N-linked glycans are required on epithelial Na⁺channel subunits for maturation and surface expression

Abstract

Epithelial Na channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 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 Fischer rat thyroid cells with αβγ-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 β-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 β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-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.

Urokinase‐type plasminogen activator (uPA) is not essential for epithelial sodium channel (ENaC)‐mediated sodium retention in experimental nephrotic syndrome

“…The band at 45 kDa represents a cleavage product of γ‐ENaC of unknown significance. To analyse the influence of N‐glycosylation of γ‐ENaC (at up to 5 sites) on the migration pattern, we treated the samples with the deglycosylating enzyme N‐glycosidase F. As shown in Figure the migration pattern changed leaving only 2 bands at 71 and 50 kDa in both healthy and nephrotic wild‐type mice that were blocked by the immunogenic peptide. Again, we could not discriminate a specific band that could correspond to fully cleaved γ‐ENaC in mouse tissue.…”

Shear force sensing of epithelial Na + channel (ENaC) relies on N -glycosylated asparagines in the palm and knuckle domains of αENaC

“…Evidence suggests that replacement of multiple glycosylated asparagines influence ENaC function or trafficking (51). To account for this possibility, amiloride-sensitive whole-cell currents as a measure of functional channels on the membrane, singlechannel open probability, and conductance of channels containing αΔN P+K were analyzed and compared to α Wt -, β-, γENaC.…”

Analyses of epithelial Na+ channel variants reveal that an extracellular β-ball domain critically regulates ENaC gating

“…Surface biotinylation was performed 24 h following transfection, as described previously (25,59). Confluent FRT cells were washed four times with cold Dulbecco's PBS with 1.0 mM CaCl 2 and 0.5 mM MgCl 2 (PBS, Corning Life Sciences).…”