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: 2884893
• 负责人: DALE J BENOS
• 金额: $ 18.64万
• 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/2884893
• 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管，其主要功能是调节全身的钠平衡。这些上皮细胞含有定位于其管腔或顶膜的钠通道，该通道可被利尿剂阿米洛利抑制。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，一个上皮性钠通道(称为ENaC)被克隆。该通道至少由三个结构上相关的亚基组成(称为α-ENaC、β-ENaC和伽马-ENaC)。这些ENaC的一级和预测的二级结构已经被描述，但在每个ENaC亚基中为结构域分配特定的功能角色才刚刚开始。该项目的长期目标是了解特定氨基酸结构域在ENaC中的功能作用。我们假设ENaC的阳离子选择性滤膜和通道孔区在空间上是不同的。此外，我们假设在每个ENaC亚基的前H_2M2区有特定的谷氨酸残基参与钙结合/抑制通道。我们还假设，在每个ENaC亚基的M2区存在特定的负电荷氨基酸(天冬氨酸和谷氨酸)，这些氨基酸构成了传导通路的一部分，即通道的孔。本应用有两个特定的目的：(1)测试两个特定的负电荷氨基酸，即位于第二大疏水结构域(H2 M2)上游的4个和12个氨基酸是α和α-rENaC的选择性过滤器内的钙离子阻断位点的假设；(2)测试每个ENaC亚单位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不可少的假设。这项研究的重要方面是将阐明ENaC独特的生化、生理和分子生物学特性；将确定参与特定通道功能的蛋白质序列区域；将对ENaC功能的总体分子结构产生新的见解。这些结果将对阿米洛利敏感钠通道的性质提供新的见解，因为涉及阳离子选择性的通道的重要结构区域、与通道的钙相互作用以及通过孔的电导将被定义。