CATION SELECTIVITY, CONDUCTION, AND CA++ BLOCK OF ENAC

ENAC 的阳离子选择性、传导性和 CA 块

• 批准号: 6381577
• 负责人: DALE J BENOS
• 金额: $ 19.77万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6381577
• 关键词: Xenopus; Xenopus oocyte; amiloride; calcium ion; cations; confocal scanning microscopy; electrophysiology; epithelium; ion transport; protein sequence; protein structure function; renal tubular transport; single cell analysis; sodium channel; tissue /cell culture; transfection

Sodium reabsorbing epithelia, such as renal distal and collecting tubules, have as their major function the regulation of whole-body sodium balance. These epithelia contain sodium channels localized in their luminal or apical membranes that are inhibited by the diuretic drug amiloride. It is at the level of these channels that the primary feedback control mechanisms necessary for the maintenance for sodium homeostasis occur. Recently, an epithelial sodium channel (termed ENaC) has been cloned. This channel, at a minimum, consists of at least three structurally related subunits (termed alpha-ENaC, beta-ENaC, and gamma-ENaC). The primary and predicted secondary structures of these ENaCs have been described, but the assignment of specific functional roles to domains within each ENaC subunit has just begun. The long term goal of this project is to understand the functional roles of specific amino acid domains within ENaC. We hypothesize that the cation selectivity filter and channel pore regions of ENaC are spatially distinct. Moreover, we hypothesize that there are specific glutamic acid residues in the pre-H2M2 region of each ENaC subunit that participates in calcium binding/inhibition of the channel. We also hypothesize that there are specific negatively charged amino acids (aspartic acid and glutamic acid) located within the M2 region of each ENaC subunit that constitute part of the conduction pathway, or pore of the channel. This application has two specific aims: (1) to test the hypothesis that two specific negatively charged amino acids, located 4 and 12 amino acids upstream from the beginning of the second large hydrophobic domain (H2M2) within the selectivity filter of alpha, beta, and gamma-rENaC, are the sites involved in Ca2+ block of both alpha and alphabetagamma-rENaC; and (2) to test the hypothesis that specific hydrophilic residues (E568, E571, and D575) within the M2 region of each ENaC subunit are essential for the conduction properties of the pore. Important aspects of this study are that unique biochemical, physiological, and molecular biological characteristics of ENaCs will be elucidated; regions of protein sequence that participate in specific channel functions will be identified; and new insights into the gross molecular architecture of a functional ENaC will be developed. These results will offer new insights into the nature of amiloride-sensitive sodium channels, in that important structural regions of the channel involved in cation selectivity, calcium interactions with the channel, and conductions through the pore will be defined.

钠重吸收上皮细胞，如肾远端小管和集合小管，具有调节全身钠平衡的主要功能。 这些上皮细胞含有位于其腔或顶膜中的钠通道，其被利尿药阿米洛利抑制。 正是在这些通道的水平上，发生了维持钠稳态所必需的主要反馈控制机制。最近，上皮钠通道（称为ENaC）已被克隆。 该通道至少由至少三个结构相关的亚基（称为α-ENaC、β-ENaC和γ-ENaC）组成。 已经描述了这些ENaC的一级和预测的二级结构，但是每个ENaC亚基内的结构域的特定功能角色的分配才刚刚开始。 该项目的长期目标是了解ENaC中特定氨基酸结构域的功能作用。 我们假设ENaC的阳离子选择性过滤器和通道孔区域在空间上是不同的。 此外，我们推测，有特定的谷氨酸残基在前H2 M2区域的每个ENaC亚基参与钙结合/抑制的通道。 我们还假设，有特定的带负电荷的氨基酸（天冬氨酸和谷氨酸）位于M2区域内的每个ENaC亚基，构成部分的传导途径，或孔的通道。 本申请有两个具体目的：（1）检验以下假设：位于α、β和γ-rENaC的选择性过滤器内第二大疏水结构域（H2 M2）起始处上游4和12个氨基酸的两个特定带负电荷的氨基酸是参与α和α-γ-rENaC的Ca 2+阻断的位点;和（2）检验每个ENaC亚基的M2区域内的特定亲水残基（E568、E571和D575）对于孔的传导性质是必需的这一假设。这项研究的重要方面是，独特的生物化学，生理和分子生物学特性的ENaC将被阐明;区域的蛋白质序列，参与特定的通道功能将被确定;和新的见解的毛分子结构的功能ENaC将被开发。 这些结果将提供新的见解阿米洛利敏感的钠通道的性质，在阳离子选择性，钙离子与通道的相互作用，并通过孔传导的重要结构区域的通道将被定义。