STRUCTURE AND REGULATION OF EPITHELIAL SODIUM CHANNELS

上皮钠通道的结构和调节

• 批准号: 6517473
• 负责人: CECILIA M CANESSA
• 金额: $ 28.36万
• 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 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6517473
• 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.

阿米洛利敏感的上皮钠通道 (ENaC) 发挥着中枢作用。 维持钠稳态的作用，它构成了主要的 紧密上皮（例如远端）中钠的重吸收途径 肾单位。 钠通道在维持功能中的重要性 这一发现强调了细胞外容量和血压 人类基因突变的活跃通道导致 高血压（利德尔综合征），或相反，降低 导致盐浪费和低血容量状态的通道 （假性醛固酮增多症）。 阐明通常情况下的机制 调节 ENaC 的功能和表达对于 生理和心理上对血容量维持的理解 病理状况。 这项工作的长期目标是 了解通道功能和调节的分子机制。 该提案的具体目标是：1）确定功能 利用形成的通道表现出的特性差异的域 由 ab 和 ag 亚基组成。 将进行功能域的映射 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。 醛固酮通过增加钠的丰度来增加钠的渗透性 钠通道并通过激活预先存在的通道。 然而， 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。 我们将检查 泛素化参与血浆内吞作用的假设 膜和细胞内区室通道的降解。 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，我们将在其中 检查功能特性、表达水平、细胞 通道生物合成和降解的分布和速率。 实验旨在检查活性和特性 通过电生理学技术的通道。 频道组合将 使用两个微电极电压钳和单电极进行研究 使用膜片钳技术的通道。 生化、免疫学、 并将应用分子生物学方法来检查状态 通道的磷酸化和泛素化。 这些研究 将识别钠通道中的重要功能域，并且 第一个探索该功能的两种新颖的调节机制 和钠通道的表达。