们还建立了一种荧光共振能量转移（FRET）检测离子传输过程中蛋白质构象变化的方法（论文提交）。我们分离了一系列突变体，在TM 4、5、10和11中每个残基都被Cys取代。对突变体的反转运蛋白活性和n -乙基马来酰亚胺的可及性的分析显示，在被Tms包围的NhaA中存在一个假定的通道状结构（J.Biol Chem.(2004)）。我们克隆了人类Na^+/H^+反转运蛋白NHE8和nhe9的新亚型，并揭示了它们在不同细胞内囊泡中的功能和细胞内分布（J.Biol.Chem.(2005)）。这些膜型反转运蛋白具有K^+/H^+反转运蛋白的功能，并调节内体ph。我们还发现了CHP的新功能。我们构建了CHP基因敲除细胞，发现NHE在敲除细胞中不易位到细胞质膜，这表明NHE在胞内到达细胞质膜需要CHP，这与Pang等人之前的观察结果不同。少

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