Endosomal SNX Proteins are Required for Recycling of the Epithelial Sodium Channel.

Abstract

The epithelial sodium channel (ENaC) facilitates Na+ absorption of polarised epithelia and is required for regulation of salt and water homeostasis. ENaC’s apical membrane population is strictly controlled, and loss of this control can lead to Liddle’s Syndrome, a form of hypertension. The retromer and retriever complexes are conserved endosome-localised protein complexes that mediate recycling of membrane proteins back to the cell surface. SNX3 and SNX17, sorting nexin proteins, are cargo-binding proteins that interact with retromer and retriever respectively, to facilitate cargo recycling. The aim of this study was to investigate whether the retromer/retriever associated SNX3 or SNX17 proteins are required for the recycling of ENaC.

Fischer rat thyroid (FRT) and mouse cortical collecting duct (mCCD) cells were transiently transfected with control, SNX3 or SNX17 siRNA. Subsequently, Western blot analysis was carried out to confirm knockdown; ‘Ussing’ chamber experiments were conducted to measure changes in ENaC’s amiloride-sensitive short circuit current (Isc-Amil), and cell surface biotinylation assays were used to measure ENaC cell surface population. Finally, co-immunoprecipitation (Co-IP) experiments were performed to detect if protein-protein interactions exist between ENaC and SNX3 or SNX17. Results were analysed using a One-sample T-test.

Significant protein knockdown was observed for both SNX proteins in the two cell lines (n = 3, P < 0.05). A significant reduction in Isc-Amil of ENaC was observed in both cell lines with a SNX3 or SNX17 siRNA knockdown (n = 9, P < 0.001). Furthermore, a significant reduction in ENaC cell surface population with either a SNX3 or SNX17 siRNA knockdown was observed (n = 3, P < 0.05), demonstrating the reduction in Isc-Amil was due to reduced ENaC cell surface population. Finally, Co-IP experiments demonstrated a protein-protein interaction between SNX17 and ENaC, however, not SNX3 and ENaC.

These results suggest that knockdown of either SNX3 and SNX17 can facilitate ENaC recycling and this may occur through different pathways. Additionally, SNX17 may specifically interact with ENaC to localise ENaC in recycling structures in the endosome. This investigation adds to our understanding of the mechanism involved in ENaC recycling, contributing to the understanding of and prevention of hypertension.