Sorting nexins associated with recycling complexes regulate epithelial sodium channel trafficking

In the kidney, the epithelial sodium channel (ENaC) facilitates sodium absorption in polarized epithelia and is required to maintain salt and water homeostasis. ENaC's apical membrane population is strictly controlled, with loss of this control leading to hyper- or hypotensive disorders such as Liddle's syndrome or pseudohypoaldosteronism type 1, respectively. Retromer and retriever are conserved endosome-localized protein trafficking complexes that mediate recycling of membrane proteins to the cell surface either directly, via recycling endosomes, or via the trans-Golgi network. Protein cargo for recycling is linked to retromer or retriever by sorting nexin (SNX) proteins. We hypothesized that SNX proteins are required for ENaC recycling. Using two epithelial cell lines [Fischer rat thyroid (FRT) and mouse cortical collecting duct clone 1 (mCCDcl1)] and the human embryonic kidney 293 (HEK293) cell line, with transient transfection of human ENaC-encoding plasmids into HEK293 and FRT epithelia, we found that siRNA knockdown of retromer- and retriever-associated sorting nexin (SNX1, 2, 3, 5, and 17) proteins reduced ENaC amiloride-sensitive short-circuit current and reduced ENaC cell surface population, observed using cell surface biotinylation. Coimmunoprecipitation experiments using transiently transfected human ENaC demonstrated a protein-protein interaction with SNX3 in FRT cells and with SNX17 in both FRT and HEK293 cells, suggesting that SNX3 and SNX17 act as cargo binding proteins between ENaC and the retromer and retriever complexes, respectively. Together, our findings suggest that SNX proteins associated with both the retromer and retriever recycling complexes play a role in maintaining ENaC cell surface populations in polarized epithelia.<b>NEW & NOTEWORTHY</b> Cell surface levels of the epithelial sodium channel, ENaC, are controlled by endocytosis and exocytosis pathways. Sorting nexin (SNX) family members facilitate the binding of protein cargo to recycling complexes for sequestration into recycling compartments. We show that knockdown of several SNX proteins decreases ENaC current and cell surface population, and both SNX3 and SNX17 coimmunoprecipitate with ENaC. Our data provide new insights into the control of ENaC cell surface levels and activity.