|dc.description.abstract||The epithelial sodium channel (ENaC) is a heterotrimeric, amiloride-sensitive Na+ channel located at the plasma membrane of various tissues, including kidneys and the vasculature. ENaC abundance and activity at the plasma membrane is pivotal for the maintenance of total body Na+, blood pressure and blood volume and is crucially controlled by the trafficking of ENaC to and from the plasma membrane. Recently, the COMMD (Copper Metabolism Murr1 Domain containing) family have been linked to the regulation of the trafficking (transport to and from the plasma membrane) of multiple membrane proteins, and thus the control of their abundance within the plasma membrane. All COMMD family members, including COMMD10, have been shown to be able to interact with ENaC and a number have also been shown to down-regulate ENaC, likely by increasing ENaC endocytosis from the plasma membrane suggesting a role for COMMD10 in ENaC regulation, however no studies have been performed to investigate COMMD10. Therefore, this study aimed to investigate whether COMMD10 is a novel regulator of protein trafficking and whether, specifically, COMMD10, similar to other COMMD members, is able to regulate ENaC.
Through immunocytochemistry experiments, with various trafficking-associated proteins it was identified that COMMD10 co-localises with Arf1 (a small GTPase localised to the endoplasmic reticulum (ER), Golgi and endosomal compartments), but not with other ER and Golgi associated proteins, suggesting an endosomal role for COMMD10. In support of this COMMD10 co-localised with the endosomal marker transferrin-546 (Tf-546) and, when knocked-down, COMMD10 reduced protein levels of the recycling, and thus endosome, associated proteins COMMD1 and Vps35 suggesting a role for COMMD10 in trafficking between endosomal compartments and the plasma membrane. In U2OS cells with a stable COMMD10 knockdown there was an increase in endocytosis of Tf-546 as well as impaired Tf-546 recycling providing further evidence of a COMMD10 role in trafficking between endosomal compartments and the plasma membrane.
A stable COMMD10 knockdown (C10 KD) of COMMD10 was developed in Fischer rat thyroid (FRT) cells using shRNA. FRT COMMD10 knockdown cells transfected with plasmids encoding αβγENaC exhibited a reduction in amiloride-sensitive short-circuit current (Isc-amil) suggesting a reduction of ENaC at the plasma membrane. Treatment of epithelia with Brefeldin-A highlighted that COMMD10 did not affect ENaC trafficking through the synthetic (i.e. ER to Golgi to plasma membrane) pathway. Results indicated that C10 KD increases the ubiquitination, and thus the endocytosis, of ENaC. In support of this FRT COMMD10 KD cells increased the endocytosis of Tf-546 from the plasma membrane providing further evidence that COMMD10 is a novel regulator of ENaC endocytosis. Preventing endocytosis in C10 KD epithelia, using Nedd4-2 siRNA or a Liddle’s mutation, however, only partially rescued Isc-amil. Studies with transferrin-546 suggested that C10 KD delays recycling of the transferrin receptor to the plasma membrane providing evidence that COMMD10 might also regulate ENaC recycling. Further support for a COMMD10 role in regulating ENaC was provided through studies with aldosterone. Aldosterone is secreted in low sodium conditions to promote sodium reabsorption through ENaC and it was identified that aldosterone regulated COMMD10, but not COMMD1, protein levels in mouse cortical collecting duct cells (M1CCD) suggesting COMMD10 regulates ENaC.
Taken together, results from this study suggest that COMMD10 is a novel regulator of ENaC trafficking by regulation of Nedd4-2 mediated ENaC ubiquitination, and possibly also of ENaC recycling from endosomal compartments to the plasma membrane.||