Epithelial Na Channels and Regulation of Na Excretion

描述(由申请人提供)： 摘要 上皮性钠通道控制着肾脏对盐的重吸收。它们对适应饮食中不同水平的钠是必不可少的，它们的调节缺陷可能会导致高血压。醛固酮是影响这些通道的主要激素因素，但其调节机制尚不完全清楚。我们将检验这一假设，即通道蛋白的加工和分布的变化是这种调节的主要因素。我们将结合使用电生理技术和使用针对3个通道亚单位α、β和伽马ENaC的抗体的免疫印迹来检测蛋白质总量、蛋白水解酶和糖基化酶对蛋白质加工过程中的变化以及细胞表面蛋白质的表达，并将这些事件与大鼠在盐限制和醛固酮挑战期间的通道活动相关联。我们将把这些对通道的影响与其他钠转运体的影响进行比较，包括氯化钠和氯化钠共转运体以及钠/氢交换器。然后，我们将研究这些蛋白质在急性盐补充过程中的行为，在这种情况下，钠的排泄可能在几个小时内发生巨大变化。在这个场景中，我们还将研究通道开放概率的降低在减少钠重吸收和促进钠排泄方面的作用。我们还将利用Xenous卵母细胞表达系统研究Na对开放概率影响的分子机制。最后，我们将通过评估缺乏丝氨酸/苏氨酸激酶基因的小鼠的通道激活来检验这一假设，即丝氨酸/苏氨酸激酶SGK是控制通道活性的关键蛋白。我们将测量荷尔蒙水平在几个小时(激素注入)、几天(短期限盐)或一周或更长时间(长期限盐、长期激素注入)内发生变化的动物的通道活动和通道蛋白。这一结果将进一步加深我们对这些通道在体内是如何调控的理解。 叙事 上皮钠通道的失调是大多数形式的单基因高血压的基础。然而，通道通常是如何被肾上腺类固醇和其他激素控制的，仍然知之甚少。这项提案中描述的工作将阐明这些控制机制是如何工作的，并将增强我们对高血压如何发展和影响其治疗方式的理解。

DESCRIPTION (provided by applicant): ABSTRACT Epithelial Na channels control the reabsorption of salt by the kidney. They are essential for adapting to different levels of sodium in the diet, and defects in their regulation can lead to hypertension. Aldosterone is the major hormonal factor influencing these channels, but the mechanisms by which regulation is exerted are not fully understood. We will test the hypothesis that changes in processing and distribution of channel protein is a major factor underlying this regulation. We will use a combination of electrophysiological techniques and immunoblotting using antibodies specific for the 3 channel subunits alpha, beta and gamma ENaC to examine changes in the total amount of protein, in the processing of protein by proteolytic and glycosylating enzymes, and the expression of protein at the cell surface, and to correlate these events with channel activity in rats during salt restriction and challenge with aldosterone. We will compare these effects on the channels with those of other Na transporters including NaCl and NaK2Cl cotransporters and the Na/H exchanger. We will then examine the behavior of these proteins during acute salt repletion, where large changes in Na excretion can occur in a few hours. In this scenario we will also examine the role of reduction in channel open probability in reducing Na reabsorption and facilitating Na excretion. We will alos examine the molecular mechanisms underlying the effects of Na on open probability using the Xenous oocyte expression system. Finally we will test the hypothesis that the serine/threonine kinase SGK is a key protein for control of channel activity by assessing the activation of channels in mice lacking the gene for this kinase. We will measure channel activity as well as channel protein in animals in which hormone levels are changed over a few hours (with hormone infusion), over a few days (short-term salt restriction) or over a week or more (long-term salt restriction, long-term hormone infusion). The results will further our understanding of how these channels are regulated in vivo. NARRATIVE Disregulation of epithelial Na channels underlies most forms of monogenic hypertension. Yet how channels are normally controlled by adrenal steroids and other hormones remains poorly understood. The work described in this proposal will elucidate how these control mechanisms work and will enhance our understanding of how hypertension develops and impact how it is treated.