Structure-Function Studies of Epithelial Sodium Channel Gating

上皮钠通道门控的结构功能研究

• 批准号: 7652670
• 负责人: Peter M Snyder
• 金额: $ 33.75万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7652670
• 关键词: Anions; Attention; Cell surface; Cleaved cell; Cysteine; Cystic Fibrosis; Data; Defect; Development; Disease; Epithelial; Epithelium; Epitopes; Equilibrium; Extracellular Domain; Family; Feedback; Goals; Homeostasis; Human; Hypertension; Inherited; Ion Channel; Ions; Kidney; Knowledge; Lung; Mediating; Modification; Molecular; Mutation; Pathogenesis; Pathway interactions; Peptide Hydrolases; Proteins; Regulation; Role; Site; Sodium Channel; Solutions; Structure; Structure-Activity Relationship; Testing; Urine; Work; absorption; blood pressure regulation; collecting tubule structure; effective therapy; epithelial Na+ channel; extracellular; innovation; novel; public health relevance

DESCRIPTION (provided by applicant): 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 1 and 3ENaC, 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 1 and 3ENaC. 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. PUBLIC HEALTH RELEVANCE: Defects in the regulation of the epithelial sodium channel (ENaC) cause diseases including hypertension and cystic fibrosis. The overall goal of this proposal is to investigate the mechanisms that control the activity of ENaC. This will provide a new understanding of the pathogenesis of these diseases which will facilitate development of more effective treatments.

描述（由申请人提供）：上皮NA通道（ENAC）形成了肾脏，肺和其他上皮中Na+吸收的途径。为了维持NA+稳态和控制血压，ENAC受到严格调节以应对Na+/体积耗竭和Na/量过量的条件。但是，该调节中的缺陷几乎负责所有已知的遗传性高血压形式，并有助于囊性纤维化的发病机理。因此，我们的长期目标是了解调节ENAC作为开发这些疾病有针对性治疗的先决条件的机制。最新发现的融合将注意力集中在调节ENAC门控的机制上。在生物合成途径和细胞表面中，蛋白酶裂解1和3ENAC的细胞外域，将不活跃的通道转化为活性Na+导导形式。此外，Na+通过细胞外（NA+自我抑制）和细胞内（NA+反馈抑制）机制调节ENAC门控，以维持稳态。其他细胞外分子也调节ENAC活性。但是，我们对基于该调节的分子机制和通道结构的知识存在关键的差距。一个关键的进步是密切相关通道ASIC1的晶体结构的最新解决方案。这已经提供了前所未有的观察，这些结构可能是对DEG/ENAC ION通道家族门控的调节的基础。利用这些进步在理解E​​NAC门口和ASIC1晶体结构方面，该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1和3ENAC的蛋白水解裂解来调节ENAC。在此目标中，我们将检验以下假设：Na+通过诱导ENAC细胞外域的构象变化来改变裂解。我们还将确定需要ENAC序列。 2。ENAC暴露于肾脏和肺中的极端pH值。在初步研究中，我们发现ENAC活性受细胞外pH的调节。在此目标中，我们将研究分子机制，并确定pH调节ENAC所需的ENAC序列。 3。ENAC还暴露于Cl浓度的显着变化。我们的初步工作表明CL-调节ENAC电流，是Na+自我抑制所必需的，Na+自我抑制是一种机制，细胞外Na+调节ENAC。在此目标中，我们的目标是了解Cl-cl- cl-改变ENAC电流的机制，并确定介导这种作用的细胞外域中的残基。通过使用创新的方法并检验新的假设，这项工作将提供对调节响应门控的机制的新理解，因此可以提供上皮NA运输和NA稳态。 公共卫生相关性：调节上皮钠通道（ENAC）的缺陷会导致包括高血压和囊性纤维化在内的疾病。该提案的总体目标是研究控制ENAC活性的机制。这将为这些疾病的发病机理提供新的了解，这将有助于发展更有效的治疗方法。