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

细胞内pH、Na~+浓度和渗透压的调节是细胞存活的最重要因素。这些调节主要由细胞质和胞内膜上的Na~+/H~+交换器NhaA或NHE来执行。在这个项目中，我们旨在阐明这些反转运蛋白的分子结构-功能关系和功能调节，包括这些分子在细菌、酵母和哺乳动物细胞中的细胞内定位。结果，取得了以下成就。(1)确定了幽门螺杆菌NhaA膜上离子转运所必需的完整结构域。此外，对Na~+和H~+结合的重要或必需残基进行了估算，并阐明了它们在膜中的疏水环境。建立了一种新的离子输运过程中NhaA构象变化的检测系统。(2)对于酵母Nhalp，确定了离子转运所必需或重要的残基。分析了与哺乳动物NHE相似的亲水性C末端半结构域的功能。膜Jaxta位的16个氨基酸残基和侧翼的38个氨基酸残基分别是NHALP与细胞膜结合和COS3p结合所必需的，COS3p是一种能促进离子交换的高贵蛋白。(3)对于哺乳动物的NHE，我们发现了两种新的异构体：NHE8和NHE9。NHE6到NHE9存在于各种内吞泡膜上，其功能是K+/H+逆向转运蛋白，而不是Na+/H+逆向转运蛋白，从而调节内吞囊泡内的pH。我们发现CHP能结合NHE1-5，还能结合其他蛋白DRAK2(凋亡蛋白激酶)和KIFIB-(驱动蛋白亚型)。这些发现将有助于了解这些蛋白的功能和结构关系及其调控，并有助于阐明不同器官和物种之间Na+/H+拮抗剂的多样性和统一性。

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.