MOLECULAR REGULATION OF THE RENAL ALPHA2 H+, K+ ATPASES

质子-钾-三磷酸腺苷酶(H+，K+-ATPase)位于 肾集合管参与血K+的维持 平衡。尽管已知醛固酮是一种主要的 K+动态平衡和尿酸的调节，没有确切的联系 已经在醛固酮和基因调控之间建立了联系 编码H+，K+-ATPase亚基或泵的活性 在肾脏里。我们研究的长期目标是获得一个 了解肾脏H+，K+-ATPase活性的调节。 至少三种含有不同α亚型的H+，K+-ATPase 子单元填充收集风管。这些H+，K+-ATPase包含 “胃”1、“结肠”2a或新的2b亚基。2bH+，K+- ATPase似乎是醛固酮反应形式的候选者 打气筒。我们建议研究2a和2b的H+，K+-的表达。 ATPas遵循两条平行的调查路线。第一, 2b亚单位的醛固酮反应性转录调控将 兔皮质集合管细胞系的体内研究 (RCCT-28A)。这将通过描述响应的特征来实现 在2a和2b的mRNAs中，蛋白质水平和H+，K+-ATPase活性 将醛固酮作用于RCCT-28A细胞。元素 控制2基因转录的将由DNase I定位 超敏反应和启动子缺失分析。回应要素 它们会在体内被映射到单核苷酸解析吗？ 足迹研究。第二，两种2H+，K+-ATPase的活性 将通过在哺乳动物细胞中表达克隆的cDNA来进行研究。 将直接比较这些活动和 表达的2a和2b H+，K+-ATPase的药理性质。 最后，这两个亚基的磷酸化将作为一种 2a和2b H~+，K~+-的差异调控机制 ATPase。

Proton-potassium adenosine triphosphatases (H+, K+-ATPases) located in the renal collecting duct are involved in maintenance of blood K+ balance. Although aldosterone is known to be one of the principal regulators of K+ homeostasis and urinary acidification, no firm linkage has been established between aldosterone and the regulation of genes encoding the subunits of the H+, K+-ATPase or the activity of the pump in the kidney. The long term goal of our research is to gain an understanding of the regulation of H+, K+-ATPase activity in the kidney. At least three H+, K+-ATPases containing distinct isoforms of the alpha subunit populate the collecting duct. These H+, K+-ATPases contain the "gastric" 1, the "colonic" 2a or the novel 2b subunit. The 2b H+, K+- ATPase appears to be a candidate for the aldosterone-responsive form of the pump. We propose to study the expression of the 2a and 2b H+, K+- ATPases following two parallel lines of investigation. First, aldosterone-responsive transcriptional regulation of the 2b subunit will be investigated in vivo in a rabbit cortical collecting tubule cell line (RCCT-28A). This will be accomplished by characterizing the response of the 2a and 2b mRNAs, protein levels and H+, K+-ATPase activity following application of aldosterone to RCCT-28A cells. The elements governing transcription from the 2 gene will be located by DNase I hypersensitivity and promoter deletion analysis. The response elements will them be mapped to single-nucleotide resolution by in vivo footprinting studies. Second, the activity of the two 2 H+, K+-ATPases will be studied by expression of the cloned cDNAs in mammalian cells. A direct comparision will be made between the activities and pharmacological properties of the expressed 2a and 2b H+, K+-ATPases. Finally, phosphorylation of the 2 subunits will be investigated as a possible mechanism for differential regulation of the 2a and 2b H+, K+- ATPases.