Structure-Function Studies of Epithelial Sodium Channel Gating

项目摘要/摘要 上皮性钠通道(ENaC)形成了肾脏、肺和其他器官吸收Na+的途径 上皮细胞。为了维持Na+稳态和控制血压，ENaC受到严格调控，以 对Na+/体积耗尽和Na/体积过剩的情况作出反应。然而，这方面的缺陷 调节是几乎所有已知的遗传性高血压的罪魁祸首，并有助于 囊性纤维化的发病机制。因此，我们的长期目标是了解 监管ENaC是开发针对这些疾病的靶向治疗的先决条件。最近 这些发现的趋同使人们的注意力集中在调节ENaC门控的机制上。在 生物合成途径，在细胞表面，蛋白酶裂解胞外区和ENaC， 将不活跃的通道转化为活跃的钠离子传导形式。此外，Na+对ENaC门控有调节作用 通过细胞外(Na+自抑制)和细胞内(Na+反馈抑制)机制 保持动态平衡。其他细胞外分子也调节ENaC的活动。然而，有一些 我们对构成这一现象的分子机制和通道结构的认识存在严重差距 监管。一个关键的进展是最近解决了一个密切相关的晶体结构 频道，ASIC1。这为人们提供了一个前所未有的视角，了解可能构成 调节DEG/ENaC离子通道家族的门控。利用这些进步在 了解ENaC门控和ASIC1晶体结构，本提案的总体目标是 了解调节ENaC门控的结构-功能关系。我们提出了三个具体目标。 1.在初步研究中，我们发现细胞内Na+通过改变蛋白水解物来调节ENaC 和ENaC的卵裂。在这个目标中，我们将检验Na+通过诱导一种 ENaC胞外区的构象变化。我们还将确定ENaC序列是 必填项。2.ENAC暴露在肾脏和肺的极端pH中。在初步研究中，我们发现 ENaC的活性受细胞外pH的调节。在这个目标中，我们将研究分子 机制，并确定pH调节ENaC所需的ENaC序列。3.ENAC是 也暴露在氯离子浓度的显著变化中。我们的初步工作表明，氯- 调节ENaC电流，是Na+自我抑制所必需的，这是细胞外Na+ 监管ENaC。在这个目标中，我们的目标是了解氯离子改变ENaC的机制(S 电流，并确定细胞外域中的残基介导这一效应。通过使用创新的 通过检验新的假说，这项工作将提供对机制的新理解 调节ENaC门控，从而调节上皮钠转运和钠动态平衡。

Project Summary/Abstract The epithelial Na channel (ENaC) forms a pathway for Na+ absorption in the kidney, lung, and other epithelia. In order to maintain Na+ homeostasis and control blood pressure, ENaC is tightly regulated to respond to conditions of Na+/volume depletion and Na/volume excess. However, defects in this regulation are responsible for nearly all of the known inherited forms of hypertension, and contribute to the pathogenesis of cystic fibrosis. Thus, our long term objective is to understand the mechanisms that regulate ENaC as a prerequisite for the development of targeted treatments for these diseases. A recent convergence of discoveries has focused attention on mechanisms that regulate ENaC gating. In the biosynthetic pathway and at the cell surface, proteases cleave the extracellular domains of ¿ and ¿ENaC, converting inactive channels into their active Na+-conducting form. Moreover, Na+ regulates ENaC gating through extracellular (Na+ self-inhibition) and intracellular (Na+ feedback inhibition) mechanisms to maintain homeostasis. Other extracellular molecules also regulate ENaC activity. However, there are critical gaps in our knowledge about the molecular mechanisms and channel structures that underlie this regulation. A critical advance is the very recent solution of the crystal structure of a closely related channel, ASIC1. This has provided an unprecedented look at the structures that may underlie the regulation of gating of the DEG/ENaC ion channel family. Taking advantage of these advances in the understanding of ENaC gating and the ASIC1 crystal structure, the overall goal of this proposal is to understand structure-function relationships that regulate ENaC gating. We propose three Specific Aims. 1. In preliminary studies, we discovered that intracellular Na+ regulates ENaC by altering proteolytic cleavage of ¿ and ¿ENaC. In this aim, we will test the hypothesis that Na+ alters cleavage by inducing a conformational change in the ENaC extracellular domain. We will also identify the ENaC sequences are required. 2. ENaC is exposed to extremes of pH in the kidney and lung. In preliminary studies, we found that ENaC activity is regulated by extracellular pH. In this aim, we will investigate the molecular mechanisms and identify the ENaC sequences that are required for pH to regulate ENaC. 3. ENaC is also exposed to significant changes in Cl- concentration. Our preliminary work indicates that Cl- modulates ENaC current and is required for Na+ self-inhibition, a mechanism by which extracellular Na+ regulates ENaC. In this aim, our goal is to understand the mechanism(s) by which Cl- alters ENaC current, and to identify residues in the extracellular domains that mediate this effect. By using innovative approaches and by testing novel hypotheses, this work will provide a new understanding of mechanisms that regulate ENaC gating, and hence, epithelial Na transport and Na homeostasis.