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Int J Mol Sci
2021 Mar 02;225:. doi: 10.3390/ijms22052500.
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Enhanced Shear Force Responsiveness of Epithelial Na+ Channel's (ENaC) δ Subunit Following the Insertion of N-Glycosylation Motifs Relies on the Extracellular Matrix.
Barth D
,
Knoepp F
,
Fronius M
.
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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.
Figure 1. Shear force (SF) activation of αβγ and δβγ ENaC. Representative current traces (A,B) and statistics of the normalised shear force response of αβγ and δβγ ENaC (C). Amiloride (a) was used to determine ENaC-mediated currents in the absence (I0) and presence (I0.2) of shear force. The shear force response (I0.2/I0) of δβγ was smaller compared with αβγ (****, p < 0.0001, unpaired t-test; I0 indicated by the dashed line).
Figure 2. Shear force effect of δβγ ENaC is not mediated by intrinsic N-glycosylation sites. (A) Sequence alignment of the extracellular domains of human α (SCNN1A, grey) and δ (SCNN1D, orange) ENaC. N-glycosylation motifs of α ENaC that facilitate the shear force response are highlighted in black, whereas intrinsic N-glycosylation motifs of δ ENaC are coloured in orange. (B) Cartoon representation of the extracellular domain of human δ ENaC (highlighted in tan) together with wt β (grey) and γ subunit (white) that are shown as surface representation. The predicted localization of glycosylated asparagines 166, 211 and 384 within δ ENaC are highlighted as spheric molecules representation and coloured in orange. (C) Solvent accessible surface representation of δ ENaC indicates that the glycosylated asparagine residues are localised at exposed positions, likely to provide connections to the ECM. (D) Individual disruption of the three intrinsic N-glycosylation sites (∆N) in δ ENaC did not affect the shear force response (I0.2/I0 normalised for each construct) when compared with channels containing the wild type δ ENaC subunit (I0.2/I0 of δwtβγ = 1 indicated by the dashed line, unpaired t-test).
Figure 3. (A) Cartoon representation of human δ ENaC’s extracellular domain (highlighted in tan) that is aligned to human α ENaC, coloured in slate grey. Extracellular domains of wt β (grey) and γ subunit (white) are shown as surface representation. The predicted localization of the inserted asparagines 292 (green) and 487 (purple) are highlighted together with the corresponding asparagines in α ENaC (N312 and 511, slate grey). (B) Solvent accessible surface representation of δ ENaC indicates that the inserted asparagine residues are localised at exposed positions.
Figure 4. Insertion of N-glycosylation sites in δ ENaC does not impair channel function. (A) ENaC mediated amiloride-sensitive whole-cell currents (Iami) of δN292βγ and δN487βγ were comparable to δwtβγ ENaC (one-way ANOVA with multiple comparisons). (B) Half-maximal amiloride concentrations for δN293 and δN487 constructs were also comparable to wt. (C) ENaC mediated currents (Iami) of δN292/N487βγ channels were similar to wt channels (unpaired t-test).
Figure 5. Single-channel properties of construct N292/N487 expressed with β and γ are comparable to wild type δβγ ENaC. (A) Patch-clamp recording in the cell-attached configuration. (B) The current-voltage relationship is similar between wt and N292/N487. Single-channel conductance (G, panel C), open probability (PO, panel D) and permeability (P, panel E) were comparable between δwtβγ ENaC and δN292/N487βγ (n ≥ 7, unpaired t-test).
Figure 6. Insertion of N-glycosylation sites into δ ENaC increases the shear force effect. (A) Representative current traces of wild type δβγ ENaC and construct N292/N487 in response to shear force (SF). (B) The insertion of N-glycosylation motifs resulted in an increased shear force response (I0.2/I0) in comparison with wild type δβγ ENaC (*, p < 0.05, **, p < 0.001, ****, p < 0.0001; with respect to wt channel indicated by dashed line, unpaired t-test). (C) Immunoblotting of whole-cell lysates from oocytes expressing HA-tagged human δ ENaC (wt, N292, N487 and N292/N487) co-expressed with βγENaC. The shift in relative molecular weight is observed between the wt (~75 kDa) and construct N487 as well as the N292/N487 construct (arrow 1). The molecular weight of construct N292 was similar to the wt δ subunit. PNGase F resulted in the complete loss of the upper band (arrow 2) and water-injected oocytes served as control. Blot is representative for n = 4. Current traces of experiments from oocytes expressing δN292βγ or δN487βγ are depicted in Figure S1.
Figure 7. Degradation of hyaluronic acid of the ECM reduces the shear force response of channels containing the N292/N487 construct. Wild type δβγENaC and δN292/N487βγ expressing oocytes were treated with hyaluronidase (hyal., grey bars). Hyaluronidase treatment had no effect on the shear force response of wild type δβγENaC, but reduced the shear force effect of δN292/N487βγ (ns, p > 0.05; *, p < 0.05; **, p < 0.01; one-way ANOVA with multiple comparisons). Representative current traces of these experiments are depicted in Figure S2.
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