The experiments were carried out with human ENaC. α-ENaC was transcribed from the pSDEasy vector, which was linearized with BglII (Cat #R0144L, New England Biolabs). β-ENaC was transcribed from the pBSK(+) Xglob construct, and γENaC from the pSD5 vector, both of which were linearized with XbaI (Cat #R0145S, NEB). The G532S mutation in γENaC was introduced using the QuikChange site-directed mutagenesis approach, utilizing the KAPA HiFi HotStart PCR polymerase (Cat #KK2501, KAPA Biosystems). Verification of the mutation’s presence was conducted by sequencing (Microsynth). Capped RNA was synthesized in vitro using the mMessage mMachine SP6 or T7 kit (Invitrogen™, Cat #AM1340 and #AM1344). Transcription of α-ENaC and γENaC was done with the SP6 RNA polymerase, while the T7 RNA polymerase was used for β-ENaC.

All procedures with Xenopus laevis frogs were approved by the Veterinarian Office of the Canton of Vaud. 1.3 g/L MS-222 (Cat #A5040-250G, Sigma) was used to anaesthetize female Xenopus laevis frogs. The oocytes were obtained via a small incision (approximately 1 cm) made in the lateral abdominal region. Subsequently, the ovary lobes were incubated with a collagenase solution (Cat #C9891, Sigma-Aldrich), which contained 1 mg/mL of the enzyme, diluted in a calcium-free modified Barth’s (MBS) solution. This treatment was conducted for a duration of 90 min at room temperature, with the aim of isolating and defolliculating oocytes of stage V and stage VI. Equal concentrations of cRNAs for α-, β- and γENaC subunits were mixed, resulting in a final concentration of 15 ng/μl, with 40 nL cRNA solution injected per oocyte. The oocytes were maintained at 19 °C in MBS solution containing (in mM): 85 NaCl, 1 KCl, 2.4 NaHCO₃, 0.33 Ca(NO₃)₂, 0.82 MgSO₄, 0.41 CaCl₂, 10 HEPES, and 4.08 NaOH. Electrophysiological measurements were conducted 20–30 h after the cRNA injection in the oocytes. Currents were recorded using the two-electrode voltage-clamp technique at a holding potential of −80 mV. A Dagan TEV200 amplifier (Minneapolis, MN) equipped with two bath electrodes was used for recordings, operated via PatchMaster (RRID: SCR_000034, HEKA Elektronik-Harvard Bioscience), and analyzed using FitMaster (RRID: SCR_016233, HEKA Elektronik-Harvard Bioscience). Each oocyte was placed in the recording chamber, penetrated by two glass electrodes, each having a resistance below 1 MΩ when filled with 1 M KCl. The recording solutions comprised (in mM): 120 NaCl, 2.5 KCl, 10 HEPES, and 1.8 CaCl₂. Variants consisted of using this solution either as it was or supplemented with 10 μM amiloride (Cat #A7410, Sigma-Aldrich) or 5 μg/mL trypsin (Cat #T1426, Sigma-Aldrich). The pH was adjusted to 7.4 with NaOH. Current sensitive to amiloride was established through the subtraction of the current measured when 10 μM amiloride was applied from the current measured in its absence. Oocytes expressing WT and γG532S-ENaC were recorded alternately to prevent bias resulting from the increase in ENaC current during the expression period.

Oocyte biotinylation and isolation of biotinylated fractions were performed as described previously (19). Control non-injected, or injected oocytes (~20 per condition) were incubated for 15 min on ice in 1 mL Biotinylation buffer (in mM, triethanolamine 10, NaCl 150 mM, CaCl₂ 2, pH 9.5, supplemented with 1 mg/mL NHS-Sulfo-S-S-Biotin (Thermo-Scientific #21331)). The residual reagent was quenched by replacing the biotinylation solution with 1 mL of MBS supplemented with (in mM) glycine 192, Tris/HCl 25, pH 7.5, and 5 min incubation at 22 °C. After one rinsing step in MBS, the drained oocytes were stored at −20 °C or used immediately.

To isolate membrane fractions, the oocytes were disrupted by pipetting in 0.75 mL of membrane isolation buffer (in mM): 50 Tris/HCl (pH 7.0 at room temperature), 150 NaCl, 5 MgCl₂, 10 N-ethylmaleimide, supplemented with cOmplete protease inhibitor cocktail (ULTRA Tablets, Mini, EDTA-free, Roche#05–892–791-001, one tablet per 10 mL), followed by centrifugation through cell shredders (Macherey and Nagel, Oensingen, Switzerland) for 3 min at 11,000 g. After removing the shredders from the collecting tubes, the lysates were further centrifuged for 45 min at 20,000 g (4 °C). The resulting pellets were resuspended in membrane solubilization solution (25 μL per oocyte) containing (in mM): 50 Tris/HCl (pH 7.0 at RT), 10 N-ethylmaleimide, and 1% (v/v) Triton X100, supplemented with protease inhibitors as indicated above. The solubilization of membrane proteins was completed by incubating the homogenates for 45–60 min on an orbital shaker at 4 °C, followed by centrifugation for 12 min as before (= membrane-enriched fraction). Samples of these Triton-soluble fractions (total membrane fractions) were mixed with 4× Sample buffer (50 mM DTT final concentration) and heated for 5 min at 72 °C for SDS-PAGE separation and Western blot analysis. After adjusting the NaCl concentration to 150 mM (from a 5 M stock solution), the Biotinylated fractions were isolated from the membrane-enriched fractions and incubated on an orbital shaker for 5–6 h at 4 °C in the presence of 25 μL (bed volume) of streptavidin-agarose beads (Thermo Scientific #20353). Non-bound fractions were discarded, the beads were washed twice with RIPA buffer (Thermo Scientific #89901), supplemented with protease inhibitors, by incubating each time for 5 min on an orbital shaker at 4 °C. Beads were rinsed once with membrane solubilization solution supplemented with NaCl (150 mM) and protease inhibitors. The drained beads were finally resuspended in 50 μL of 2xSample buffer/DTT (50 mM final). Bound fractions were eluted by heating for 7 min at 72 °C. Triton-soluble fractions (1% of total) and neutravidin-bound fractions were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blocked in 2% (w/v) skimmed milk powder in 1xTBS. Nitrocellulose membranes were subsequently incubated overnight at 4 °C in the presence of antibodies (see Table 1) diluted in 1% (w/v) skimmed milk in 1xTBS. After three rounds of washing in 1xTBS, 0.05% Tween-20, membranes were incubated for 1 h at RT in the presence of HRP-conjugated anti-rabbit immunoglobulins (AffiniPure Fab Fragment Goat Anti-Rabbit, Jackson ImmunoResearch #111–007-003), diluted 1/12,000 in 1% (w/v) skimmed milk, in 1xTBS. After washing, the HRP signal was revealed using Western Bright Quantum detection reagent (Advansta, Menlo Park, CA, #K12042-D20) and detected using a Fusion Solo imaging system (Vilber Lourmat, Marne-la-Vallée, France). The exposure time was adjusted to avoid saturation of the recorded bands. Band intensities were measured from 16-bit, grayscale, uncompressed TIF images using ImageJ software (ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, United States)².

To correct for differences in protein recovery, the intensities of bands in membrane preparations and biotinylated fractions, corresponding to each of the ENaC subunits, were normalized to that of the endogenous Na⁺/K⁺-ATPase α subunit.

Statistical analysis was performed using GraphPad Prism, version 10 (RRID: SCR_002798). To compare two groups, the Student’s unpaired t-test or Mann Whitney test was used. For comparisons involving more than two groups, the Kruskal–Wallis test followed by Dunn’s multiple comparisons test was performed. The data are presented as mean ± standard error of the mean (SEM), showing the individual data points.

The γENaC-Gly532 residue is highly conserved among ENaC/degenerin channels. Although the high-resolution 3D structures of ENaC do not resolve the parts containing this residue (9, 10), alignment to ASIC sequences indicate that this residue is located in the transmembrane α-helix 2 (TM2) which lines the channel pore. We examined here the functional consequences of the αβγG532S mutation. αβγWT or αβγG532S ENaC (i.e., αβγ ENaC containing the mutation G532S in the γ subunit) was expressed in Xenopus laevis oocytes, and channel function was measured with two-electrode voltage-clamp. Recordings were started in the presence of the ENaC inhibitor amiloride at a concentration of 10 μM, to establish the baseline. Subsequently, the oocytes were exposed for 20 s to an amiloride-free solution (control) and were then switched back to the amiloride-containing solution, to measure the amplitude of the ENaC current (left traces of Figure 1A). After repeating this protocol once, oocytes were exposed to the control solution for 20 s, followed by exposure to 5 μg/mL trypsin for 2 min (middle traces in Figure 1A). Trypsin, a serine protease, cleaves extracellular parts of α and γ subunits and removes inhibitory segments, thereby increasing ENaC activity (20–22). After washing out trypsin, the amiloride-sensitive current was measured again (traces on the right in Figure 1A). The amiloride-sensitive current amplitude of oocytes expressing the αβγG532S mutation was 37 ± 4% relative to that of oocytes expressing the αβγWT ENaC when measured before trypsin exposure (p < 0.0001; Figures 1B,C). In this analysis, the current amplitudes measured by individual αβγWT and αβγG532S ENaC oocytes were normalized with respect to the average amplitude of the αβγWT currents of the same oocyte batch.

A comparison of the current recorded before and after trypsin exposure provides an estimate of the open probability of ENaC channels under basal (unstimulated) conditions, under the assumption that after trypsin exposure, ENaC is fully active (8, 21, 23). αβγWT showed in many studies an open probability of ≤0.5 (24). Trypsin increased the ENaC αβγWT currents, as shown by the ratio of current after/before trypsin of 1.77 ± 0.14 (n = 31, Figures 1B,D). Assuming full activation after trypsin exposure, this indicates a basal open probability of ENaC αβγWT of 0.56. In contrast, the current ratio after/before trypsin was 1.2 ± 0.04 with αβγG532S (Figure 1D). The ratio is therefore 32% lower in the mutant (p ≤ 0.0001), suggesting an increased basal open probability in αβγG532S. Upon exposure to trypsin, both ENaC αβγWT and αβγG532S oocytes exhibited a transient current peak, followed by a stable plateau phase (Figure 1A), as observed in 78% of WT- and 90% of mutant channel-expressing oocytes (considering transient currents if I_peak/I_plateau ≥ 1.5; Figure 1E). The transient peak current was 7 ± 1 times greater than the plateau current in αβγWT-expressing oocytes, and 26 ± 7 times greater in αβγG532S-expressing oocytes (Figure 1F). The trypsin-induced plateau, but not the transient current amplitude was significantly different between the WT and the mutant (Figure 1E). Taken together, the functional analysis shows a 63% current reduction in the mutant, which appears not to be caused by a lower open probability.

To assess whether the reduction in Na⁺ currents observed with the mutated form of the γENaC was a result of a decrease in total or cell surface expression of ENaC at the protein level, Western blot analysis was carried out from membrane-enriched fractions (= total expression) and from plasma membrane-resident channels isolated by cell surface biotinylation of intact oocytes, after injection with either αβγWT or αβγG532S cRNAs. Western blots of the membrane-enriched fractions showed that the oocytes expressing the mutated γ-ENaC displayed, when compared to the WT, a lower expression of not only this subunit, but also of the α and β subunits (Figures 2A,B). This decrease of expression at the protein level was confirmed on membrane-enriched fractions of oocytes injected only with cRNAs for either the wild type or the mutated γENaC subunit (Supplementary Figure S1), indicating that the effect of the mutation takes place before the formation of the three-subunit complex. Surprisingly, αENaC expression at the cell-surface was increased in the mutant αβγG532S (Figures 2A,C). Consistent with their total expression pattern and in contrast to αENaC, the expression at the cell surface of β- and γENaC was decreased in αβγG532S relative to αβγWT (Figures 2A,C). The biochemical analysis indicated the occurrence of cleavage of the α and γ subunits (Figure 2A). Although there may be an indication of lower abundance of the cleaved forms in oocytes expressing αβγG532S, the low expression of the mutant precluded a quantitative analysis of subunit cleavage.