Enhanced Shear Force Responsiveness of Epithelial Na+ Channel’s (ENaC) δ Subunit Following the Insertion of N-Glycosylation Motifs Relies on the Extracellular Matrix

Abstract

Members of the Degenerin/epithelial Na+ channel (ENaC) protein family and the extracellular cell matrix (ECM) form a mechanosensitive complex. A core feature of this complex are tethers, which connect the channel with the ECM, however, knowledge about the nature of these tethers is scarce. N-glycans of α ENaC were recently identified as potential tethers but whether N-glycans serve as a ubiquitous feature for mechanosensation processes remains unresolved. The purpose of this study was to reveal whether the addition of N-glycans to δ ENaC—which is less responsive to shear force (SF)—increases its SF-responsiveness and whether this relies on a linkage to the ECM. Therefore, N-glycosylation motifs were introduced via site-directed mutagenesis, the resulting proteins expressed with β and γ ENaC in Xenopus oocytes, and SF-activated currents measured by two-electrode voltage-clamp. The insertion of N-glycosylation motifs increases δ ENaC’s SF responsiveness. The inclusion of a glycosylated asparagine (N) at position 487 did increase the molecular mass and provided a channel whose SF response was abolished following ECM degradation via hyaluronidase. This indicates that the addition of N-glycans improves SF-responsiveness and that this effect relies on an intact ECM. These findings further support the role of N-glycans as tethers for mechanotransduction.

Detrimental or beneficial: Role of endothelial ENaC in vascular function

“…However, no additional effect of amiloride on the SF‐induced response was observed when combined with hyaluronidase (Knoepp et al, 2020). In another set of experiments, where they replaced the α subunit with the less SF‐sensitive δ subunit, they could also detect an enhanced amiloride‐sensitive response upon SF challenge when N‐glycosylation motifs were introduced into the δ subunit (Barth et al, 2021). This response was also largely diminished when cells were treated with hyaluronidase.…”

Epithelial Sodium Channel δ Subunit Is Expressed in Human Arteries and Has Potential Association With Hypertension

“…For example, the δ-ENaC-containing channels have high open probability and large conductance but have reduced shear force sensitivity compared with the α subunit-containing channels. 15,17,18 However, due to the apparent absence of the δ subunit in rats and mice and the absence of reliable δ subunit-specific blockers, the function of the δ/δβγ-ENaC channel is still not well understood in native tissue. 17,19 Furthermore, the expression of δ-ENaC mRNA has been detected in human umbilical vein endothelial cells, 20 suggesting their potential function in human arteries.…”

Epithelial Na+ channel and the glycocalyx: a sweet and salty relationship for arterial shear stress sensing

“…The interdependence of this effect was demonstrated in a subsequent study investigating the δ-ENaC subunit. Channels constituting δ-ENaC (instead of α) are less responsive to shear stress and this coincides with the lack of glycosylated asparagines in comparison to α-ENaC [88 ▪ ]. The introduction of glycosylation motifs into δ-ENaC did increase the shear stress response of the resulting channels.…”

“…The introduction of glycosylation motifs into δ-ENaC did increase the shear stress response of the resulting channels. Importantly, the elevated shear stress response of this modified channel was dependent on the intact glycocalyx as treatment with hyaluronidase reversed the increased shear stress response [88 ▪ ]. Last but not least, the role of the N-glycans for arterial shear stress responsiveness was also observed in mice transduced with viral vectors.…”