N-linked Glycans are Required on Epithelial Na+ Channel Subunits for Maturation and Surface Expression.

Epithelial Na+ channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an alpha beta gamma complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse alpha-, beta-, and gamma-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with alpha beta gamma-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na+. The lack of N-linked glycans on the beta-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the beta-subunit had the largest effect on total levels, with the lack of N-linked glycans on the gamma- and alpha-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type beta-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the beta-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.