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

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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.