REGULATION AND FUNCTION OF THE ALPHA SUBUNIT OF ENAC

这项建议的目的是研究阿米洛利敏感的上皮钠通道(ENaC)的阿尔法亚单位的调节和功能。这一通道的活动决定了远端肾单位钠重吸收和钾分泌的程度。醛固酮、糖皮质激素和cAMP对通道活动有重要的调节作用。我们最近鉴定了多个受不同启动子调控的人αENaC(HalphaENaC)转录本，并编码两个功能性N末端变异蛋白。这项提议有两个广泛的目标：第一个是研究肾脏和呼吸道上皮细胞中halphaENaC亚型的调节。我们假设皮质类固醇和cAMP通过转录调控αENaC的表达来调节对阿米洛利敏感的钠转运。第二个是研究α-ENaC的N-末端在蛋白质-蛋白质相互作用和赋予ENaC复合体功能特性中的作用。我们推测，ENaC转运上皮细胞的功能异质性可能部分来自ENaC复合体的分子变体，胞液效应器与αENaC的每一种N-末端异构体的作用不同。该提案提供的数据包括人αENaC替代转录本的鉴定和特征，以及皮质类固醇对其在肾和肺细胞系中的转录调控。在halphaENaC的5‘侧翼区发现了一个功能性激素反应元件，突变的alphaENaC蛋白被证明重建了一个功能性的阿米洛利敏感离子通道。其具体目的是：(1)检测肾脏集合管和呼吸道上皮细胞中α-ENaC基因表达的转录调控；(2)鉴定调节α-ENaC表达的转录因子；(3)确定α-ENaC的N端在蛋白质-蛋白质相互作用中的作用以及赋予ENaC复合体功能特性的作用。长期目标是了解ENaC在跨上皮钠转运中的调节和功能。

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.