Mutated KCNJ5 activates the acute and chronic regulatory steps in aldosterone production

Namita G Hattangady; Shigehiro Karashima; Lucy Yuan; Daniela Ponce-Balbuena; José Jalife; Celso E Gomez-Sanchez; Richard J Auchus; William E Rainey; Tobias Else

doi:10.1530/JME-15-0324

Mutated KCNJ5 activates the acute and chronic regulatory steps in aldosterone production

¹Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, Michigan, USA
²Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA
³Center for Arrhythmia Research, University of Michigan, Ann Arbor, Michigan, USA
⁴G. V. (Sonny) Montgomery VA Medical Center and Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi, USA
⁵Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA

Correspondence should be addressed to T Else; Email: telse{at}umich.edu

Abstract

Somatic and germline mutations in the inward-rectifying K⁺ channel (KCNJ5) are a common cause of primary aldosteronism (PA) in aldosterone-producing adenoma and familial hyperaldosteronism type III, respectively. Dysregulation of adrenal cell calcium signaling represents one mechanism for mutated KCNJ5 stimulation of aldosterone synthase (CYP11B2) expression and aldosterone production. However, the mechanisms stimulating acute and chronic production of aldosterone by mutant KCNJ5 have not been fully characterized. Herein, we defined the effects of the T158A KCNJ5 mutation (KCNJ5^T158A) on acute and chronic regulation of aldosterone production using an adrenal cell line with a doxycycline-inducible KCNJ5^T158A gene (HAC15-TRE-KCNJ5^T158A). Doxycycline incubation caused a time-dependent increase in KCNJ5^T158A and CYP11B2 mRNA and protein levels. Electrophysiological analyses confirm the loss of inward rectification and increased Na⁺ permeability in KCNJ5^T158A-expressing cells. KCNJ5^T158A expression also led to the activation of CYP11B2 transcriptional regulators, NURR1 and ATF2. Acutely, KCNJ5^T158A stimulated the expression of total and phosphorylated steroidogenic acute regulatory protein (StAR). KCNJ5^T158A expression increased the synthesis of aldosterone and the hybrid steroids 18-hydroxycortisol and 18-oxocortisol, measured with liquid chromatography-tandem mass spectrometry (LC-MS/MS). All of these stimulatory effects of KCNJ5^T158A were inhibited by the L-type Ca²⁺ channel blocker, verapamil. Overall, KCNJ5^T158Aincreases CYP11B2 expression and production of aldosterone, corticosterone and hybrid steroids by upregulating both acute and chronic regulatory events in aldosterone production, and verapamil blocks KCNJ5^T158A-mediated pathways leading to aldosterone production.