ATP2B3, and KCNJ5 Mutations in Aldosterone-Producing Adenomas

Aldosterone ‐ producing adenomas (APAs) cause a sporadic form of primary aldosteronism and somatic mutations in the KCNJ5 gene, which encodes the G ‐ protein–activated inward rectifier K + channel 4, GIRK4, account for ≈ 40% of APAs. Additional somatic APA mutations were identified recently in 2 other genes, ATP1A1 and ATP2B3 , encoding Na + /K + ‐ ATPase 1 and Ca 2+ ‐ ATPase 3, respectively, at a combined prevalence of 6.8%. We have screened 112 APAs for mutations in known hotspots for genetic alterations associated with primary aldosteronism. Somatic mutations in ATP1A1 , ATP2B3 , and KCNJ5 were present in 6.3%, 0.9%, and 39.3% of APAs, respectively, and included 2 novel mutations (Na + /K + ‐ ATPase p.Gly99Arg and GIRK4 p.Trp126Arg). CYP11B2 gene expression was higher in APAs harboring ATP1A1 and ATP2B3 mutations compared with those without these or KCNJ5 mutations. Overexpression of Na + /K + ‐ ATPase p.Gly99Arg and GIRK4 p.Trp126Arg in HAC15 adrenal cells resulted in upregulation of CYP11B2 gene expression and its transcriptional regulator NR4A2. Structural modeling of the Na + /K + ‐ ATPase showed that the Gly99Arg substitution most likely interferes with the gateway to the ion binding pocket. In vitro functional assays demonstrated that Gly99Arg displays severely impaired ATPase activity, a reduced apparent affinity for Na + activation of phosphorylation and K + inhibition of phosphorylation that indicate decreased Na + and K + binding, respectively. Moreover, whole cell patch ‐ clamp studies established that overexpression of Na + /K + ‐ ATPase Gly99Arg causes membrane voltage depolarization. In conclusion, somatic mutations are common in APAs that result in an increase in CYP11B2 gene expression and may account for the dysregulated aldosterone production in a subset of patients with sporadic primary aldosteronism. mutation is likely to cause a steric clash with an impact on surrounding residues, including Glu334 that binds K + . Consistently, we show that the p.Gly99Arg mutant displayed minimal ATPase activity, reduced Na + affinity, and markedly reduced K + affinity when compared with the wild ‐ type ATPase as observed in phosphorylation studies. These results are in agreement with a disturbed gating mechanism, in particular, affecting K + binding to the mutated pump. Adrenal zona glomerulosa cells display a distinctive high resting K + conductance that maintains the high negative membrane potential ( − 80 mV) of these cells. 26 We demonstrate by whole cell patch ‐ clamp studies that the p.Gly99Arg mutation results in the depolarization of the membrane voltage. In adrenal glomerulosa cells, this depolarization results in the opening of voltage ‐ gated Ca 2+ channels. 2,8 In accordance with an increase in Ca 2+ influx, we show that the overexpression of p.Gly99Arg or p.Leu104Arg in adrenal cells results in an increase in CYP11B2 gene expression and also, for the p.Gly99Arg mutation, NR4A2 . Finally, the expression of CYP11B2 in APAs harboring ATPase mutations was significantly higher compared with those without these or GIRK4 mutations; an effect of heterozygous ATPase mutations on glomerulosa cells is consistent with the unique sensitivity of these cells to small changes in intracellular K + and membrane potential. 26,27 Taken together, these observations are in agreement with a pathophysiological link between the presence of these mutations and the dysregulated aldosterone secretion in patients with APA.