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

维持细胞内离子浓度的稳态是每一个活细胞最基本的要求。这种机制是由位于细胞膜上的各种离子转运蛋白支持的。Na^+/H^+反向转运蛋白在维持细胞内Na^+和H^+浓度方面起着重要作用，分别被细菌和哺乳动物命名为Nha或NHE。本研究的最终目标是通过对细菌、酵母和哺乳动物细胞中Na ^2/H^+反向转运蛋白的结构、功能和调控的分析，阐明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。在为期两年的项目期间，我们成功地获得了Na^+/H^2反向转运蛋白的几个新信息：（1）阐明了离子转运所必需的跨膜结构域和pH传感所需的结构域。我们证明了H. pylori反向转运蛋白具有pH特异性结构 关于我们 与E.大肠杆菌抗蛋白酶我们还成功地纯化了H。pylon反向转运蛋白，并在体外重建反向转运蛋白活性。(2)通过克隆这些基因，我们发现各种不同的酵母物种具有相似的Na^+/H^+（Nhalp）。我们还发现Nhalp具有两个结构域的结构，包括疏水的膜区域和亲水的胞质区域，其整体结构与哺乳动物NHE接近。在这一胞质区域内，我们发现，结构域的重要反向转运和细胞内定位存在。此外，我们确定了一种新的膜蛋白结合到该结构域，并增强反向转运蛋白的活性。(3)我们先前鉴定了一种能够与NHE结合的新型Ca^<2+>蛋白（CHP）。在本项目中，我们鉴定了一种新的蛋白激酶和一种新的驱动蛋白作为CHP的结合靶点。本文研究了CHP在体内外的功能意义，并对CHP的多功能性进行了探讨。本项目在Na^+/H^+反向转运蛋白方面的几条不同的研究路线为今后的研究开辟了新的方向。研究结果以6篇独立论文的形式发表。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