Introduction
There are four epithelial sodium channel (ENaC) subunits (α, β, γ, δ) which form heterotrimeric αβγ- or δβγ‑ENaCs that are vital in the salt and water homeostasis in vertebrates. As they mature through the secretory pathway, the ENaC subunits undergo N-linked glycosylation. αβγ-ENaC open probability is tightly coupled to the processing of its α- and γ-ENaC subunits by both intracellular and extracellular proteases. We have previously shown that guinea pig or Xenopus laevis αβγ-ENaC is potently activated by chymotrypsin, whereas δβγ‑ENaC is not (Gettings et al., 2021; Wichmann et al., 2018). Since ENaC activation by chymotrypsin requires cleavage of the γ-ENaC subunit, it is unknown how the presence of the δ-ENaC subunit prevents ENaC activation by the protease. Glycosylation of ENaC subunits has been shown to impact the channel’s activation by proteases (Kashlan et al., 2018). As such, we aimed to investigate whether glycans in the δ-ENaC subunit contribute to the observed differences in the activation of ENaC isoforms by chymotrypsin.
Methods
N-linked glycosylation sites were predicted on guinea pig δ‑ENaC using the NetNGlyc 1.0 server. With site-directed mutagenesis, the glycosylated asparagines (N) in the δ‑ENaC subunit extracellular domain were substituted with alanine (A). We heterologously expressed wild type αβγ-ENaC, wild type δβγ-ENaC or δβγ‑ENaC containing the N/A substituted δ-ENaC subunits in Xenopus laevis oocytes. ENaC activity was measured using the two-electrode voltage-clamp technique at – 60 mV.
Results
Extracellular chymotrypsin (2 µg/ml) significantly stimulated αβγ-ENaC currents by 2.8 ± 0.34-fold (n=11) in comparison with control experiments without chymotrypsin (0.82 ± 0.07-fold, n=12; p<0.0001, unpaired t-test). There was no difference in the fold-activation of δβγ-ENaC currents without (0.97 ± 0.02-fold, n=10) and with chymotrypsin (0.95 ± 0.03-fold, n=13, p=0.6, unpaired t-test). The NetNGlyc 1.0 server predicted three glycosylated asparagines in the extracellular loop of the guinea pig δ‑ENaC subunit: N129, N174 and N344. When these asparagines were substituted one at a time by alanine, none of the channels were activated by chymotrypsin (δN129Aβγ-ENaC: with chymotrypsin 0.92 ± 0.03-fold, n=12, without chymotrypsin 0.84 ± 0.06, n=8; p=0.2080, unpaired t-test; δN174Aβγ-ENaC: with chymotrypsin 0.97 ± 0.05-fold, n=9, without chymotrypsin 0.87 ± 0.04, n=8; p=0.1482, unpaired t-test; and δN344Aβγ-ENaC: with chymotrypsin 0.91 ± 0.04-fold, n=8, without chymotrypsin 0.89 ± 0.05, n=9; p=0.8438, unpaired t-test). When all three asparagines were substituted, extracellular chymotrypsin significantly stimulated the mutant δN129,174,344Aβγ-ENaC by 1.72 ± 0.14-fold (n=15) in comparison with the control experiments without chymotrypsin (0.9 ± 0.03-fold, n=7; p=0.0007, unpaired t-test).
Conclusion
Taken together, these data indicate that removal of glycans in the δ-ENaC subunit renders guinea pig δβγ‑ENaC sensitive to activation by chymotrypsin. N-glycans in the δ-ENaC subunit might alter the accessibility of the γ‑ENaC subunit to proteases.