Mechanisms of Regulation of NHE-1

描述（申请人提供）：这是一份两次修订的R 01延续申请，目的是研究快速调节1型钠质子交换剂NHE-1（也称为SLC 9A 1的产物，溶质载体家族9A，1型）的分子机制。NHE-1是普遍存在的，几乎在每个哺乳动物细胞的质膜上表达。它介导细胞外Na+与细胞内H+的1：1交换，从而维持细胞内pH。NHE-1还在细胞体积维持、有丝分裂发生、细胞周期调节、细胞凋亡和许多其他细胞功能中发挥细胞特异性作用。NHE-1还涉及临床相关病症，如高血压、左心室肥大和缺血-再灌注损伤。尽管其在哺乳动物细胞中的普遍表达及其潜在的临床意义，但关于这种重要蛋白质调控的分子机制仍有许多有待了解。我们寻求更好地了解NHE-1被激活的机制。我们提出了一个模型的基础上的想法，磷酸化的NHE-1和结合的钙调素NHE-1，破坏静电系绳闭塞的质子传感和运输区域的NHE-1在其基础状态。系链的破坏允许环境质子进入NHE-1的质子感测和运输区域，从而导致其活化。CA-II可能促进活化，CA-II可以在“代谢子”中与NHE-1偶联，并且可以通过滑动到静电系链被破坏时产生的口袋中来进入NHE-1的质子感测和运输区域。该模型将在三个具体目标中进行测试：目标#1：我们将研究Jak 2诱导的CaM酪氨酸磷酸化在NHE-1活化中的作用。目的#2：我们将研究碳酸酐酶2型（CA-II）作为增加NHE-1活性的关键调节蛋白的作用。目标#3：我们将研究涉及NHE-1羧基末端的静电相互作用在其活化中的作用。我们认为，该提案已得到显着改善，重点是评估这种潜在的统一机制的激活NHE-1，这是一个自然的产物，我们以前的工作在这一领域，并可以作为基础的发展的分子模型的激活NHE-1。这一建议也可以为未来利用NMR和/或晶体学方法进行结构实验奠定基础。 公共卫生相关性 该项目与公共卫生有关，因为NHE-1参与调节血压、肾功能和心肌收缩。这项工作可能会导致高血压，充血性心力衰竭，心肌病和肾衰竭的新疗法。

DESCRIPTION (provided by applicant): This is a twice-amended R01 renewal application, the purpose of which is to study the molecular mechanisms of the rapid regulation of the type-1 sodium-proton exchanger, NHE-1 (also known as product of SLC9A1, solute carrier family 9A, type 1). NHE-1 is ubiquitous, being expressed on the plasma membrane of virtually every mammalian cell. It mediates the 1:1 exchange of extracellular Na+ for intracellular H+, thereby maintaining intracellular pH. NHE-1 also plays cell-specific roles in cell volume maintenance, mitogenesis, cell- cycle regulation, apoptosis and a host of other cellular functions. NHE-1 has also been implicated in clinically relevant conditions such as hypertension, left ventricular hypertrophy, and ischemia-reperfusion injury. Despite its ubiquitous expression in mammalian cells and its potential clinical relevance, much remains to be learned regarding the molecular mechanisms through which this important protein is regulated. We seek a better understanding of the mechanisms through which NHE-1 is activated. We propose a model based on the idea that phosphorylation of NHE-1 and binding of CaM to NHE-1, disrupt electrostatic tethers that occlude the proton sensing and transport regions of NHE-1 in its basal state. Disruption of the tethers allows access of ambient protons to the proton sensing and transport regions of NHE-1, thus resulting in its activation. The activation is potentially facilitated by CA-II, which could couple to NHE-1 in a "metabolon", and which could gain access to the proton sensing and transport regions of NHE-1 by sliding into a pocket created when the electrostatic tethers are disrupted. This model will be tested in three specific aims: Aim #1: We will examine the role of Jak2-induced tyrosine phosphorylation of CaM in the activation of NHE-1. Aim #2: We will examine the role of carbonic anhydrase type 2 (CA-II) as a key regulatory protein that increases the activity of NHE-1. Aim #3: We will examine the role of electrostatic interactions involving the carboxyl terminus of NHE-1 in its activation. We believe that the proposal has been significantly improved by focusing on evaluating this potentially unifying mechanism of activation of NHE-1, which is a natural outgrowth of our previous work in this area, and which could serve as the foundation for the development of a molecular model for the activation of NHE-1. This proposal could also lay the foundation for future structural experiments utilizing NMR and/or crystallography methods. PUBLIC HEALTH RELEVANCE This project is relevant to public health in that NHE-1 is involved in the regulation of blood pressure, kidney function and heart muscle contraction. The work could lead to new therapies for hypertension, congestive heart failure, cardiomyopathies and kidney failure.