Molecular basis for regulation of intracellular environment and function of ion transporting proteins

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Intracellular concentrations of ions including pH and Na^+ are maintained at certain values. Na^+/H^+ antiporters required for these regulations exit in cytoplasmic and endocytic membranes as well and play a central role. In this study we aimed to clarify ion transport mechanisms including the binding sites of the trasporting ions in the antiporters. Main results of this study are classified into the following three groups. (1) The structure and function relationship of H.pylori Na^+/H^+ antiporter NhaA (HPNhaA) has been studied by means of biochemical and molecular biological approaches. The residues involved in the ion binding and transport have been newly identified for HPNhaA and the results were published in Biochemistry ( two other manuscripts are in preparation for publication). To determine the atomic structure of NhaA, a large scale of purified HPNhaA was successfully prepared to make a crystal. Further a new approach using FRET analysis has been successfully applied to detect … More conformational changes expected for ion transporting process (JBC (2005)). (2) Three new isoforms of human Na^+/H^+ antiporter (NHE) have been identified and revealed to localize in endocytic membranes. These isoforms were clarified to function as a K^+/H^+ antiporter and localize in different compartments depending on isoform type (JBC(2005)). DT40 cells with knocked out gene for CHP which can bind to NHE1 has been established. In these cells, CHP is missing and NHE1 activity was reduced to less than 90 % of the wild type. Surprisingly NHE was degraded in these cells, indicating that CHP is essentail for stabilizing NHE. This gives us a new concept of CHP1 with NHE (Amer.J.Phys (2007)). Further we determined crystal structure of CHP1. The crystal structure suggested a binding mechanism of CHP1 wuth NHE1 (JBC(2005)). (3) We have established a new procedure to detect Nhalp activity of S. cereviciae. Application of this method gave us stoichiometry of ions (Na^+/H^+) being electrogenic (BBA(2005)). The Nhalp was found to form a dimer which is essential in functioning (BBA(2005)).Our achievements were presented in several meeting including 2006 International Biochemistry Meeting, European Bioenergetics meeting, and so on. Less