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

质子-钾腺苷三磷酸酶（H+，K+-ATP酶）位于 肾集合管参与血K+的维持 平衡 虽然醛固酮是已知的主要的 K+稳态和尿酸化的调节剂，没有牢固的联系 醛固酮和基因调控之间的关系已经建立 编码H+，K+-ATP酶的亚基或泵的活性 在肾脏。 我们研究的长期目标是获得 了解肾脏中H+，K+-ATP酶活性的调节。 至少三种H+，K+-ATP酶，含有不同的α亚型， 子单元填充收集管道。 这些H+，K+-ATP酶含有 “胃”1、“结肠”2a或新的2b亚基。 2b H+，K+- ATP酶似乎是一个候选的醛固酮反应形式的 泵。 我们建议研究2a和2b H+，K+- ATP酶沿着两条平行的研究路线。 第一、 2b亚基的醛固酮反应性转录调节将 在兔皮质集合管细胞系中进行体内研究 （RCCT-28A）。 这将通过表征响应来实现 2a和2b mRNA、蛋白水平和H+，K+-ATP酶活性 在将醛固酮应用于RCCT-28 A细胞后。 的元素 从2基因的控制转录将被DNA酶I定位 超敏性和启动子缺失分析。 的响应元素 它们是否会被映射到单核苷酸分辨率， 足迹研究 第二，两种2 H+，K+-ATP酶的活性 将通过克隆的cDNA在哺乳动物细胞中的表达来研究。 一个直接的比较将在活动之间进行， 表达的2a和2b H+，K+-ATP酶的药理学性质。 最后，将研究2个亚基的磷酸化， 2a和2b H+，K+-差异调节的可能机制 ATP酶

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.