Unity and diversity of ion transport mechanisms and regulation of Na+/H+ antiporters

细胞内pH值、Na^+浓度和渗透压摩尔浓度的调节是细胞存活的最重要因素。这些调节主要是通过细胞质膜和内吞膜上的Na^+/H^+交换剂NhaA或NHE完成的。本研究旨在阐明这些反向转运蛋白的分子结构与功能的关系以及它们的功能调控，包括这些分子在细菌、酵母和哺乳动物细胞中的胞内定位。因此，取得了以下成就。(1)对于幽门螺杆菌NhaA膜的离子转运所必需的整合结构域进行了鉴定。此外，还对Na^+和H^+结合的重要或必需残基进行了估计，并阐明了它们在膜内的疏水环境。建立了一种新的离子迁移过程中NhaA构象变化的检测体系。(2)对于酵母Nhalp离子运输的必要或重要的残留物进行了鉴定。分析了与哺乳动物NHE相似的亲水性C端半结构域的功能。膜jaxta位置的16个氨基酸残基和其侧翼的38个氨基酸残基被发现是必需的目的地Nhalp的细胞质膜，并结合Cos 3 p，一个高贵的蛋白质，能够增强离子交换，分别。(3)对于哺乳动物NHE，我们确定了两种新的亚型，NHE 8和9。NHE 6 - 9亚型存在于各种内吞囊泡的膜上，其功能是K+/H+反向转运蛋白，而不是Na^+/H^+反向转运蛋白，从而调节内吞囊泡内的pH值。我们发现的能够结合NHE 1 -5的CHP被发现结合其他蛋白质，DRAK 2（凋亡蛋白激酶）和KIFIB□（驱动蛋白同种型）。本研究结果将有助于了解这些蛋白质的功能、结构关系和调控规律，并有助于阐明Na+/H+转运蛋白在不同器官和物种间的多样性和统一性。

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.