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

包括H^+和Na^+在内的每种离子的胞内离子条件都维持在一定范围内。即使在细胞外盐度较高的条件下，细胞也会从细胞内清除Na~+和Li~+等碱性阳离子，以保持体内的动态平衡。这种从细胞内排出有毒碱性阳离子的过程是由Na+/H+逆向转运蛋白完成的，这种转运蛋白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条件的适应机制，需要阐明Na~+/H~+逆向转运蛋白的分子机制。在本研究中，我们主要通过诱变和生化研究分析了大肠杆菌和幽门螺杆菌的Na~+/H~+拮抗剂NhaA的分子结构。结果表明，离子转运的基本跨膜片段涉及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))。建立了FRET(荧光共振能量转移…)的检测方法更多的NSFER)，以检测离子转运过程中蛋白质构象的变化(提交的手稿)。我们分离了一系列突变株，TM4，5，10和11的每个残基都替换了Cys。对突变株的反转运蛋白活性和N-乙基马来酰亚胺的可及性的分析表明，TMS包围的NhaA内存在一个可能的通道状结构(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，并揭示了它们的功能和在不同细胞内小泡中的分布(J.Biol.Chem.(2005))。这些内膜型逆向转运蛋白具有K~+/H~+逆向转运蛋白的功能，调节内膜pH。我们还揭示了CHP的一个新功能，我们之前已经发现了NHE1的结合伙伴。我们构建了CHP基因的敲除细胞，发现敲除细胞的NHE没有移位到细胞膜，这表明CHP是NHE到达细胞膜所必需的，这与Pang等人以前的观察不同。较少

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