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

• 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
• 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' -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

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）：在代谢应激条件下，许多膜转运蛋白的表达和活性受到抑制，从而限制了离子梯度的消散，并保留了维持离子梯度所需的能量。然而，这种抑制的机制尚不清楚。我们已经建立了AMP激活的激酶（AMPK），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性之间的重要潜在联系。我们最近发现，AMPK抑制上皮Na+通道（ENaC），肾盐重吸收的限速途径和总身体容量状态和血压的主要调节器，通过减少在质膜上的通道表达。然而，AMPK在体外不结合或磷酸化ENaC，表明AMPK通过其他信号通路间接抑制ENaC。一些证据表明，Nedd 4 -2，一种促进ENaC内化和降解的泛素-蛋白连接酶，在AMPK调节ENaC中起核心作用。AMPK依赖性ENaC抑制在表达不结合Nedd 4 -2的ENaC突变体的卵母细胞中或在共表达ENaC和显性阴性或组成型活性Nedd 4 -2突变体的卵母细胞中被阻止。此外，AMPK使Nedd 4 -2磷酸化，这表明AMPK依赖性调节Nedd 4 -2并因此调节ENaC活性的可能机制。我们的中心假设是AMPK通过调节重要的转运蛋白如ENaC，在上皮转运与细胞代谢状态的偶联中起着至关重要的作用。我们进一步提出AMPK通过Nedd 4 -2功能的AMPK磷酸化依赖性调节来抑制ENaC。本项目的具体目标是：（1）通过测量ENaC在质膜上的半衰期、内吞作用和递送速率（作为AMPK活化的函数）来确定AMPK依赖性抑制ENaC在质膜上表达的机制;（2）检查AMPK依赖性Nedd 4 -2磷酸化在ENaC调节中的作用;和（3）确定AMPK在响应化学诱导的代谢应激的ENaC抑制中的作用。从这些研究中获得的结果应该提供具体的机制的见解ENaC是如何调节AMPK和更广泛的见解耦合离子转运细胞代谢。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，以及常见疾病的病理生理学，如高血压和囊性纤维化，这些都是相关的公共卫生问题。