Role of palmitoylation in regulating the epithelial sodium channel (ENAC)

棕榈酰化在调节上皮钠通道 (ENAC) 中的作用

基本信息

批准号：
10688447
负责人：
Andrew J Nickerson
金额：
$ 6.91万
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：
2023
资助国家：
美国
起止时间：
2023-09-03 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10688447
关键词：
Affect Aldosterone Amiloride Binding Binding Sites Biological Blood Blood Pressure CRISPR/Cas technology Cell membrane Cells Clinical Colon Complex Cysteine Cytoplasm Distal Duct (organ) structure Electrolytes Epithelium Exhibits Extracellular Domain Extracellular Fluid Failure Future Genetic Diseases Genetic Transcription Homeostasis Hypoaldosteronism Impairment In Vitro Ion Channel KAI1 gene Kidney Knowledge Liddle syndrome Maintenance Measures Mediating Membrane Metabolic Modeling Molecular Mucous Membrane Mus Mutation N-terminal Natriuresis Nephrons Organ Palmitic Acylation Site Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phospholipids Physiological Physiology Plasma Play Probability Process Regulation Role Site Sodium Chloride Surface System Testing Tissues Urine Variant Whole Organism Xenopus oocyte absorption analog apical membrane deprivation dietary dietary salt disease phenotype epithelial Na+ channel experimental study extracellular gain of function in vivo insight male novel palmitoylation patch clamp pharmacologic receptor response salt sensitive hypertension sex skills

项目摘要

Abstract: Epithelial Na+ channels (ENaCs) play a major role in controlling extracellular fluid volume by facilitating Na+ absorption across transporting epithelial tissues, such as the kidney and distal colon. In the distal nephron, K+ secretion also depends on ENaC-induced apical membrane depolarization. ENaCs are heterotrimeric channels composed of homologous ,  and  subunits, each regulated by various transcriptional and post- translational mechanisms. Previous studies using heterologous expression systems identified cys-palmitoylation of ENaC  and  subunits as a major regulator of channel activity, and two key sites of palmitoylation in -ENaC’s N-terminus (C33,C41) are required for normal channel function in vivo. CRISPR/Cas9 was used to generate mice lacking these palmitoylation sites (C33A,C41A, “No-P” mice; No Palmitoylation site). We found that ENaC activity in the aldosterone-sensitive distal nephron (ASDN) is significantly reduced, compared to littermate controls. Studies proposed in Aim 1 will assess ENaC-dependent renal Na+ and K+ transport in the ASDN of control versus No-P mice at baseline, and in response to dietary salt challenges. ENaC-mediated Na+ absorption in the distal colon will be assessed by short-circuit current (ISC). Blood and urine analyses will provide novel information regarding the effects of ENaC palmitoylation on electrolyte homeostasis. Phosphatidylinositol 4,5 bis-phosphate (PIP2) is a well-known ENaC activator, and key -subunit palmitoylation sites reside near identified (PIP2) binding sites. Studies proposed in Aim 2 will examine whether palmitoylation facilitates PIP2 binding by promoting an interaction between the N-terminus and the plasma membrane. These proposed studies will provide novel in vivo insights into the functional importance of ENaC palmitoylation. Completion of this project will equip the applicant with the necessary knowledge and skills to investigate mechanisms of epithelial transport and renal physiology in future endeavors.

摘要：上皮 Na+ 通道（ENaCs）通过促进细胞外液体积在控制细胞外液体积方面发挥着重要作用。 Na+ 穿过运输上皮组织（例如肾脏和远端结肠）的吸收。 K+ 分泌还取决于 ENaC 诱导的顶膜去极化 ENaC 是异三聚体。由同源的 、 和  亚基组成的通道，每个亚基均受各种转录和后转录调节先前使用异源表达系统的研究鉴定了半胱氨酸棕榈酰化。 ENaC  和  亚基作为通道活动的主要调节因子，以及 -ENaC 中棕榈酰化的两个关键位点 N 末端（C33、C41）是体内正常通道功能所必需的，使用 CRISPR/Cas9 来生成。缺乏这些棕榈酰化位点的小鼠（C33A，C41A，“No-P”小鼠；没有棕榈酰化位点）。与同窝动物相比，醛固酮敏感的远端肾单位 (ASDN) 的活性显着降低目标 1 中提出的研究将评估 ASDN 中 ENaC 依赖性肾 Na+ 和 K+ 转运。对照组与基线时的 No-P 小鼠相比，以及对 ENaC 介导的 Na+ 吸收的反应。通过短路电流（ISC）对远端结肠进行评估，血液和尿液分析将提供新颖的结果。有关 ENaC 棕榈酰化对电解质稳态影响的信息 4,5。双磷酸 (PIP2) 是一种众所周知的 ENaC 激活剂，关键的  亚基棕榈酰化位点位于已识别的附近 (PIP2) 结合位点。目标 2 中提出的研究将检查棕榈酰化是否通过以下方式促进 PIP2 结合。这些拟议的研究将促进 N 末端和质膜之间的相互作用。为 ENaC 棕榈酰化的功能重要性提供新颖的体内见解完成该项目。将为申请人提供必要的知识和技能来研究上皮运输机制肾脏生理学在未来的努力。