STRUCTURE AND REGULATION OF EPITHELIAL SODIUM CHANNELS

上皮钠通道的结构和调节

• 批准号: 6177646
• 负责人: CECILIA M CANESSA
• 金额: $ 26.74万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177646
• 关键词: Xenopus oocyte; aldosterone; chimeric proteins; epithelium; hormone regulation /control mechanism; immunologic assay /test; immunoprecipitation; laboratory rabbit; phosphorylation; polymerase chain reaction; protein structure function; saluresis; sodium channel; transfection; ubiquitin; voltage /patch clamp

The amiloride-sensitive epithelial sodium channel (ENaC) plays a central role in the maintenance of sodium homeostasis, it constitutes the main pathway for reabsorption of sodium in tight epithelia such as the distal nephron. The importance of the sodium channel in the maintenance of extracellular volume and blood pressure is underscored by the finding of mutations in the human genes that either active channels leading to hypertension (Liddle's syndrome), or conversely, decrease activity of channels leading to salt-wasting and hypovolemic states (pseudohypoaldosteronism). Elucidation of the mechanisms that normally regulate the function and expression of ENaC is of major importance for the understanding of blood volume maintenance in physiological and pathological conditions. The long term objectives of this work are to understand the molecular mechanisms of channel function and regulation. The specific goals of this proposal are: 1) to identify functional domains using the differences in properties exhibited by channels formed by ab and ag subunits. Mapping of functional domains will be performed using chimeras generated between the b and g subunits; 2) to elucidate the mechanics by which aldosterone mediates phosphorylation of ENaC. Aldosterone increase sodium permeability by increasing the abundance of sodium channels and by activating pre-existing channels. However, the mechanism(s) that mediate the activation of channels is still unknown. We propose that aldosterone induced phosphorylation is one of the mechanisms that activates pre-existing channels; 3) to understand the regulation of expression of ENaC by ubiquitination. We will examine the hypothesis that ubiquitination participates in endocytosis at the plasma membrane and in degradation of channels in intracellular compartments. Injected Xenopus oocytes and transfected cells will provide the expression systems for wild-type and mutant channels in which we will examine the functional properties, levels of expression, cellular distribution and rates of biosynthesis and degradation of channels. Experiments are designed to examine the activity and properties of channels by electrophysiologycal techniques. Ensembles of channels will be studied using the two micro-electrode voltage clamp and single channels using the patch-clamp technique. Biochemical, immunological, and molecular biological approaches will be applied to examine the state of phosphorylation and ubiquitination of the channel. These studies will identify important functional domains in the sodium channel, and are the first to explore two novel regulatory mechanisms of the function and expression of sodium channels.

阿米洛利敏感性上皮钠通道（ENaC）是一种中枢钠通道。 在维持钠稳态的作用，它构成了主要的 钠在紧密上皮细胞中的重吸收途径， 肾单位 钠离子通道在维持心肌细胞功能中的重要性 细胞外容积和血压的关系 人类基因的突变要么是导致 高血压（Liddle综合征），或相反， 导致盐耗和低血容量状态的通道 （假性醛固酮减少症）。 阐明通常 调节ENaC的功能和表达对于 对生理和血容量维持的理解 病理条件。 这项工作的长期目标是 了解通道功能和调节的分子机制。 本建议的具体目标是：1）确定功能 域利用所形成的通道所表现出的性质差异 由AB和AG亚基组成。 将进行功能域的定位 使用B和g亚基之间产生的嵌合体; 2）阐明 醛固酮介导ENaC磷酸化的机制。 醛固酮增加钠渗透性通过增加 钠通道和激活预先存在的通道。 但 介导通道激活的机制仍然未知。 我们认为，醛固酮诱导的磷酸化是其中之一， 激活预先存在的渠道的机制; 3）了解 通过泛素化调节ENaC的表达。 我们会研究 泛素化参与血浆内吞作用假说 膜和细胞内室中的通道降解。 注射的非洲爪蟾卵母细胞和转染的细胞将提供 野生型和突变型通道的表达系统，我们将 检查功能特性，表达水平，细胞 通道的生物合成和降解的分布和速率。 设计实验来检查活性和性质， 通过电生理技术建立通道。 频道组合将 采用双微电极电压钳和单 使用膜片钳技术的通道。 生化免疫学 分子生物学方法将被应用于检查状态 磷酸化和泛素化的通道。 这些研究 将确定钠通道中的重要功能域， 是第一个探索两种新的调节机制的功能 和钠通道的表达。