Regulation of ENaC Trafficking and Activity in the Kidney

ENaC 贩运和肾脏活动的监管

• 批准号: 10363434
• 负责人: LAWRENCE G PALMER
• 金额: $ 42.96万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10363434
• 关键词: Adrenal Glands; Affect; Agonist; Aldosterone; Antibodies; Apical; Binding; Biological Assay; Biotinylation; Blood Pressure; C-terminal; Cell membrane; Cell physiology; Cells; Cytoplasm; Data; Disease model; Distal; Early Endosome; Electrophysiology (science); Endoplasmic Reticulum; Enzymes; Epithelial Cells; Event; Excretory function; Extracellular Fluid; Glucocorticoids; Hormones; Human; Hypertension; In Situ; Intake; Kidney; Kidney Diseases; Liddle syndrome; Liver diseases; Location; Measurement; Measures; Mediating; Modeling; Modification; Mouse Strains; Mus; Mutation; Nephrons; Organism; Patients; Peptide Hydrolases; Phosphotransferases; Physiological; Predisposition; Process; Protein Subunits; Proteins; Proteolytic Processing; Purinoceptor; Rattus; Regulation; Renal tubule structure; Serum; Signal Transduction; Sodium Chloride; Standard Model; Steroids; Surface; Techniques; Testing; Time; Ubiquitin; Ubiquitination; Urine; Water; Western Blotting; Work; apical membrane; body volume; dietary; epithelial Na+ channel; exosome; immunocytochemistry; predictive modeling; renal epithelium; residence; response; steroid hormone; trafficking; ubiquitin ligase; uptake; urinary

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.

概括 肾脏通过调节钠的排泄来控制细胞外液量和血压。 膳食钠的摄入量和机体的整体生理需求是醛固酮的一种关键激素。 为了调节这一过程，肾上腺会增加分泌以应对细胞外液量的减少。 这种类固醇的成分，进而向部分肾小管（所谓的醛固酮敏感的远端肾单位）发出信号 增加钠的重吸收，这至少部分是通过刺激从尿液中吸收钠来实现的。 然而，这种现象如何通过上皮钠通道（ENaC）穿过顶膜尚不完全清楚。 据了解，在该过程最普遍的模型中，醛固酮会刺激关键酶的合成， 血清和糖皮质激素诱导激酶 (SGK1) 然后磷酸化泛素连接酶 Nedd4-2， 然后抑制其与 ENaC 的相互作用并降低通道内化的速率。 由于顶端表面的通道停留时间增加而增强。 有证据表明，这不是激素发挥作用的主要机制。 通过截短 ENaC 表面的 C 端来抑制 Nedd4-2 与 ENaC 的结合（模仿 Liddle 的 人类综合症）与醛固酮水平升高的综合症具有协同作用（5,10）。 两种操作影响相同的细胞过程。其次，分析 ENaC 在细胞和细胞中的分布。 尿外泌体中 ENaC 蛋白的排泄表明醛固酮的主要作用是增加 最后，测量 ENaC 的泛素化状态。 表明当醛固酮刺激运输时，泛素化亚基的数量增加， 而标准模型预测会减少。在本申请描述的工作中，我们将检查一个。 修订后的 ENaC 运输模型与这些数据更加一致，此处表达的亚基蛋白为 即使在运输需求极小时的基础条件下也会过量 这是主要的激素刺激事件。 ENaC 蛋白从 ER 转运至质膜，到达顶膜后暴露。 尿蛋白酶的通道，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/或它们的寿命。 该模型将使用各种实验技术进行测试，包括定量西方技术。 印迹来估计亚基数量，免疫细胞化学，原位生物素化和蛋白质印迹来评估 ENaC 亚基的裂解状态、测量整体通道功能的电生理学以及泛素检测 评估通道的修饰是亚细胞区室。