Renal Na+ reabsorption, facilitated by the epithelial Na+ channel (ENaC), is subject to multiple forms of control to ensure optimal body blood volume and pressure through altering both the ENaC population and activity at the cell surface. Here, the focus is on regulating the number of ENaCs present in the apical membrane domain through pathways of ENaC synthesis and targeting to the apical membrane as well as ENaC removal, recycling, and degradation. Finally, the mechanisms by which ENaC trafficking pathways are regulated are summarized.

Optimal function of the epithelial sodium channel (ENaC) in the distal nephron is key to the kidney’s long-term control of salt homeostasis and blood pressure. Multiple pathways alter ENaC cell surface populations, including correct processing and trafficking in the secretory pathway to the cell surface, and retrieval from the cell surface through ubiquitination by the ubiquitin ligase Nedd4-2, clathrin-mediated endocytosis, and sorting in the endosomal system. Members of the Copper Metabolism Murr1 Domain containing (COMMD) family of 10 proteins are known to interact with ENaC. COMMD1, 3 and 9 have been shown to down-regulate ENaC, most likely through Nedd4-2, however, the other COMMD family members remain uncharacterized. To investigate the effects of the COMMD10 protein on ENaC trafficking and function, the interaction of ENaC and COMMD10 was confirmed. Stable COMMD10 knockdown in Fischer rat thyroid epithelia decreased ENaC current and this decreased current was associated with increased Nedd4-2 protein, a known negative regulator of ENaC. However, inhibition of Nedd4-2’s ubiquitination of ENaC was only able to partially rescue the observed reduction in current. Stable COMMD10 knockdown results in defects both in endocytosis and recycling of transferrin suggesting COMMD10 likely interacts with multiple pathways to regulate ENaC and therefore could be involved in the long-term control of blood pressure.

The epithelial sodium channel, ENaC, located at the apical membrane in many epithelia, is the rate-limiting step for sodium reabsorption. Tight regulation of the plasma membrane population of ENaC is required as hyper- or hypotension may result if too many or too few ENaCs are present. Endocytosed ENaC travels to the early endosome and is then either trafficked to the lysosome for degradation or recycled back to the plasma membrane. Recently, the retromer recycling complex, located at the early endosome, has been implicated in plasma membrane protein recycling pathways. We hypothesized that retromer is required for recycling of ENaC. Stabilization of retromer function with the retromer stabilizing chaperone R55 increased ENaC current, while knockdown or overexpression of individual retromer and associated proteins altered ENaC current and cell surface population of ENaC. KIBRA was identified as an ENaC binding protein allowing ENaC to link to Snx4 to alter ENaC trafficking. Knockdown of the retromer-associated cargo-binding Snx27 protein did not alter ENaC current, whereas CCDC22, a CCC-complex protein, coimmunoprecipitated with ENaC and CCDC22 knockdown decreased ENaC current and population at the cell surface. Together our results confirm that retromer and the CCC complex play a role in recycling of ENaC to the plasma membrane.

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.Support or Funding InformationDepartment of Physiology Funding for Research Conferences, Workshops or Scientific VisitsDivision of Health Sciences Funding for Conference TravelThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.