Epithelial Na Channels: Structure-Function Studies

描述（由申请方提供）：上皮钠通道（ENaCs）与基底外侧Na+/K+-ATP酶一起介导跨上皮细胞层的重吸收钠转运。这些通道在调节尿Na+重吸收、细胞外液容量稳态和血压中起关键作用。ENaC依赖的跨气道上皮的Na+重吸收在调节气道表面液体的体积和粘膜纤毛清除中具有重要作用。已经确定了重要的功能域，包括影响通道门控、运输和阳离子选择性的位点。本申请中提出的研究将解决有关ENaC结构-功能关系的问题。我们最近确定了α和γ亚基细胞外结构域内的位点，其中突变影响通道门控动力学并通过外部Ni 2+、Zn 2+和Na+（Na+自抑制）改变通道调节。目标1中提出的实验将使用定点诱变来探索这些胞外位点内的残基的作用以及高度保守的半胱氨酸残基在响应胞外因子调节通道活性中的作用。目标2中提出的实验将检查通道的选择性滤波器的结构组织。目标3中提出的实验将表征α亚基的羧基末端内的区域，该区域不同于PY基序并调节ENaC运输。本申请中提出的研究的成功完成将提供对上皮钠通道的关键结构特征的更详细的理解。

DESCRIPTION (provided by applicant): Epithelial sodium channels (ENaCs) mediate reabsorptive sodium transport across epithelial cell layers in conjunction with a basolateral Na+/K+-ATPase. These channels have a key role in the regulation of urinary Na+ reabsorption, extracellular fluid volume homeostasis and blood pressure. ENaC-dependent Na+ reabsorption across airway epithelia has a significant role in regulating the volume of airway surface liquids and mucociliary clearance. Important functional domains have been identified, including sites that affect channel gating, trafficking and cation selectivity. Studies proposed in this application will address questions regarding ENaC structure-function relationships. We recently identified sites within alpha- and gammasubunit extracellular domains where mutations affect channel gating kinetics and alter channel regulation by external Ni2+, Zn2+ and Na+ (Na+ self-inhibition). Experiments proposed in Aim 1 will use site-directed mutagenesis to explore the role of residues within these extracellular sites and the role of highly conserved cysteine residues in modulating channel activity in response to extracellular factors. Experiments proposed in Aim 2 will examine the structural organization of the channel's selectivity filter. Experiments proposed in Aim 3 will characterize a region within the carboxyl-terminus of the alpha subunit that is distinct from the PY motif and regulates ENaC trafficking. Successful completion of the studies proposed in this application will provide a more detailed understanding of key structural features of epithelial sodium channels.