Structure and Function of Epithelial Sodium Channels

上皮钠通道的结构和功能

• 批准号: 8918334
• 负责人: Isabelle Rhyssa Joe Eduria Baconguis
• 金额: $ 38.5万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-19 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8918334
• 关键词: Address; Affect; Amiloride; Architecture; Baculoviruses; Binding Sites; Biochemical; Biological Assay; Blood Pressure; Cell membrane; Cells; Clinical; Crystallography; Data Set; Detection; Disease; Electrophysiology (science); Environment; Epithelial; Equilibrium; Event; Experimental Designs; Extracellular Domain; Extracellular Fluid; Fluorescence; Foundations; Future; Genes; Genetic; Genetic Transcription; Goals; Homeostasis; Human body; Hyperactive behavior; Hypertension; Hypotension; Integral Membrane Protein; Ion Channel; Ion Channel Gating; Ions; Kidney; Light; Mammalian Cell; Maps; Measures; Mediating; Membrane; Membrane Proteins; Methods; Molecular; Molecular Sieve Chromatography; Nephrotic Syndrome; Peptides; Physiological; Play; Population; Production; Proteins; Proteolysis; Pseudohypoaldosteronism; Regulation; Regulation of Proteolysis; Research; Research Proposals; Resolution; Rest; Role; Rupture; Site; Sodium Channel; Stimulus; Structure; Structure-Activity Relationship; Syndrome; Technology; Therapeutic; Water; Work; base; biophysical analysis; blood pressure reduction; channel blockers; epithelial Na+ channel; human disease; insight; ion channel blocker; member; milligram; mutant; novel; protein expression; public health relevance; research study; three dimensional structure; trafficking; voltage

DESCRIPTION (provided by applicant): Epithelial sodium channels (ENaCs) are members of the ubiquitous ENaC/DEG superfamily of trimeric voltage-independent, Na+-selective and amiloride-sensitive ion channels. Highly expressed in the kidneys, ENaCs assemble as heterotrimers that harbor an exquisitely Na+-selective pore that is critical in the fine tuning of Na+ and K+ balance. ENaC function is regulated at multiple levels from transcription of the ENaC genes, to the trafficking and the proteolytic activation of the ion channel. Abnormal regulation in the functional activity of ENaCs contributes importantly to human disease, and especially to hypertension (high blood pressure), a condition that affects about 1 billion people worldwide. Furthermore, although proteolysis is integral in the activation of ENaCs, aberrant proteolysis of ENaCs contributes to NaCl retention that characterizes nephrotic syndrome. Despite such clinical importance, detail into the mechanism of ENaC function at atomic resolution has remained elusive and the lack of x-ray crystal structures of ENaC has been a barrier to progress in the field. The objective of this research application is to resolve molecula mechanisms underlying ENaC assembly, gating, ion permeation, and allosteric modulation utilizing methods of x-ray crystallography, electrophysiology, and other biochemical and biophysical assays. At present, there are no methods for producing large quantities of heterotrimeric ENaCs for biochemical and biophysical experiments. The aim of this research application is to develop the technology to express milligram quantities of ENaC that demonstrate a homogenous population suitable for functional and structural assays. Central to this application is to determine the x-ray crystal structures of ENaC at different physiological states and these studies will provide the first atomic-level presentation of these Na+-selective channels in their resting, closed and open states, thus contributing to the understanding of the molecular basis of ENaC function. More importantly, these structural studies will serve as blueprints for future therapeutic strategies.

描述（由申请人提供）：上皮钠通道（ENaCs）是无处不在的ENaC/DEG超家族的成员，是三聚体电压无关，Na+选择性和酰胺敏感的离子通道。ENaCs在肾脏中高度表达，聚集成异源三聚体，其中包含一个精致的Na+选择性孔，对Na+和K+平衡的微调至关重要。从ENaC基因的转录，到离子通道的运输和蛋白水解激活，ENaC功能在多个水平上受到调节。ENaCs功能活动的异常调节对人类疾病，特别是影响全球约10亿人的高血压（高血压）起着重要作用。此外，尽管ENaCs的蛋白水解是其活化过程中不可或缺的一部分，但ENaCs的异常蛋白水解会导致肾病综合征特征的NaCl滞留。尽管具有如此重要的临床意义，但在原子分辨率上详细了解ENaC功能的机制仍然难以捉摸，缺乏ENaC的x射线晶体结构一直是该领域进展的障碍。本研究应用的目的是利用x射线晶体学、电生理学和其他生化和生物物理分析方法，解决ENaC组装、门控、离子渗透和变构调制的分子机制。目前，还没有大量制备异三聚体ENaCs用于生化和生物物理实验的方法。本研究申请的目的是开发一种技术来表达毫克量的ENaC，这种ENaC显示出适合于功能和结构分析的同质群体。该应用程序的核心是确定ENaC在不同生理状态下的x射线晶体结构，这些研究将首次在原子水平上展示这些Na+选择通道在静息、关闭和打开状态下的结构，从而有助于理解ENaC功能的分子基础。更重要的是，这些结构研究将为未来的治疗策略提供蓝图。