Regulation of ENaC Trafficking and Activity in the Kidney

摘要 肾脏通过调节钠的排泄来控制细胞外液的量和血压，以匹配 膳食钠的摄入量和机体的整体生理需求。醛固酮是一种关键激素，有助于 来调解这一过程。作为对细胞外液容量减少的反应，肾上腺增加分泌。 这种类固醇的作用，进而向部分肾小管(所谓的对醛固酮敏感的远端肾单位)发出信号。 以增加钠的重吸收。这至少部分是通过刺激尿液中钠的吸收而发生的 通过上皮钠通道(ENaC)穿过根尖膜。然而，这是如何发生的，还不完全清楚。 明白了。在这个过程中最流行的模型中，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(SGK1)。然后SGK1将泛素连接酶Nedd4-2磷酸化， 抑制其与ENaC的相互作用，降低通道内化的速率。然后是NA重吸收 由于槽道在根尖表面的停留时间增加而增强。然而，有几行 有证据表明，这并不是荷尔蒙的主要作用机制。首先，它的影响 通过截断ENaC表面的C-末端抑制Ned4-2与ENaC的结合 与那些升高的醛固酮水平(5，10)具有协同作用。如果出现以下情况，则不会出现这种情况 两种操作会影响相同的细胞过程。第二，分析了ENaC在细胞内的分布和 尿外切体中ENaC蛋白的排泄表明，醛固酮的主要作用是增加 前向运输至根尖膜(18)。最后，测定ENaC的泛素化状态 表明当醛固酮刺激转运时，泛素化亚基的数量增加， 而标准模型预测的是下降。在本应用程序中描述的工作中，我们将检查一个 修订后的ENaC贩运模型与这些数据更加一致。在这里，亚单位蛋白表达在 即使在运输需求最小的基本条件下也是过剩的。荷尔蒙刺激的主要事件 是ENaC蛋白从内质网到质膜的运输。到达根尖膜暴露出 尿液蛋白水解酶的通道，它介导了通道的最终蛋白水解性处理和激活。 这增加了亚基对泛素化的敏感性，限制了它们在表面和/或它们的寿命 活动状态。该模型将使用各种实验技术进行测试，包括定量Western 印迹法估计亚单位数，免疫细胞化学，原位生物素化和蛋白质印迹法评估 ENaC亚基的切割状态、用于测量整体通道功能的电生理学和泛素分析 要评估通道的改变是亚细胞亚室。

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.