CLCN2 chloride channel mutations in familial hyperaldosteronism type II

Abstract

Primary aldosteronism, a common cause of severe hypertension ¹ , features constitutive production of the adrenal steroid aldosterone. We analyzed a multiplex family with familial hyperaldosteronism type II (FH-II) ² and 80 additional probands with unsolved early-onset primary aldosteronism. Eight probands had novel heterozygous variants in CLCN2, including two de novo mutations and four independent occurrences of a mutation encoding an identical p.Arg172Gln substitution; all relatives with early-onset primary aldosteronism carried the CLCN2 variant found in the proband. CLCN2 encodes a voltage-gated chloride channel expressed in adrenal glomerulosa that opens at hyperpolarized membrane potentials. Channel opening depolarizes glomerulosa cells and induces expression of aldosterone synthase, the rate-limiting enzyme for aldosterone biosynthesis. Mutant channels show gain of function, with higher open probabilities at the glomerulosa resting potential. These findings for the first time demonstrate a role of anion channels in glomerulosa membrane potential determination, aldosterone production and hypertension. They establish the cause of a substantial fraction of early-onset primary aldosteronism.

Figure 1. Kindreds with Hypertension and Primary Aldosteronism with CLCN2 Mutations

(a) Pedigrees of eight kindreds with primary aldosteronism and hypertension, with indicated novel CLCN2 variants. Filled black symbols denote subjects with primary aldosteronism, and filled grey symbols denote subjects with early-onset hypertension or borderline elevated ARRs. Genotypes are shown beneath each symbol, M/+ denotes the indicated novel CLCN2 variant in the heterozygous state, and +/+ denotes homozygous wildtype sequence. (b) Position of the indicated variants in the N-terminus, D helix, K helix and C-terminus of the ClC-2 chloride channel encoded by CLCN2. Red ellipses represent C-terminal CBS domains. (c) Conservation of the respective amino acid positions among orthologous species (H. sapiens, human; M. musculus, mouse; G. gallus, chicken; X. tropicalis, western clawed frog; D. rerio, zebrafish; C. intestinalis, vase tunicate; D. mojavensis, fruit fly; C. elegans, roundworm).

Figure 2. Expression of ClC-2 in Human Adrenal Gland

(a) Section of human adrenal cortex (C, capsule; G, glomerulosa; F, fasciculata; R, reticularis) stained by immunohistochemistry and counterstained with hematoxylin. One of two technical replicates is shown. Left, anti-ClC-2; middle, control preincubation of the anti-ClC-2 antibody with the antigenic peptide; right, anti-Dab2 as marker of the adrenal zona glomerulosa. The comparison of the three panels demonstrates specific expression of ClC-2, predominantly in the zona glomerulosa. Scale bars represent 200 µm. (b) Higher magnifications of the zona glomerulosa, scale bars represent 100 µm.

Figure 3. CLCN2 Mutations Increase Excitatory Anion Efflux by Modifying the Voltage Dependence of Channel Opening

(a) Representative mouse adrenal gland section loaded with MQAE at 37°C. Short lifetimes (red, 1 ns) indicate high, long lifetimes (blue, 4 ns) low intracellular chloride concentrations. Scale bar, 10 µm. (b) Insert, calibration curve of MQAE fluorescence lifetimes in cells with preset intracellular chloride concentrations. A kernel density estimation of intracellular chloride concentrations for glomerulosa cells was obtained (Gaussian kernel, bandwidth=8.0 as determined by Scott’s rule). Median intracellular chloride concentration was 74.7 mM (300 cells, 12 slices, five animals). Box, interquartile range; whiskers, 1.5× interquartile range; line, median. (c) Whole-cell patch clamp recordings of representative ClC-2^WT and ClC-2^MUT and voltage protocol (150 mM [Cl⁻]_out, 75 mM [Cl⁻]_int, see Online Methods for solutions) are shown. (d) Time constants for representative ClC-2^WT and ClC-2^MUT with mean values ± 95% confidence intervals are shown (Supplementary Table 8). (e) and (f) Mean relative open probabilities (e) and common gate open probabilities (f) were fit with a Boltzmann function (bold lines, Supplementary Table 8) with 95% confidence intervals as calculated from a bootstrap resampling (translucent areas). Open probabilities of mutant channels are significantly higher at the glomerulosa resting potential of ~−80 mV (WT, 0.22±0.02 (n=11); p.Arg172Gln, 0.45±0.02 (n=13; p<0.001 vs. WT); p.Ser865Arg, 0.33±0.02 (n=12; p<0.001 vs. WT); all mean±SEM, one-way ANOVA; F=36.307; d.f.=5). The insert in (e) demonstrates the shift in voltage activation as assessed by the point of half maximal activation (Supplemental Table 8). **, p<0.01.

Figure 4. ClC-2 Increases Aldosterone Synthase Expression in H295R cells

(a) RNA sequencing of H295R cells transfected with CLCN2 (WT or p.Arg172Gln), and vector control (heatmap). FPKM, fragments per kilobase of transcript per million fragments mapped. CLCN2 and CYP11B2 show the largest increase in expression versus control. Genes involved in adrenal function or calcium pathway are highlighted. (b) Relative expression levels of CYP11B2 (box, interquartile range; whiskers, 1.5× interquartile range; line, median) in the H295R cell line after transfection with empty vector (control), CLCN2^WT (blue), and CLCN2^MUT (yellow). Parallel transfections and real-time PCRs were performed in each group. CYP11B2 expression significantly increases after transfection of CLCN2^MUT (see Supplementary Table 8 for statistical analysis). (c) Resting membrane potential (plots as in b) of HAC15 cells stably expressing CLCN2 (WT or p.Arg172Gln) and untransfected controls. WT and p.Arg172Gln cause significant depolarization versus control, and p.Arg172Gln causes significant depolarization versus WT (see Supplementary Table 8 for statistical analysis). (d) Model of ClC-2 function in human adrenal glomerulosa. Resting cells are hyperpolarized. Angiotensin II (AngII) and hyperkalemia cause depolarization, activation of voltage-dependent calcium channels, calcium influx, and increased CYP11B2 expression via the transcription factor NR4A2 (NURR1). ClC-2^MUT causes increased CYP11B2 expression by membrane depolarization via increased Cl⁻ efflux. **, p<0.01; ***, p<0.001; ****, p<0.0001.