Epithelial Na Channels and Regulation of Na Excretion

描述（由申请人提供）： 摘要 上皮Na通道控制肾脏对盐的重吸收。它们对于适应饮食中不同水平的钠是必不可少的，其调节的缺陷可能导致高血压。醛固酮是影响这些通道的主要激素因子，但其调节机制尚不完全清楚。我们将测试这一假设，即通道蛋白的加工和分布的变化是这种调节的主要因素。我们将使用电生理技术和免疫印迹的组合，使用3个通道亚基α，β和γ ENaC的特异性抗体，以检查蛋白质总量的变化，蛋白质的蛋白水解和糖基化酶的处理，以及蛋白质在细胞表面的表达，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性相关联。我们将比较这些影响的通道与其他钠转运蛋白，包括NaCl和NaK 2Cl cotransporters和Na/H交换。然后，我们将研究这些蛋白质在急性盐饱期间的行为，其中钠排泄的大变化可以在几个小时内发生。在这种情况下，我们还将研究通道开放概率降低在减少钠重吸收和促进钠排泄中的作用。我们还将利用异种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，我们将测试的假设，丝氨酸/苏氨酸激酶SGK是一个关键的蛋白质控制的通道活性，通过评估缺乏这种激酶的基因的小鼠的通道的激活。我们将测量通道活性以及通道蛋白在动物中，其中激素水平在几个小时内（激素输注），几天内（短期盐限制）或一周或更长时间内（长期盐限制，长期激素输注）发生变化。这些结果将进一步我们了解这些通道是如何在体内调节。 叙事 上皮Na通道的失调是大多数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。本提案中描述的工作将阐明这些控制机制如何工作，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.