Epithelial Sodium Channel (ENaC) Trafficking and COMMD Proteins
Authors: Rasulov Sahib , Ware Adam William , McDonald Fiona J 
Published: June 2021

The epithelial sodium channel (ENaC), is assembled as a heterotrimer composed of three homologous subunits α or δ, β, and γ and is selectively permeable to Na+ ions and plays an essential role in the regulation of sodium transport in the renal connecting tubule and collecting duct. Mutations in ENaC subunits cause various disorders including the low and high blood pressure conditions of pseudohypoaldosteronism‐I and Liddle syndrome respectively. In renal principal cells, ENaC cell surface population is tightly regulated by many trafficking pathways and aldosterone. In this project, we study the role of retromer in mediating trafficking of ENaC. Retromer is formed by a number of sub‐complexes including the CCC protein complex (COMMD1‐10/CCDC22/CCDC93) where the COMMD (COMM (Copper Metabolism gene MURR1) domain containing) family proteins interact with ENaC. The aim of this study was to investigate the role of the COMMD10 protein in ENaC trafficking and cell surface population and the effect of aldosterone on COMMD mRNA and protein expression.

Fischer rat thyroid (FRT) cells with stable knockdown (KD) of COMMD10, or a control line, were transiently transfected with αβγ‐ENaC for this study. ENaC cell surface activity was studied by Ussing assay measuring ENaC amiloride‐sensitive current (Isc‐Amil). Then ENaC cell surface population was determined by cell surface biotinylation assay. Endogenous ENaC cell surface activity in a mouse cortical collecting duct (mCCDcl1) cell line was also measured by Ussing assay. For aldosterone regulation of COMMDs, mCCDcl1 cells were treated with aldosterone (300nM) for 0.5, 3 and 24 hours which increased endogenous ENaC expression. mRNA and protein levels of COMMDs and SGK1 (as a positive control for aldosterone stimulation) were determined by RT‐qPCR and Western blotting assays.

ENaC Isc‐Amil was decreased by 55% (P=0.0004, one‐sample t‐test) in FRT COMMD10 KD epithelia compared to control KD epithelia suggesting that the ENaC cell surface population was decreased in COMMD10 KD epithelia. Cell surface ENaC quantification confirmed COMMD10 KD decreased the ENaC cell surface population by 32% (P=0.02, onesample t‐test). Aldosterone increased mRNA level of SGK1 by 3.8, 33.5 and 48.1 fold (P=0.02, P=0.007, P=0.003 respectively; one‐sample t‐test) with 0.5, 3 and 24 hours of treatment, respectively whilst having no effect on COMMD1‐10 mRNA levels. Aldosterone increased the protein level of SGK1 ~ 1.5 fold with 30 min aldosterone treatment. Interestingly, aldosterone decreased COMMD10 protein level (0.71 ± 0.07, P=0.03, one‐sample t‐test) with 30‐minute treatment significantly but not with 3 and 24‐hour treatment.

Therefore, COMMD10 regulates the cell surface population of ENaC, probably through altering ENaC trafficking, however short or longterm aldosterone treatment does not alter the mRNA level of any COMMD family member. Understanding this pathway could lead to advancing knowledge in diseases like hypertension.

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This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.