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Elucidating the molecular mechanism of ENaC function

阐明 ENaC 功能的分子机制

基本信息

批准号：
10593943
负责人：
Isabelle Rhyssa Joe Eduria Baconguis
金额：
$ 34.65万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-05 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10593943
关键词：
3-Dimensional Acceleration Address Affect Baculoviruses Binding Biochemical Biological Assay Biophysics Blood Pressure Blood Volume Cell physiology Characteristics Classification Cloning Complex Computer Analysis Cryoelectron Microscopy Detection Development Disease Distal Drug Design Electrophysiology (science)Equilibrium Excision Extracellular Domain Fluorescence Foundations Future Genetic Goals Heterogeneity Homeostasis Human Hypertension Integral Membrane Protein Investigation Ion Channel Ions Kidney Kidney Diseases Length Life Ligand Binding Ligands Link Liquid substance Mammals Maps Mass Spectrum Analysis Measures Mediating Membrane Proteins Methodology Methods Modification Molecular Molecular Conformation Molecular Sieve Chromatography Nephrons Peptide Hydrolases Peptides Persons Physiology Positioning Attribute Potassium Channel Proteins Proteolysis Publishing Radiolabeled Regulation Research Resolution Rest Sampling Signal Transduction Site Sodium Channel Sodium Chloride Structure System Therapeutic Transmembrane Domain Water Work absorption biophysical analysis blood pressure control epithelial Na+ channel glycosylation insight molecular modeling novel therapeutic intervention particle patch clamp protein expression stoichiometry therapeutic target three dimensional structure voltage

项目摘要

Project summary The epithelial sodium channel (ENaC) fine-tunes Na+ and water balance and is the primary regulator of blood pressure and volume through Na+ reabsorption in the distal nephron. Thus, ENaC is a critical therapeutic target for kidney diseases and blood pressure control. Here we propose to develop a molecular model for ENaC function by determining high-resolution structures of ENaC in different functional states. Molecular studies of ENaC have proven difficult largely because ENaCs are labile, multimeric integral membrane proteins that are activated by proteolysis of inhibitory domains located in the extracellular domain; how these peptidyl tracts exert their effects remains unresolved. Our goal is to develop methods for expression and purification of ENaC in the uncleaved and cleaved conformations. We will apply these methods to elucidate structure-based mechanisms underpinning ENaC gating, ion permeation, and allosteric modulation by combining single-particle cryo-electron microscopy and electrophysiology, together with judiciously chosen biochemical and biophysical assays. The structures will offer key insight into the molecular basis of proteolytic activation of ENaC providing positions of the inhibitory domains and identifying critical regions that mediate ENaC acitivity yielding the first accurate blueprints for rational, structure-based therapeutic strategies.

项目概要上皮钠通道 (ENaC) 微调 Na+ 和水平衡，是通过远端肾单位的 Na+ 重吸收来主要调节血压和容量。因此，ENaC 是肾脏疾病和血压控制的关键治疗靶点。这里我们建议通过确定高分辨率来开发 ENaC 功能的分子模型 ENaC在不同功能状态下的结构。 ENaC 的分子研究已被证明是困难的主要是因为 ENaC 是不稳定的多聚体膜蛋白，可被以下物质激活：位于细胞外结构域的抑制结构域的蛋白水解；这些肽基束是如何形成的发挥其作用仍悬而未决。我们的目标是开发表达和表达的方法纯化未切割和切割构象的 ENaC。我们将应用这些方法阐明支持 ENaC 门控、离子渗透和通过结合单粒子冷冻电子显微镜和电生理学，以及明智选择的生化和生物物理测定。这结构将为了解 ENaC 蛋白水解激活的分子基础提供关键见解提供抑制域的位置并识别介导 ENaC 的关键区域活动为合理的、基于结构的治疗策略提供了第一个准确的蓝图。