ENaC Regulation by Cell Surface Associated SGK1

细胞表面相关 SGK1 对 ENaC 的调节

• 批准号: 8734591
• 负责人: ALAN C PAO
• 金额: $ 14.1万
• 依托单位: PALO ALTO VETERANS INSTIT FOR RESEARCH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-19 至 2015-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8734591
• 关键词: Aldosterone; Apical; Binding; Blood Pressure; Cell membrane; Cell surface; Cells; Chinese Hamster Ovary Cell; Data; Diet; Duct (organ) structure; Epithelial; Equilibrium; Extracellular Fluid; Hypertension; Hypotension; Ion Channel; Knockout Mice; Knowledge; Laboratories; Mediating; Molecular; Mus; Mutagenesis; Mutation; Pathogenesis; Pathway interactions; Peptides; Phosphorylation; Phosphotransferases; Physiological; Point Mutation; Probability; Proteins; Publishing; Reagent; Regulation; Renal tubule structure; Research; Residencies; Role; Sgk protein; Signal Pathway; Site; Site-Directed Mutagenesis; Sodium Channel; Testing; Therapeutic Intervention; Wild Type Mouse; apical membrane; base; design; epithelial Na+ channel; feeding; in vivo; insight; kidney cell; mutant; novel; patch clamp; public health relevance; trafficking; urinary; wasting

DESCRIPTION (provided by applicant): Epithelial sodium channel (ENaC) activity in the collecting duct is essential for maintaining extracellular fluid volume and blood pressure. Serum and glucocorticoid regulated kinase 1 (sgk1) has emerged as a key component of the aldosterone signaling pathway that stimulates ENaC in the collecting duct. The most studied mechanism by which sgk1 increases ENaC activity involves an increase in the number of channels in the apical cell membrane. However, our laboratory has recently demonstrated that sgk1 can also stimulate the open probability (Po) of ENaC, which represents a novel means by which sgk1 acts. There remain critical gaps in our knowledge about the mechanisms that underlie sgk1 regulation of ENaC, including questions about: 1) how does sgk1 stimulate ENaC Po, and 2) whether this mode of ENaC regulation is physiologically important. The objective of the proposed research is to define novel mechanisms by which sgk1 stimulates ENaC Po in the collecting duct and determine whether this mode of ENaC regulation is important in vivo. We hypothesize that sgk1 interacts with and phosphorylates ENaC to stimulate channel Po. This mechanism of sgk1 action is distinct from sgk1-mediated regulation of Nedd4-2, a major regulator of cell surface ENaC expression. We propose three Specific Aims. In Aim 1, we will test whether sgk1 stimulates ENaC Po independent of Nedd4-2. We will introduce mutations in sgk1 or ENaC that: 1) disrupt Nedd4-2-mediated regulation of ENaC; or 2) disrupt ENaC gating. We will then test whether sgk1 can still increase channel Po under these conditions. We will also test whether sgk1 can stimulate ENaC Po in split-open collecting ducts from Nedd4-2 knockout mice. In Aim 2, we will test whether sgk1 stimulates ENaC Po through channel phosphorylation. We will use site- directed mutagenesis to identify sites in ENaC that are phosphorylated by sgk1. We will then test whether sgk1 can stimulate Po of these mutant channels. In Aim 3, we will test whether sgk1 stimulates ENaC Po through channel interaction. We will use site-directed mutagenesis and blocking peptides to identify sites in sgk1 responsible for mediating interaction with ENaC. We will use these sgk1 mutants and cell-permeant peptides to disrupt the sgk1-ENaC interaction and test whether these reagents will impair sgk1-mediated stimulation of ENaC Po and activity in collecting duct cells and split-open collecting ducts from wild type mice. The proposed research is expected to expand the current paradigm of sgk1 action and provide novel insights into how sgk1 stimulates ENaC Po in the collecting duct. We expect that this research will delineate the mechanisms by which sgk1 stimulates ENaC Po and establish the physiological importance of this mode of ENaC regulation in vivo. The impact of the proposed research may provide new insights into the pathogenesis of hypertension and suggest new targets (e.g. sgk1-ENaC interaction) for therapeutic intervention.

描述（由申请方提供）：集合管中的上皮钠通道（ENaC）活性对于维持细胞外液量和血压至关重要。血清和糖皮质激素调节激酶1（sgk 1）已成为醛固酮信号通路的关键组成部分，刺激集合管中的ENaC。sgk 1增加ENaC活性的研究最多的机制涉及顶端细胞膜中通道数量的增加。然而，我们的实验室最近证明，sgk 1也可以刺激ENaC的开放概率（Po），这代表了sgk 1发挥作用的一种新方法。在我们对sgk 1调控ENaC的机制的了解中仍然存在关键的空白，包括以下问题：1）sgk 1如何刺激ENaC Po，以及2）ENaC调控的这种模式是否具有生理学重要性。这项研究的目的是确定sgk 1刺激集合管中ENaC Po的新机制，并确定这种ENaC调节模式在体内是否重要。我们假设sgk 1与ENaC相互作用并磷酸化ENaC以刺激Po通道。这种sgk 1作用机制与sgk 1介导的Nedd 4 -2调节不同，Nedd 4 -2是细胞表面ENaC表达的主要调节因子。我们提出三个具体目标。在目标1中，我们将测试sgk 1是否独立于Nedd 4 -2刺激ENaC Po。我们将在sgk 1或ENaC中引入突变：1）破坏Nedd 4 -2介导的ENaC调控;或2）破坏ENaC门控。然后，我们将测试在这些条件下sgk 1是否仍然可以增加通道Po。我们还将测试sgk 1是否可以刺激Nedd 4 -2敲除小鼠的分裂开放收集管中的ENaC Po。在目标2中，我们将测试sgk 1是否通过通道磷酸化刺激ENaC Po。我们将使用定点突变来鉴定ENaC中被sgk 1磷酸化的位点。然后，我们将测试sgk 1是否可以刺激这些突变通道的Po。在目标3中，我们将测试sgk 1是否通过通道相互作用刺激ENaC Po。我们将使用定点突变和阻断肽来确定sgk 1中负责介导与ENaC相互作用的位点。我们将使用这些sgk 1突变体和细胞渗透肽来破坏sgk 1-ENaC相互作用，并测试这些试剂是否会损害sgk 1介导的ENaC Po刺激以及野生型小鼠集合管细胞和分裂开放集合管中的活性。这项研究有望扩展目前sgk 1作用的范式，并为sgk 1如何刺激集合管中的ENaC Po提供新的见解。我们希望这项研究将描绘sgk 1刺激ENaC Po的机制，并建立这种ENaC调节模式在体内的生理重要性。拟议研究的影响可能为高血压的发病机制提供新的见解，并为治疗干预提供新的靶点（例如sgk 1-ENaC相互作用）。