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 αβγ complex. Six, thirteen and five consensus sites (Asn-x-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α, β and γ 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 Fisher rat thyroid cells with αβγENaC 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 β subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-link 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 β subunit had the largest effect on total levels, with the lack of N-linked glycans on the α and γ subunits having intermediate and modest effects, respectively. Finally, channels with wild type β subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression and function of the channel.