A Missense Mutation in the Extracellular Domain of α ENaC Causes Liddle Syndrome

Abstract

Liddle syndrome is an autosomal dominant form of hypokalemic hypertension due to mutations in the β- or γ-subunit of the epithelial sodium channel (ENaC). Here, we describe a family with Liddle syndrome due to a mutation in αENaC. The proband was referred because of resistant hypokalemic hypertension, suppressed renin and aldosterone, and no mutations in the genes encoding β- or γENaC. Exome sequencing revealed a heterozygous, nonconservative T>C single-nucleotide mutation in αENaC that substituted Cys479 with Arg (C479R). C479 is a highly conserved residue in the extracellular domain of ENaC and likely involved in a disulfide bridge with the partner cysteine C394. In oocytes, the C479R and C394S mutations resulted in similar twofold increases in amiloride-sensitive ENaC current. Quantification of mature cleaved αENaC in membrane fractions showed that the number of channels did not increase with these mutations. Trypsin, which increases open probability of the channel by proteolytic cleavage, resulted in significantly higher currents in the wild type than in C479R or C394S mutants. In summary, a mutation in the extracellular domain of αENaC causes Liddle syndrome by increasing intrinsic channel activity. This mechanism differs from that of the β- and γ-mutations, which result in an increase in channel density at the cell surface. This mutation may explain other cases of patients with resistant hypertension and also provides novel insight into ENaC activation, which is relevant for kidney sodium reabsorption and salt-sensitive hypertension.

Keywords: ENaC; electrophysiology; genetic renal disease; hypertension; hypokalemia.

Graphical abstract

Figure 1.

The novel αENaC mutation is characterized clinically by hypertension, suppressed plasma renin and aldosterone, and an exaggerated natriuretic response to an ENaC blocker. (A) Pedigree showing three generations of the family with Liddle syndrome. Generation II was analyzed by genotyping and biochemical profiling. The arrow indicates the proband. (B) Sequence chromatogram. (C) The C479R mutation segregated with suppressed plasma renin and aldosterone but not with hypertension. Renin and aldosterone were measured in the absence of interfering drugs. Dashed lines represent lower limits of normal. HT, hypertension; NT, normotension; WT, wild type. (D) Results of a standardized diuretic test showing the natriuretic response to a single dose of the ENaC blocker triamterene in the proband and subject II-4 in comparison with healthy volunteers. *Proband; ^#subject II-4.

Figure 2.

C479 is a highly conserved Cys that forms a disulfide bond with C394. (A) Sequence comparison of hαENaC and rat αENaC (SCAA), βENaC (SCAB), and γENaC (SCAG) subunit isoforms with human hASIC1 and chicken cASIC1. (B) Crystal structure of a cASIC1 subunit with the disulfide bonds in the extracellular domain labeled in green for the first cysteine-rich domain (CRD1) and yellow for CRD2. The Cys366 (red) corresponding to Cys479 in the human αENaC (hSCAA) makes a disulfide bond with Cys291 (purple) corresponding to C394 in hSCAA (inset).

Figure 3.

αENaC C479R is a gain of function mutation. (A) Amiloride-sensitive current increase of αC479R ENaC mutant and the Cys partner, αC394S, mutant. Current values were normalized for the average INa of the wild-type (wt) control obtained in oocytes of each independent batch (n≥4). Bars represent mean±SD for hαENaC wt (n=225), hαENaC C479R (n=231), and hαENaC C394S (n=18). (B) Normalized amiloride-sensitive current values as in A with hαENaC wt (n=14) and hαENaC C479R expressed alone (n=13) or together with hαENaC wt at a cRNA weight ratio of 1:1 (n=13). (C) Normalized amiloride-sensitive current values for rat αENaC wt (n=12), C507S (n=12), and C422S (n=12) corresponding to C479R and C394S in the human αENaC sequence. *P < 0.05.

Figure 4.

C479R does not increase channel surface density. (A) Anti-HA tag (red; left panel) and anti-γENaC (green; right panel) Western blot analysis of Triton-soluble fractions from Xenopus oocytes noninjected (n.i.) or injected with cRNAs for hα-HA alone or with β- and γENaC cRNAs together with either the wild type (wt) or C479R mutant hα-HA. (B) The intensities of the bands corresponding to CL hα- and hγENaC were normalized to the amount of 2,2,2-Trichloroethanol–labeled total protein obtained for each lane on the blot. The ratios between the thus-calculated values for hα- and hγENaC in the hα_wtβγENaC and those for hα_C479RβγENaC are shown in the graph. Data correspond to mean±SEM (ten blots from seven independent experiments); differences are NS. (C, right panel) Anti-HA immunoblot analysis of neutravidin-bound fractions isolated from control (−) or cell surface biotinylated (+) Xenopus oocytes n.i. or injected with either α_wt-HA/β/γ or α_C479R-HA/β/γ cRNAs. (C, left panel) Inputs corresponding to 1% of the Triton-soluble preparations from biotinylated oocytes used in the pull-down experiments. (D) Values (mean±SD) of CL + FL band intensities of inputs or neutravidin-bound fractions corresponding to experiments shown in C. Results from four blots with samples of four independent experiments. Differences are NS.

Figure 5.

Lower trypsin sensitivity of C479R and C394S than wild type αENaC suggests higher intrinsic channel activity. (A) Amiloride-sensitive current (microampere) of hENaC wild-type (wt; n=17), hαENaC C479R (n=17), and hαENaC C394S (n=18) in the absence (−) or presence (+) of trypsin. The magnitude of the currents in the absence of trypsin was significantly higher for C479R and C394S compared with wt (P<0.01 by one-way ANOVA), whereas the currents in oocytes expressing the mutant forms were no longer significantly higher after trypsin treatment. (B) Relationship between baseline INa in the absence of trypsin and fold increase in INa after the addition of trypsin in wt and mutant human ENaC (C479R and C394S). Current values for a single oocyte (filled symbols) and means±SD (open symbols) are shown (P<0.01 by ANOVA). (C) Correlation between current INa (microampere) values in the absence and presence of trypsin. Linear regression analysis gives the following best fit values for the slopes hα_wt (4.08; 95% confidence interval, 3.82 to 4.35), hα_C479R (2.28; 95% confidence interval, 2.14 to 2.42), and hα_C394S (2.23; 95% confidence interval, 2.03 to 2.42). Supplemental Figure 3 shows original traces. INa, Na⁺ current.

Figure 6.

Rat αENaC mutations C507S and C422S are also less sensitive to trypsin than wild type and the βENaC Liddle mutation Y618C. (A) Relationship between the baseline INa in the absence of trypsin and the fold increase in INa after the addition of trypsin for wild-type (wt) rat ENaC, two αENaC mutants (C507S and C422S), and the βENaC Liddle mutant Y618A. Current values for a single oocyte (filled symbols) and means±SD (open symbols) are shown (P<0.01 by ANOVA). (B) Correlation between current INa values (microampere) in the absence and presence of trypsin. Linear regression analysis gives best fit values for the slopes corresponding to rα_wt/rβ/rγ, rα_C507S/rβ/rγ, rα_C422S/rβ/rγ, and rα_wt/rβ/rγ_Y618A, and the values were 4.07 (95% confidence interval, 3.92 to 4.23), 2.09 (95% confidence interval, 2.0 to 2.18), 1.92 (95% confidence interval, 1.74 to 2.09), and 4.04 (95% confidence interval, 3.79 to 4.30), respectively. INa, Na⁺ current.