Functional characterization of two novel germline mutations of the KCNJ5 gene in hypertensive patients without primary aldosteronism but with ACTH-dependent aldosterone hypersecretion

Abstract

Background: Germline mutations of the KCNJ5 gene encoding Kir3·4, a member of the inwardly rectifying K⁺ channel, have been identified in 'normal' adrenal glands, patients with familial hyperaldosteronism (FH) type III, aldosterone-producing adenomas (APAs) and sporadic cases of primary aldosteronism (PA).

Objective: To present two novel KCNJ5 gene mutations in hypertensive patients without PA, but with Adrenocorticotropic hormone (ACTH)-dependent aldosterone hypersecretion.

Design and patients: Two hypertensive patients without PA, who exhibited enhanced ACTH-dependent response of aldosterone secretion, underwent genetic testing for the presence of the CYP11B1/CYP11B2 chimeric gene and KCNJ5 gene mutations. Genomic DNA was isolated from peripheral white blood cells, and the exons of the entire coding regions of the above genes were amplified and sequenced. Electrophysiological studies were performed to determine the effect of identified mutation(s) on the membrane reversal potentials. Structural biology studies were also carried out.

Results: Two novel germline heterozygous KCNJ5 mutations, p.V259M and p.Y348N, were detected in the two subjects. Electrophysiological studies showed that the Y348N mutation resulted in significantly less negative reversal potentials, suggesting loss of ion selectivity, while the V259M mutation did not affect the Kir3.4 current. In the mutated structural biology model, the N348 mutant resulted in significant loss of the ability for hydrogen bonding, while the M259 mutant was capable of establishing weaker interactions. The CYP11B1/CYP11B2 chimeric gene was not detected.

Conclusions: These findings expand on the clinical spectrum of phenotypes associated with KCNJ5 mutations and implicate these mutations in the pathogenesis of hypertension associated with increased aldosterone response to ACTH stimulation.

Figure 1

Sequencing chromatograms showing the two novel mutations detected in the KCNJ5 gene. (A) G to A substitution at nucleotide 775 (c.775G>A), altering codon 259 from Valine to Methionine p.Val259Met, in exon 2. (B) T to A substitution at nucleotide 1042 (c. 1042T>A), altering codon 348 from Tyrosine to Asparagine, p.Tyr348Asn in exon 3.

Figure 2

Uniprot multiple alignment of the KCNJ5 protein sequence in different species, indicating conservation of the residues Val258 (A) and Tyr348 (B) affected by these mutations.

Figure 3

Kir3.4 currents from HEK 293T cells expressing the KCNJ5 wild-type (WT) channels and the two mutants KCNJ5^V259M and KCNJ5^Y348N. (A) Examples of current traces evoked by −100 to +60mV voltage steps (1s, 20mV increment; scale bars: 100pA/100ms). (B) KCNJ5 mutations result in depolarization of current reversal potential. Reversal potentials are plotted for the wild-type (WT) Kir3.4(KCNJ5) channels and each Kir3.4 (KCNJ5) mutant. WT channels (black box) show a negative reversal potential. The mutant KCNJ5^Y348N (green box) shows significantly less negative reversal potentials compared with the WT channel (P <0.05), suggesting a loss of ion selectivity. The mutant KCNJ5^V259M (orange box) does not affect the Kir3.4 current significantly. Numbers in parentheses represent the number of the cells recorded.

Figure 4

Molecular interactions of the wild-type and the mutated model of KCNJ5 protein. (A) The p.Val259Met position, where only Valine is capable of hydrogen bonding to Ile262. (B) The p.Tyr348Asn position, where the less balky Asparagine is not capable of hydrogen bonding to Asp228 as the wild-type Tyrosine does.

Figure 5

Superimposed snapshot of the wild-type (orange) and mutant (blue) model of the KCNJ5 protein. (A) The p.Val259Met mutation, where the –CH3 moiety stands in the mid-space between the 259 residue position and the network of beta-sheets. (B) The p.Tyr348Asn mutation, where the Asparagine residue (N) is less balkier than the Tyrosine (Y) wild-type amino acid, thus preventing its hydrogen bonding interactions with amino acids in the nearby beta-sheets.