Regulation of ENaC Trafficking and Activity in the Kidney

总结 肾脏通过调节Na的排泄来控制细胞外液量和血压，以与细胞外液的浓度相匹配。 膳食钠摄入量和机体的总体生理需求。醛固酮是一种关键的激素， 来调节这个过程。作为对细胞外液量减少的反应，肾上腺增加分泌 这种类固醇，这反过来又信号肾小管（所谓的醛固酮敏感性远端肾单位）的部分 以增加钠重吸收。这至少部分是通过刺激从尿中摄取Na而发生的 通过上皮Na通道（ENaC）穿过顶膜。然而，这是如何发生的，是不完全的。 明白在这一过程的最普遍模型中，醛固酮刺激一种关键酶的合成， 血清和糖皮质激素诱导的激酶（SGK 1）。然后SGK 1磷酸化泛素连接酶Nedd 4 -2， 抑制其与ENaC的相互作用并降低通道内化的速率。钠重吸收 由于通道在顶端表面的停留时间增加而增强。然而，几行 有证据表明，这不是激素发挥作用的主要机制。第一，影响 通过截短ENaC表面的C-末端（模仿Liddle’s 人类综合征）与醛固酮水平升高具有协同作用（5，10）。这是不期望的，如果 两种操作影响相同的细胞过程。第二，分析ENaC在细胞中的分布和 尿外泌体中ENaC蛋白的排泄表明，醛固酮的主要作用是增加 向前运输至顶膜（18）。最后，测量ENaC的泛素化状态， 表明当运输被醛固酮刺激时，泛素化亚基的数量增加， 而标准模型预测下降。在本申请中描述的工作中，我们将检查 经修订的ENaC贩运模式与这些数据更加一致。这里亚基蛋白表达在 即使在基础条件下，当运输的需要是最小的。主要的地震刺激事件 是ENaC蛋白从内质网到质膜的转运。到达顶膜时， 尿蛋白酶介导最终的蛋白水解加工和通道的激活。 这增加了亚基对泛素化的敏感性，限制了它们在表面的寿命和/或它们的蛋白质。 活跃的国家。该模型将使用各种实验技术进行测试，包括定量Western blot。 印迹法估计亚基数量，免疫细胞化学，原位生物素化和Western印迹法评估 ENaC亚基的裂解状态、测量总体通道功能的电生理学和泛素分析 评估通道的修饰是亚细胞区室。

Summary The kidneys control extracellular fluid volume and blood pressure by adjusting the excretion of Na to match the dietary Na intake and the overall physiological needs of the organism. Aldosterone is a key hormone that helps to mediate this process. In response to a reduction in extracellular fluid volume the adrenals increase secretion of this steroid, which in turn signals parts of the renal tubule (the so-called aldosterone-sensitive distal nephron) to increase Na reabsorption. This occurs at least in part through stimulation of the uptake of Na from the urine across the apical membrane through epithelial Na channels (ENaC). How this occurs, however, is incompletely understood. In the most prevalent model of this process, aldosterone stimulates the synthesis of a key enzyme, the serum and glucocorticoid induced kinase (SGK1). SGK1 then phosphorylates the ubiquitin ligase Nedd4-2, inhibiting its interaction with ENaC and diminishing the rate of channel internalization. Na reabsorption is then enhanced due to increased residence times of the channels at the apical surface. However, several lines of evidence suggest that this is not the main mechanism through which the hormone operates. First, the effects of inhibiting the binding of Nedd4-2 to ENaC by truncating the C-terminal of the ENaC surface (mimicking Liddle’s syndrome in humans) are synergistic with those of elevated aldosterone levels (5,10). This is not expected if the two manipulations affect the same cellular processes. Second, analysis of ENaC distribution in the cell and the excretion of ENaC protein in urinary exosomes suggests that the major effect of aldosterone is to increase forward trafficking to the apical membrane (18). Finally, measurement of the ubiquitination state of ENaC indicates that when transport is stimulated by aldosterone the number of ubiquitinated subunits increases, whereas the standard model predicts a decrease. In the work described in this application, we will examine a revised model of ENaC trafficking that is more consistent with these data. Here subunit protein is expressed in excess even under basal conditions when the need for transport is minimal. The main hormone-stimulated event is transport of ENaC protein from the ER to the plasma membrane. Arrival at the apical membrane exposes the channels to urinary proteases, which mediate the final proteolytic processing and activation of the channels. This increases the susceptibility of the subunits to ubiquitination, limiting their lifetime at the surface and/or their active states. This model will be tested using a variety of experimental techniques including quantitative Western blots to estimate subunit numbers, immunocytochemistry, in situ biotinylation and Western blotting to assess the cleavage states of ENaC subunits, electrophysiology to measure overall channel function, and ubiquitin assays to assess the modification of channels is subcellular compartments.