Structure, function and regulation of Na^+/H^+ antiporters and intracellular localization mechanism.

维持细胞内离子浓度的动态平衡是每个活细胞最基本的要求。这一机制得到了位于细胞膜上的各种离子转运蛋白的支持。离子转运蛋白中的Na^/H^逆向转运蛋白在维持细胞内Na^和H^浓度方面起着核心作用，对细菌和哺乳动物细胞分别命名为NHA或NHE。在本研究中，我们通过分析细菌、酵母和哺乳动物细胞的Na^2/H^逆向转运蛋白的结构、功能和调控，最终确定了活细胞对各种离子环境，特别是Na^和H^的适应机制的分子基础。在两年的时间里，我们成功地获得了以下几个Na^/H^2逆向转运蛋白的新信息：(1)阐明了离子转运所必需的跨膜结构域以及pH敏感所需的结构域。我们发现幽门螺杆菌反向转运蛋白对pH…有特定的结构不同于大肠杆菌抗蛋白的感受性更强。我们还成功地纯化了幽门螺杆菌的反向转运蛋白，并在体外重组了其反向转运蛋白的活性。(2)克隆了不同酵母的Na^/H^(Nhalp)基因，结果表明不同的酵母具有相似的Na^/H^(Nhalp)。我们还发现，NHALP具有两个结构域，由疏水的膜区和亲水性的细胞质区域组成，总体结构接近哺乳动物的NHE。在这个细胞质区域内，我们发现了对反转运和细胞内定位具有重要意义的区域。此外，我们还发现了一种新的膜蛋白结合到该结构域并增强其反向转运蛋白的活性。(3)我们发现了一种新的能与NHE结合的钙蛋白(CHP)。在本项目中，我们确定了一种新的蛋白激酶和一种新的激动素作为CHP的结合靶点。我们研究了CHP在体外和体内的功能意义，并对CHP的多功能进行了讨论。在未来的研究中，CHP的细胞内调控功能还需要进一步的研究。本项目中关于Na^/H+逆向转运蛋白的几条不同的结果线开启了新的未来研究。研究结果以6篇独立全文发表。较少

Maintaining homeostasis of intracellular ion concentrations is the most basic requirement for every living cells. This mechanism is supported by various ion transpoting proteins located in cellular membranes. Na^+/H^+ antiporters among the ion-transporting proteins play a central role to maintain intracellular Na^+ and H^+ concentrations and named Nha or NHE, for bacteria or mammalian cells, respectively. In the present study, we set a final goal of this project to clarify the molecular basis of adaptation mechanism of living cells to various ion environments, especially Na^+ and H^+, by analyzing structure, function and regulation of Na^2/H^+ antiporters from bacteria, yeast and mammalian cells. During 2 years project term, we successfully obtained several new information of Na^+/H^2 antiporters as follows: (1) We clarified transmembrane domains essential for ion transport as well as domains required for pH sensing. We showed that H. pylori antiporter have a specific structure for pH … More sensing which is different from E. coli antiproter. We also successfully purified H. pylon antiporters and reconstituted the antiporter activity in vitro. (2) We showed that various different yeast species have similar Na^+/H^+ (Nhalp) by cloning the genes. We also found that Nhalp has a two-domain structure, comprised of hydrophobic membrane region, and hydrophilic cytoplasmic region which overall structure is close to mammalian NHE. Within this cytoplasmic region, we revealed that domains important for antiport and also intracellular localization exist. Further we identified that a novel membrane protein binds to the domain and enhance the antiporter activity. (3) We identified a novel Ca^<2+> protein (CHP) capable of binding to NHE previously. In this project we identified a new protein kinase and a new kinesin as the binding target of CHP. We studied functional significance of CHP in vitro and in vivo and discussed the multifunction of CHP. Further study is required for understanding intracellular regulatory function of CHP in the future study.The several different lines of result in terms of Na^+/H^+ antiporters in this project opened a new future study. The results were published as 6 independent full papers. Less