Mechanisms and Relevance of ENaC Regulation by AMP-Activated Kinase

AMP 激活激酶调节 ENaC 的机制和相关性

基本信息

批准号：
7333256
负责人：
KENNETH R HALLOWS
金额：
$ 23.77万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-02-01 至 2012-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7333256
关键词：
14-3-3 Proteins 5&apos -AMP-activated protein kinase Affect Affinity Binding Biological Assay Blood Pressure Carrier Proteins Cell membrane Cell physiology Cells Condition Coupling Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Disease Dominant-Negative Mutation Duct (organ) structure Endocytosis Epithelial Functional disorder Half-Life Homeostasis Hypertension Hypoxia In Vitro Injury Ion Transport Ischemia Kidney Link Maintenance Measurement Measures Mediating Membrane Membrane Transport Proteins Metabolic Metabolic stress Metabolism Molecular Mus Mutate Oocytes Pathway interactions Phosphorylation Phosphotransferases Play Process Production Public Health RNA Interference Rate Regulation Role Secondary to Signal Pathway Site Sodium Chloride Surface Testing Tissues Transmembrane Transport Ubiquitin-Protein Ligase Complexes Ubiquitination body volume epithelial Na+ channel inhibitor/antagonist insight mutant prevent response salt balance sensor trafficking ubiquitin ligase ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): The expression and activity of many membrane transport proteins are inhibited under conditions of metabolic stress, thereby limiting the dissipation of ionic gradients and preserving the energy required to maintain them. However, the mechanisms involved in this inhibition are unclear. We have established the AMP-activated kinase (AMPK), a metabolic sensor and key regulator of cellular energy homeostasis, as an important potential link between cellular metabolism and ion transport activity. We recently found that AMPK inhibits the epithelial Na+ channel (ENaC), the rate-limiting pathway for renal salt reabsorption and a major regulator of total body volume status and blood pressure, by decreasing channel expression at the plasma membrane. However, AMPK does not bind or phosphorylate ENaC In vitro, suggesting that AMPK inhibits ENaC indirectly through other signaling pathways. Several lines of evidence suggest that Nedd4-2, a ubiquitin-protein ligase that promotes ENaC internalization and degradation, plays a central role in the regulation of ENaC by AMPK. AMPK-dependent ENaC inhibition is prevented in oocytes expressing an ENaC mutant that does not bind Nedd4-2, or in oocytes co-expressing ENaC and dominant-negative or constitutively active Nedd4-2 mutants. Moreover, AMPK phosphorylates Nedd4-2, suggesting a possible mechanism for AMPK-dependent modulation of Nedd4-2 and thus ENaC activity. Our central hypothesis is that AMPK plays a crucial role in the coupling of epithelial transport to cellular metabolic status through its regulation of important transport proteins such as ENaC. We further propose that ENaC inhibition by AMPK occurs via AMPK phosphorylation-dependent modulation of Nedd4-2 function. The specific aims of this project are to: (1) determine the mechanism of AMPK-dependent inhibition of ENaC expression at the plasma membrane by measuring ENaC half-life, endocytosis and delivery rates at the plasma membrane as a function of AMPK activation; (2) examine the role of AMPK-dependent Nedd4-2 phosphorylation in the regulation of ENaC; and (3) determine the role of AMPK in the inhibition of ENaC in response to chemically induced metabolic stress. Results obtained from these studies should provide specific mechanistic insights into how ENaC is regulated by AMPK and broader insights into the coupling of ion transport to cellular metabolism. They should also promote our understanding of the consequences and pathogenic details of ischemic tissue injury, and the pathophysiology of common diseases, such as hypertension and cystic fibrosis, which are all relevant public health concerns.

描述（由申请人提供）：许多膜转运蛋白的表达和活性在代谢应激条件下受到抑制，从而限制离子梯度的消散并保留维持离子梯度所需的能量。然而，这种抑制作用的机制尚不清楚。我们已经建立了 AMP 激活激酶（AMPK），一种代谢传感器和细胞能量稳态的关键调节器，作为细胞代谢和离子转运活动之间的重要潜在联系。我们最近发现 AMPK 通过降低质膜上的通道表达来抑制上皮 Na+ 通道 (ENaC)，这是肾盐重吸收的限速途径，也是总体容量状态和血压的主要调节因子。然而，AMPK 在体外不会结合或磷酸化 ENaC，这表明 AMPK 通过其他信号通路间接抑制 ENaC。多项证据表明，Nedd4-2（一种促进 ENaC 内化和降解的泛素蛋白连接酶）在 AMPK 对 ENaC 的调节中发挥着核心作用。在表达不结合 Nedd4-2 的 ENaC 突变体的卵母细胞中，或在共表达 ENaC 和显性失活或组成型活性 Nedd4-2 突变体的卵母细胞中，AMPK 依赖性 ENaC 抑制被阻止。此外，AMPK 磷酸化 Nedd4-2，这表明 AMPK 依赖性调节 Nedd4-2 以及 ENaC 活性的可能机制。我们的中心假设是，AMPK 通过调节 ENaC 等重要转运蛋白，在上皮转运与细胞代谢状态的耦合中发挥着至关重要的作用。我们进一步提出 AMPK 对 ENaC 的抑制是通过 AMPK 磷酸化依赖性调节 Nedd4-2 功能而发生的。该项目的具体目标是：（1）通过测量 ENaC 半衰期、内吞作用和质膜递送率与 AMPK 激活的关系，确定 AMPK 依赖性抑制质膜 ENaC 表达的机制； (2)考察AMPK依赖性Nedd4-2磷酸化在ENaC调节中的作用； (3) 确定 AMPK 在抑制 ENaC 响应化学诱导的代谢应激中的作用。从这些研究中获得的结果应该提供有关 ENaC 如何受 AMPK 调节的具体机制见解，以及离子转运与细胞代谢耦合的更广泛见解。它们还应该促进我们对缺血性组织损伤的后果和致病细节的理解，以及高血压和囊性纤维化等常见疾病的病理生理学，这些都是相关的公共卫生问题。