Epithelial Na Channels: Structure-Function Studies

描述（由申请人提供）：上皮钠通道（ENaCs）与基底侧Na+/K+- atp酶一起介导钠在上皮细胞层之间的重吸收转运。这些通道在尿钠离子重吸收、细胞外液容量平衡和血压的调节中起关键作用。enact依赖性的Na+通过气道上皮的重吸收在调节气道表面液体的体积和纤毛粘膜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。我们最近在α -和γ亚基胞外区域内发现了突变影响通道门控动力学和改变外部Ni2+， Zn2+和Na+ （Na+自抑制）通道调节的位点。Aim 1中提出的实验将使用位点定向诱变来探索这些细胞外位点内残基的作用，以及高度保守的半胱氨酸残基在响应细胞外因子调节通道活性中的作用。在目标2中提出的实验将检查通道的选择性滤波器的结构组织。Aim 3中提出的实验将表征α亚基羧基末端的一个区域，该区域与PY基序不同，并调节ENaC的运输。本申请中提出的研究的成功完成将提供对上皮钠通道关键结构特征的更详细的了解。

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.