REGULATION AND FUNCTION OF THE ALPHA SUBUNIT OF ENAC

本研究的目的是研究amiloride敏感上皮Na+通道(ENaC) α亚基的调控和功能。该通道的活性决定了远端肾元Na+重吸收和K+分泌的程度。醛固酮、糖皮质激素和cAMP对通道活性有重要的调节作用。我们最近鉴定并鉴定了多种人类alphaENaC （halphaENaC）转录本，这些转录本由不同的启动子调控，并编码两种功能性n端变异蛋白。本研究有两大目标：第一是研究肾和气道上皮细胞中halphaENaC异构体的调控。我们假设皮质类固醇和cAMP通过转录调节alphaENaC的表达来调节酰胺敏感的Na+转运。第二是研究α ENaC的n端在蛋白-蛋白相互作用和赋予ENaC复合物功能特性中的作用。我们假设ENaC转运上皮细胞的功能异质性可能部分来自ENaC复合物的分子变异，并且细胞质效应物与alphaENaC的每个n端异构体的相互作用不同。本研究提出的数据包括鉴定和表征人alphaENaC替代转录本及其在肾和肺细胞系中皮质类固醇的转录调节。在halphaENaC的5'侧区发现了一个功能性的激素反应元件，变异的alphaENaC蛋白被证明可以重建一个功能性的酰胺敏感离子通道。具体目的是：(1)研究肾集管和气道上皮中alphaENaC基因表达的转录调控；(2)表征调节alphaENaC表达的转录因子；(3)确定α ENaC的n端在蛋白-蛋白相互作用和赋予ENaC复合物功能特性中的作用。长期目标是了解ENaC在上皮Na+转运中的调控和功能。

The goals of this proposal are to examine the regulation and function of the alpha subunit of the amiloride-sensitive epithelial Na+ channel (ENaC). The activity of this channel determines the extent of Na+ reabsorption and K+ secretion in the distal nephron. Aldosterone, glucocorticoids and cAMP have important regulatory influences on channel activity. We have recently identified and characterized multiple human alphaENaC (halphaENaC) transcripts that are regulated by distinct promoters and encode two functional N-terminal variant proteins. This proposal has two broad goals: The first is to examine the regulation of halphaENaC isoforms in renal and airway epithelial cells. We hypothesize that corticosteroids and cAMP regulate amiloride-sensitive Na+ transport by transcriptional regulation of alphaENaC expression. The second is to examine the role of the N-terminus of alphaENaC in protein-protein interactions and in conferring functional properties to the ENaC complex. We hypothesize that functional heterogeneity of ENaC transporting epithelia may be in part from molecular variants of the ENaC complex and that cytosolic effectors interact differently with each of the N-terminal isoforms of alphaENaC. The data presented with this proposal includes the identification and characterization of human alphaENaC alternate transcripts and their transcriptional regulation by corticosteroids in renal and lung cell lines. A functional hormone response element in the 5' flanking region of halphaENaC is discovered and variant alphaENaC proteins are shown to reconstitute a functional amiloride- sensitive ion channel. The specific aims are: (1) to examine transcriptional regulation of alphaENaC gene expression in kidney collecting duct and airway epithelia; (2) to characterize transcription factors that regulate alphaENaC expression; and (3) to determine the role of the N-terminus of alphaENaC in protein- protein interactions and in conferring functional properties to the ENaC complex. The long-term objectives are to understand the regulation and function of ENaC in transepithelial Na+ transport.