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

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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.