Sorting Nexins 1, 2, 3 and 17 Regulate Epithelial Sodium Channel Trafficking

Abstract

The epithelial sodium channel (ENaC) facilitates sodium absorption of polarized epithelia and therefore, is required for regulation of 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 back to the cell surface either directly, via recycling endosomes or via the trans‐Golgi network. Both retromer and retriever associate with accessory proteins including sorting nexins (SNX) proteins which play a role in the establishment of a tubulation complex leading to the formation of endosomal tubule‐shaped extensions which contain the cargo selected for recycling. We hypothesized that retromer and/or retriever are required for ENaC recycling. Using three epithelial cell lines (Fischer rat thyroid (FRT), mouse cortical collecting duct (mCCD) or human embryonic kidney cells (HEK293), we found that siRNA knockdown of retromer and retriever associated SNX proteins reduced ENaC amiloride‐sensitive short circuit current (Isc‐Amil). SNX1 and SNX2 are reported to be essential for cargo recycling through retromer. Individually, knockdown of SNX1 (n=6) or SNX2 (n=8) reduced Isc‐Amil by 38±9% and 39±8% compared with control FRT epithelia, respectively. Double‐knockdown of those SNXs reduced Isc‐Amil by 47±1% (n=6) compared with control FRT epithelia. SNX3 is known to bind directly with the retromer complex. Knockdown of SNX3 decreased Isc‐Amil by 47±5% FRT (n=9) and 35±4% in mCCD (n=9) epithelia compared with control epithelia. Additionally, it is known that SNX17 mediates cargo retrieval via the retriever complex. Knockdown of SNX17 lowered Isc‐Amil by 56±1% (n=9) in FRT and 35±6% (n=9) in mCCD epithelia compared with control epithelia. With cell surface biotinylation experiments and using FRT cells, knockdown of SNX3 and SNX17 reduced the ENaC cell surface population by 68±25% (n=3) and 77±17% (n=3), respectively, thus, attributing to the reduced Isc‐Amil. Furthermore, co‐immunoprecipitation experiments of αβHAγENaC overexpressed in HEK293 cells, demonstrated a protein‐protein interaction between retriever associated SNX17 and ENaC (n=3) suggesting SNX17 may act as a cargo binding protein between ENaC and the retriever complex. However, there was no evidence for an interaction between SNX3 and ENaC (n=3). Together, our findings suggest that SNX protein complexes play a role in ENaC recycling to the plasma membrane in polarized epithelia. Additionally, these data suggest that both the retromer and retriever recycling complexes may aid in the trafficking of ENaC to the plasma membrane.Support or Funding InformationThis work was supported by a grant from the New Zealand Lottery Health Board. Further support was provided by the School of Biomedical Sciences and the Department of Physiology of the University of Otago.