Regulation of ENaC by sgk1

描述（由申请人提供）：肾远端小管的钠重吸收对维持细胞外体积和血压至关重要。醛固酮、抗利尿激素和胰岛素通过上调ENaC活性刺激钠重吸收。越来越多的证据表明，sgkl（血清和糖皮质激素诱导的激酶）介导这些激素的作用；然而，sgkl所使用的信号通路和修饰ENaC活性的分子机制尚未阐明。我们的工作假设源于一个模型，其中sgkl主要定位于上皮细胞的基底外膜，靠近胰岛素或AVP等进入的激动剂。通过这些和/或其他迄今未知的刺激，sgkl的磷酸化触发信号级联，到达顶膜，增加ENaC的丰度和/或活性。该资助提案的具体目标是：1)确定sgkl对ENaC影响的机制，特别是研究通道流量的变化，从根尖膜插入和/或恢复，以及通道动力学的变化。2)确定sgkl对醛固酮、抗利尿激素和胰岛素反应的贡献；3)确定与sgkl相关的分子，将其连接到膜上，调节其活性或构成sgkl信号级联的底物。我们已经开发了工具并设计了生化（ENaC亚基的转换、磷酸化测定、修饰的Ras募集系统）和电生理（膜片钳研究和阻滞剂诱导的噪声分析）实验，这些实验将在体外（在四环素控制下修饰A6细胞系以表达各种形式的sgkl和sirna）和大鼠组织中进行，共同阐明提出的目标。sgkl在许多信号通路整合中的核心作用使其成为介导与高血压疾病相关的钠潴留的候选者。因此，了解这一信号通路的分子机制对高血压的诊断和发展新的治疗策略具有潜在的意义。

DESCRIPTION (provided by applicant): Sodium reabsorption in the distal tubule of the kidney is essential for maintenance of extracellular volume and blood pressure. Aldosterone, vasopressin and insulin stimulate sodium reabsorption by upregulating the activity of ENaC. There is growing evidence that sgkl (serum- and glucocorticoid-induced kinase) mediates the effects of these hormones; however, the signaling pathways employed by sgkl and the molecular mechanisms that modify ENaC activity have not been elucidated. Our working hypothesis stems from a model in which sgkl localizes predominantly at the basolateral membrane of epithelial cells in close proximity with incoming agonists such as insulin or AVP. Phosphorylation of sgkl by these and/or other stimuli to date unknown triggers a signal cascade that reaches the apical membrane to increase the abundance and/or activity of ENaC. The specific aims of this grant proposal are to: 1) Identify the mechanism underlying the effects of sgkl on ENaC in particular, to investigate changes in the traffic of channels, insertion and/or retrieval from the apical membrane, and changes in channel kinetics. 2) Determine the contribution of sgkl to aldosterone, vasopressin and insulin responses, and 3) To identify molecules that by associating with sgkl tether it to membranes, modulate its activity or constitute substrates of the sgkl-signaling cascade. We have developed tools and designed biochemical (turnover of ENaC subunits, phosphorylation assays, modified Ras recruitment system) and electrophysiological (patch-clamp studies and blocker-induced noise analysis) experiments to be conducted in vitro (A6 cell lines modified to express various forms of sgkl and siRNAs under the control of tetracycline) and in rat tissues that together will elucidate the proposed aims. The central role of sgkl in the integration of many signaling pathways makes it a candidate to mediate sodium retention associated with hypertensive disorders. Therefore, understanding the molecular mechanisms of this signaling pathway has potential implications for the diagnosis and development of new treatment strategies for the correction of high blood pressure.