CACNA1HM1549V Mutant Calcium Channel Causes Autonomous Aldosterone Production in HAC15 Cells and Is Inhibited by Mibefradil

Abstract

We recently demonstrated that a recurrent gain-of-function mutation in a T-type calcium channel, CACNA1HM1549V, causes a novel Mendelian disorder featuring early-onset primary aldosteronism and hypertension. This variant was found independently in five families. CACNA1HM1549V leads to impaired channel inactivation and activation at more hyperpolarized potentials, inferred to cause increased calcium entry. We here aimed to study the effect of this variant on aldosterone production. We heterologously expressed empty vector, CACNA1HWT and CACNA1HM1549V in the aldosterone-producing adrenocortical cancer cell line H295R and its subclone HAC15. Transfection rates, expression levels, and subcellular distribution of the channel were similar between CACNA1HWT and CACNA1HM1549V. We measured aldosterone production by an ELISA and CYP11B2 (aldosterone synthase) expression by real-time PCR. In unstimulated cells, transfection of CACNA1HWT led to a 2-fold increase in aldosterone levels compared with vector-transfected cells. Expression of CACNA1HM1549V caused a 7-fold increase in aldosterone levels. Treatment with angiotensin II or increased extracellular potassium levels further stimulated aldosterone production in both CACNA1HWT- and CACNA1HM1549V-transfected cells. Similar results were obtained for CYP11B2 expression. Inhibition of CACNA1H channels with the T-type calcium channel blocker Mibefradil completely abrogated the effects of CACNA1HWT and CACNA1HM1549V on CYP11B2 expression. These results directly link CACNA1HM1549V to increased aldosterone production. They suggest that calcium channel blockers may be beneficial in the treatment of a subset of patients with primary aldosteronism. Such blockers could target CACNA1H or both CACNA1H and the L-type calcium channel CACNA1D that is also expressed in the adrenal gland and mutated in patients with primary aldosteronism.