A mutation of the epithelial sodium channel associated with atypical cystic fibrosis increases channel open probability and reduces Na+ self inhibition

Abstract

Increased activity of the epithelial sodium channel (ENaC) in the respiratory airways contributes to the pathophysiology of cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some patients suffering from atypical CF a mutation can be identified in only one CFTR allele. We recently identified in this group of CF patients a heterozygous mutation (W493R) in the alpha-subunit of ENaC. Here, we investigate the functional effects of this mutation by expressing wild-type alpha beta gamma ENaC or mutant alpha(W493R)beta gamma ENaC in Xenopus oocytes. The alpha W493R mutation stimulated amiloride-sensitive whole-cell currents (Delta I(ami)) by approximately 4-fold without altering the single-channel conductance or surface expression of ENaC. As these data suggest that the open probability (P(o)) of the mutant channel is increased, we investigated the proteolytic activation of ENaC by chymotrypsin. Single-channel recordings revealed that chymotrypsin activated near-silent channels in outside-out membrane patches from oocytes expressing wild-type ENaC, but not in membrane patches from oocytes expressing the mutant channel. In addition, the alpha W493R mutation abolished Na(+) self inhibition of ENaC, which might also contribute to its gain-of-function effects. We conclude that the alpha W493R mutation promotes constitutive activation of ENaC by reducing the inhibitory effect of extracellular Na(+) and decreasing the pool of near-silent channels. The resulting gain-of-function phenotype of the mutant channel might contribute to the pathophysiology of CF in patients carrying this mutation.

Figure 1. αW493R mutation stimulates ENaC-mediated Na⁺ currents

Oocytes were injected with 0.5 ng cRNA per subunit and incubated in ND96. Amiloride-sensitive whole-cell currents (ΔI_ami) were measured with the two-electrode voltage-clamp technique 2 days after cRNA injection. A, representative whole-cell current traces of a αβγENaC (wt) and a α_W493RβγENaC (αW493R)-expressing oocyte at a holding potential of −60 mV. Amiloride (2 μm) was present in the bath to specifically inhibit ENaC as indicated by the black bars. Washout of amiloride revealed a sizeable inward current component which corresponds to the ENaC-mediated Na⁺ inward current. Reapplication of amiloride returned the whole-cell current toward the initial baseline level. At the time points indicated by asterisks current responses from voltage-step pulses were removed for clarity. B, summary of similar experiments as shown in A performed in wild-type and mutant ENaC-expressing oocytes on the wild-type αT663 and the polymorphic αA663 background. To pool data from different batches of oocytes, the individual ΔI_ami values were normalized to the mean ΔI_ami value of the corresponding wild-type ENaC-expressing control group. C, average amiloride concentration–response curves measured in wild-type and mutant ENaC-expressing oocytes (9 oocytes per group). Error bars are partly covered by symbols. IC₅₀ inhibitory constants of amiloride are indicated. N indicates number of different batches of oocytes. ***P < 0.001.

Figure 2. The stimulatory effect of the αW493R mutation is independent of Na⁺ feedback inhibition

αβγENaC (wt)- and α_W493RβγENaC (αW493R)-expressing oocytes were incubated in parallel in high Na⁺ and low Na⁺ solution. A, representative whole-cell current traces of individual oocytes at a holding potential of −60 mV. Amiloride (2 μm) was present in the bath as indicated by the black bars. B, summary of similar experiments as shown in A performed in oocytes from 3 different batches. C, to illustrate the relative effect of Na⁺ feedback inhibition on wild-type and mutant ENaC the individual ΔI_ami values summarized in B were normalized for each batch to the mean ΔI_ami value measured in the corresponding group of low Na⁺ oocytes. Numbers in or above columns indicate individual number of oocytes, N indicates number of different batches of oocytes. **P < 0.01, ***P < 0.001.

Figure 3. The stimulatory effect of the αW493R mutation is not caused by an increase in channel surface expression

Oocytes were injected with cRNA for αβ_FLAGγENaC (wt) or α_W493Rβ_FLAGγENaC (αW493R) and incubated for 2 days in a low Na⁺ solution. The insertion of a FLAG reporter epitope in the extracellular loop of the β-subunit enabled the detection of channel surface expression by a chemiluminescence assay (filled columns). For control experiments the amount of αβ_FLAGγENaC cRNA injected per oocyte was tripled (3× wt). ΔI_ami was determined in parallel in oocytes from the same experimental groups (open columns). To pool data from different batches of oocytes, individual current and surface expression values were normalized to the corresponding mean values of wild-type control oocytes. Numbers in columns indicate individual number of oocytes. N indicates number of different batches of oocytes. n.s., not significant. ***P < 0.001.

Figure 4. The αW493R mutation does not alter the single-channel conductance of ENaC

A, representative single-channel current recordings obtained at a holding potential of −70 mV from an outside-out patch of an oocyte expressing wild-type (upper trace) or mutant ENaC (lower trace). NaCl or LiCl were present in the bath solution as indicated above the trace by white and grey bars, respectively. B and C, in similar experiments as shown in A single-channel current voltage (I–V) values were obtained at different holding potentials (V_hold) with NaCl or LiCl in the bath solution from outside-out patches of wt (B, n= 4, N= 2) and mutant ENaC (C, n= 6, N= 2)-expressing oocytes. The I–V data were fitted using the Goldman–Hodgkin–Katz equation.

Figure 5. The αW493R mutation reduces the stimulatory effect of chymotrypsin on ΔI_ami

Oocytes were injected with cRNA (0.2 ng per subunit) for αβγENaC (wt) or α_W493RβγENaC (αW493R) and incubated for 2 days in low Na⁺. A, representative current traces demonstrating the effect of chymotrypsin (2 μg ml⁻¹) on wild-type and mutant ENaC. B, summary of ΔI_ami values determined before and after exposure to chymotrypsin in similar experiments as shown in A. C, same data as in B but with ΔI_ami values after chymotrypsin application normalized to the corresponding control value before chymotrypsin application. Numbers in columns indicate individual number of oocytes. N indicates number of different batches of oocytes. *P < 0.05, ***P < 0.001.

Figure 6. Chymotrypsin fails to activate near-silent channels in outside-out patches expressing the mutant α_W493RβγENaC

A and B, representative single-channel current recordings obtained at a holding potential of −70 mV from an outside-out patch of oocytes expressing wild-type (A) or mutant ENaC (B). Amiloride (Ami, 2 μm) and chymotrypsin (2 μg ml⁻¹) were present in the bath solution as indicated above the trace by black and grey bars, respectively. The current level at which all channels are closed (C) was determined in the presence of amiloride. The different channel open levels are indicated by horizontal lines. Time periods used to calculate NP_o values are indicated by broken lines below the traces. C, amplitude histograms were used to calculate NP_o values from similar outside-out patch-clamp recordings as shown in A and B. D, the maximal number of apparent channel levels was determined by visual inspection of single-channel current traces as shown in A and B. C and D summarize data from n= 13 and n= 14 outside-out patches with wild-type or mutant ENaC, respectively. Please note that some open circles and lines represent 2–3 individual experiments. *P < 0.05, **P < 0.01, n.s., not significant.

Figure 7. The αW493R mutation does not alter endogenous channel cleavage

Oocytes were injected with 2 ng per subunit of cRNA for wt and mutant (αW493R) ENaC and incubated in low Na⁺ for 2 days. A, surface-expressed protein was isolated by biotinylation and separated by PAGE. Full length αENaC (at ∼95 kDa) and expected cleavage products (Rossier & Stutts, 2009) of the α-subunit were detected by using an N-terminal HA-tag (left blot) or a C-terminal V5-tag (right blot). Non-injected (non inj.) oocytes served as control. B, cleavage was quantified by densitometric analysis. The N-terminal ∼24 kDa and the ∼95 kDa bands were used to determine the amount of cleaved and non-cleaved ENaC, respectively. N represents number of blots from different batches of oocytes.

Figure 8. The αW493R mutation abolishes Na⁺ self inhibition

Oocytes were injected with 0.5 ng per subunit of cRNA for αβγENaC (wt) or α_W493RβγENaC (αW493R). After 2 days of incubation in low Na⁺, oocytes were clamped at −100 mV. The bath solution initially contained 1 mm Na⁺ and was subsequently changed to a bath solution containing 110 mm Na⁺ as indicated. After 60 s, amiloride (2 μm) was applied to determine the ENaC-mediated current component. A, representative current traces of a wild-type (left) and a mutant ENaC (right)-expressing oocyte. Downward deflections represent inward currents. B, averaged current traces from similar experiments as shown in A, starting from the change to 110 mm Na⁺. Lines represent mean, error bars s.e.m. n indicate number of oocytes.

Figure 9. Amino acid substitution at residue α493 stimulates ENaC

Oocytes were injected with cRNA for αβγENaC (wt), α_W493RβγENaC (αW493R), α_W493AβγENaC (αW493A), α_W493CβγENaC (αW493C) or α_W493EβγENaC (αW493E). A, representative whole-cell current traces of individual oocytes at a holding potential of −60 mV. Amiloride (2 μm) was present in the bath as indicated by the black bars. B, summary of similar experiments as shown in A. To pool data from different batches of oocytes, individual ΔI_ami values were normalized to the mean ΔI_ami of the corresponding wild-type ENaC-expressing control oocytes. N indicates number of different batches of oocytes. Numbers in columns indicate total number of oocytes. ***P < 0.001.