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

细胞内的离子浓度，包括pH和Na~+，都保持在一定的值。这些调控所需的Na^+/H^+逆向转运蛋白也存在于细胞质和内细胞膜中，并发挥核心作用。在本研究中，我们旨在阐明离子转运机制，包括转运离子在反转运体中的结合位置。本研究的主要成果分为以下三组。(1)用生化和分子生物学方法研究了幽门螺杆菌Na~+/H~+逆向转运蛋白NhaA的结构和功能关系。最近在HPNhaA中发现了与离子结合和转运有关的残基，结果发表在《生物化学》杂志上(另外两篇手稿正在准备发表)。为了确定NhaA的原子结构，成功地制备了大量纯化的HPNhaA晶体。此外，利用FRET分析的新方法已经成功地应用于…的检测离子传输过程预计会有更多的构象变化(JBC(2005))。(2)人类Na~+/H~+逆向转运蛋白(NHE)的三种新亚型已被发现并定位于细胞内膜。这些异构体被阐明为K^+/H^+逆向转运蛋白，并根据异构体类型而定位于不同的区室(JBC(2005))。已建立了能与NHE1结合的CHP敲除基因DT40细胞。在这些细胞中，CHP缺失，NHE1活性降低到不到野生型的90%。令人惊讶的是，NHE在这些细胞中被降解，表明CHP是稳定NHE所必需的。这给了我们一个新的CHP1和NHE的概念(amer.J.Phys(2007))。进一步确定了CHP1的晶体结构。晶体结构表明CHP1与NHE1的结合机制(JBC(2005))。(3)建立了检测酿酒酵母NHALP活性的新方法。应用这种方法给出了离子(Na~+/H~+)是电生的化学计量(BBA(2005))。Nhalp被发现形成了一种在功能上必不可少的二聚体(BBA(2005))。我们的成果被介绍在几次会议上，包括2006年国际生物化学会议，欧洲生物能量学会议等。较少

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