Regulation of ENaC by phosphatidylinositide 3-kinase and phospholipids

描述(由申请人提供)： 调节远端肾单位的Na+重吸收，在一定程度上控制着人类和其他陆生脊椎动物的血压。醛固酮敏感的上皮性Na+通道(ENaC)的活性限制了Na+在这里的运输。ENaC的功能障碍和不适当的调节会导致血压紊乱和肾脏、结肠和肺对盐的不当处理。这项提案是对R01的竞争性更新，该R01正在调查ENaC的醛固酮调节，该调节在最初的资助期取得了很大成功。这一初始资助期的结果以及最新的初步结果有力地表明，包括磷脂酰肌醇3-激酶(PI3-K)在内的磷脂激酶及其产物磷脂酰肌醇是ENaC活性的关键决定因素。两种重要的生理激素，醛固酮和胰岛素，可增加远端肾单位上皮细胞的P3-K活性及其产物磷脂酰肌醇3，4，5-三磷酸(PI(3，4，5)P3)的合成。支撑这一增长的细胞机制仍不清楚。此外，PI3-K在多大程度上是醛固酮和胰岛素对ENaC作用的主要仲裁者仍未被探索。此外，PI(3，4，5)P3调节ENaC的可能机制和生理后果尚未得到充分认识。类似地，ENaC中响应PI(3，4，5)P3信号的假定分子决定因素仍有待确定。PI3-K及其磷脂酰肌醇产物是调节ENaC的核心这一普遍假设统一了这一提议的实验和想法。这条研究路线是我早期研究的合乎逻辑的延伸，我希望对其进行测试会得出新的和有意义的发现。在这里，我针对三个具体目标：1)描述并赋予针对ENaC的PI3-K和PI(3，4，5)P3信号通路的生理意义；2)确定PI(3，4，5)P3调节ENaC的细胞机制；3)建立PI(3，4，5)P3调节ENaC的分子决定因素。我用一个全面的实验设计来验证我的假设，该设计由互补的实验组成，以提供从分子到整个组织的PI3-K对ENaC调控的综合理解。在人类和其他陆生脊椎动物中，肾远端肾单位适当的Na+重吸收是调节全身Na+平衡从而调节血压的中心。在这里，醛固酮敏感的上皮性Na+通道(ENaC)的活性限制了Na+的重吸收。目前的建议继续我们对醛固酮反应控制ENaC活性的细胞和分子机制的研究。

DESCRIPTION (provided by applicant): Regulated Na+ reabsorption at the distal nephron, in part, controls blood pressure in humans and other terrestrial vertebrates. Activity of the aldosterone-sensitive epithelial Na+ channel (ENaC) is limiting for Na+ transport here. Dysfunction and inappropriate regulation of ENaC result in blood pressure disorders and improper salt handling by the kidney, colon and lungs. This proposal is a competitive renewal of an R01 investigating aldosterone regulation of ENaC that has had much success during its initial funding period. Results from this initial funding period, as well as, newer preliminary results strongly suggest that phospholipid kinases, including phosphatidylinositide 3-kinase (PI3-K), and their product phosphatidylinositides are critical determinants of ENaC activity. Two physiologically important hormones that increase ENaC activity, aldosterone and insulin, increase P3-K activity and synthesis of its product phosphatidylinositol, phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), in distal nephron epithelia. The cellular mechanism underpinning this increase remains unknown. The extent to which PI3-K is the primary arbiter of aldosterone and insulin actions on ENaC, in addition, remains unexplored. Moreover, possible mechanisms and physiological consequences of PI(3,4,5)P3 modulation of ENaC are not fully appreciated. Putative molecular determinants within ENaC responsive to PI(3,4,5)P3 signaling, similarly, remain to be identified. The general hypothesis that PI3-K and its phosphatidylinositide products are central to regulation of ENaC unites the experiments and ideas of this proposal. This line of inquiry is a logical extension of my earlier studies, and I expect testing it to result in novel and significant findings. Here, I address three specific aims: 1) Delineate and assign physiological significance to PI3-K and PI(3,4,5)P3 signaling pathways targeting ENaC; 2) Determine the cellular mechanism of PI(3,4,5)P3 regulation of ENaC; and 3) Establish the molecular determinants within ENaC of PI(3,4,5)P3 regulation. I test my hypotheses with a comprehensive experimental design structured with complementary experiments to provide an integrative understanding of ENaC regulation by PI3-K from the molecule to the whole tissue. Proper Na+ reabsorption at the distal renal nephron in humans and other terrestrial vertebrates is central to regulation of systemic Na+ balance and thus, blood pressure. Activity of the aldosterone-sensitive epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption here. The current proposal continues our investigation of the cellular and molecular mechanisms controlling ENaC activity in response to aldosterone.