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 Kidneys通过调整NA的极端以匹配细胞外液体体积和血压 饮食中的摄入量和生物体的整体物理需求。醛固酮是一种钥匙马，有助于 调解此过程。响应减少细胞外液体积，肾上腺增加了分泌 这种类固醇又向肾管的部分（所谓的醛固酮敏感的肾小管）发出信号 增加NA的重吸收。至少部分是通过刺激尿液的摄取而发生的 穿过上皮NA通道（ENAC）的顶端膜。但是，这种情况的发生方式不完全 理解齿。在此过程的最普遍模型中，醛固酮刺激了关键酶的合成， 血清和糖皮质激素诱导的激酶（SGK1）。然后SGK1磷酸化泛素连接酶Nedd4-2， 抑制其与ENAC的相互作用，并降低通道内在化速率。那时是Na的重吸收 由于顶部表面的通道的停留时间增加而增加。但是，几行 有证据表明，这不是霍森运作的主要机制。首先，效果 通过截断ENAC表面的C末端（模仿Liddle的），抑制NEDD4-2与ENAC的结合 人类综合征）与醛固酮水平升高的综合症（5,10）是协同的。如果这是不期望的 两个操纵影响相同的细胞过程。其次，分析细胞中的ENAC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 向前贩运顶膜（18）。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， 而标准模型预测的减少。在本应用程序中描述的工作中，我们将检查 经过修订的ENAC运输模型与这些数据更一致。在这里，亚基蛋白在 即使在基本条件下，运输需求最少也是多余的。主要激素刺激的事件 是ENAC蛋白从ER到质膜的转运。到达顶部膜，暴露了 尿蛋白酶的通道，介导通道的最终蛋白水解处理和激活。 这增加了亚基对泛素化的敏感性，限制了它们在表面和/或它们的一生中的敏感性 活跃状态。该模型将使用包括定量西部的各种实验技术进行测试 印迹以估计亚基数，免疫细胞化学，原位生物素化和蛋白质印迹以评估 ENAC亚基的切割状态，测量整体通道功能的电生理学和泛素测定法 评估通道的修饰是亚细胞室。