Trafficking and cell surface stability of the epithelial sodium ion channel expressed in epithelial MDCK cells

The apically located epithelial Na+ channel (αβγENaC) plays a key role in the regulation of salt and fluid transport in the kidney and other epithelia. We generated epithelial Madin-Darby canine kidney (MDCK) cells stably expressing αβγENaC, where each subunit is differentially epitope tagged. ENaC expression was verified by immunoblotting and patch clamp analysis showed the tagged channel is functional. Moreover, we demonstrated apical membrane localization of ENaC in these cells. The glycosylation pattern of the intracellular pool of ENaC revealed sensitivity to endoglycosidase H. The cell surface pool of ENaC was also core glycosylated and lacked detectable endoglycosidase H-resistant channels. Extraction in Triton X-100 demonstrated that both intracellular and cell surface pools of ENaC are largely detergent-soluble. Moreover, flotation assays showed that both intracellular and cell surface pools of this channel are not associated with rafts. The surface pool of ENaC has a short half-life (t_1/2 ∼ 1hr) in these cells.