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

翻译后摘要：上皮Na+通道（ENaCs）发挥了重要作用，在控制细胞外液量，通过促进 Na+吸收穿过转运上皮组织，如肾脏和远端结肠。在远端肾单位， K+分泌也依赖于ENaC诱导的顶端膜去极化。ENaC是异源三聚体 通道由同源的α，β和β亚基组成，每个亚基都受各种转录和后 翻译机制以前的研究使用异源表达系统鉴定了cys-棕榈酰化 ENaC β和β亚基作为通道活性的主要调节剂，以及β-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。CRISPR/Cas9用于产生 缺乏这些棕榈酰化位点的小鼠（C33 A，C41 A，“No-P”小鼠;无棕榈酰化位点）。我们发现ENaC 与同窝仔相比，醛固酮敏感性远端肾单位（ASDN）的活性显著降低 对照目的1中提出的研究将评估ENaC依赖性肾脏Na+和K+转运在ASDN中的作用， 对照组与No-P小鼠在基线时以及对饮食盐挑战的响应。ENaC介导的Na+吸收 将通过短路电流（ISC）评估远端结肠中的电流。血液和尿液分析将提供新的 关于ENaC棕榈酰化对电解质稳态的影响的信息。磷脂酰肌醇4，5 二磷酸（PIP 2）是一种众所周知的ENaC激活剂，关键的β-亚基棕榈酰化位点位于鉴定的 （PIP 2）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PIP 2结合： 促进N-末端和质膜之间的相互作用。这些拟议的研究将 为ENaC棕榈酰化的功能重要性提供了新的体内见解。完成本项目 将使申请人具备必要的知识和技能，以调查上皮运输机制 以及肾脏生理学的研究

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.