a Novel Y152C KCNJ5 mutation responsible for familial hyperaldosteronism type III

Abstract

Context: Primary aldosteronism is a heterogeneous group of disorders comprising both sporadic and familial forms. Mutations in the KCNJ5 gene, which encodes the inward rectifier K(+) channel 4 (G protein-activated inward rectifier K(+) channel 4, Kir3.4), cause familial hyperaldosteronism type III (FH-III) and are involved in the pathogenesis of sporadic aldosterone-producing adenomas.

Objective: The objective of the study was to characterize the effects of a newly described KCNJ5 mutation in vitro.

Patients and methods: The index case is a 62-year-old woman affected by primary aldosteronism, who underwent left adrenalectomy after workup for adrenal adenoma. Exon 1 of KCNJ5 was PCR amplified from adrenal tissue and peripheral blood and sequenced. Electrophysiological and gene expression studies were performed to establish the functional effects of the new mutation on the membrane potential and adrenal cell CYP11B2 expression.

Results: KCNJ5 sequencing in the index case revealed a new p.Y152C germline mutation; interestingly, the phenotype of the patient was milder than most of the previously described FH-III families. The tyrosine-to-cysteine substitution resulted in pathological Na(+) permeability, cell membrane depolarization, and disturbed intracellular Ca(2+) homeostasis, effects similar, albeit smaller, to the ones demonstrated for other KCNJ5 mutations. Gene expression studies revealed an increased expression of CYP11B2 and its transcriptional regulator NR4A2 in HAC15 adrenal cells overexpressing KCNJ5(Y152C) compared to the wild-type channel. The effect was clearly Ca(2+)-dependent, because it was abolished by the calcium channel blocker nifedipine.

Conclusions: Herein we describe a new germline mutation in KCNJ5 responsible for FH-III.

Figure 1.

(A) Sequences of tumor cDNA, adjacent adrenal tissue, and peripheral blood gDNA of KCNJ5 codons 151–153 showing the c.455A>G substitution resulting in the p.Y152C mutation. (B) Real-time PCR analysis of CYP11B2 and NR4A2 in HAC15 cells overexpressing KCNJ5^Y152C compared with cells overexpressing KCNJ5^WT and pcDNA3.1 empty vector. Each bar represents the mean ± SE of relative fold change of gene expression in three independent experiments. Each assay was performed in triplicate, and glyceraldehyde-3-phosphate dehydrogenase was used as endogenous control. *, P < .05 compared with WT. §, P < .05 compared with KCNJ5^Y152C not treated with nifedipine.

Figure 2.

Basic characteristics of the KCNJ5^Y152C mutant channel. (A, B) Representative current traces of a wild-type KCNJ5/KCNJ3 (J5^WT/J3; left panel) and of a mutant KCNJ5^Y152C/KCNJ3 (J5^Y152C/J3; right panel) expressing HEK293 cell are shown. Current traces were recorded at 50 mM extracellular K⁺ (K⁺ 50), 5 mM extracellular K⁺ (con), and after replacement of extracellular Na⁺ by NMDG (Na⁺-free). (C–E) I/V curves of similar whole cell experiments as shown in (A) and (B). (F) Effect of Na⁺ replacement on the membrane voltage. *, Significant differences compared to control conditions. (G) Na⁺-dependent conductance (calculated from the conductance between −120 and −90 mV from the data shown in [C–E]) was highest in KCNJ5^Y152C/KCNJ3 cells. Effects of extracellular Ca²⁺ concentration on cytosolic Ca²⁺ concentration. (Fura-2 fluorescence). (H) Mean values of the Fura-2 ratio (340 nm/380 nm) ± SEM in cells expressing KCNJ5^WT/KCNJ3 (WT), KCNJ5^G151E/KCNJ3 (G151E), and KCNJ5^Y152C/KCNJ3 (Y152C) at various extracellular Ca²⁺ concentrations. Numbers of experiments are shown in parentheses. (I) Initial rate of Fura-2 ratio changes induced by 5 mM extracellular Ca²⁺. The period labeled with red dots in (H) was used for the linear fitting and values were normalized to that of the wild type. Values are mean values ± SEM. *, Significant differences compared to wild-type channels expressing cells.