Reimer Esther N, Walenda Gudrun, Seidel Eric, Scholl Ute I
Department of Nephrology, Medical School, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany.
Endocrinology. 2016 Aug;157(8):3016-22. doi: 10.1210/en.2016-1170. Epub 2016 Jun 3.
We recently demonstrated that a recurrent gain-of-function mutation in a T-type calcium channel, CACNA1H(M1549V), causes a novel Mendelian disorder featuring early-onset primary aldosteronism and hypertension. This variant was found independently in five families. CACNA1H(M1549V) 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, CACNA1H(WT) and CACNA1H(M1549V) 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 CACNA1H(WT) and CACNA1H(M1549V). We measured aldosterone production by an ELISA and CYP11B2 (aldosterone synthase) expression by real-time PCR. In unstimulated cells, transfection of CACNA1H(WT) led to a 2-fold increase in aldosterone levels compared with vector-transfected cells. Expression of CACNA1H(M1549V) caused a 7-fold increase in aldosterone levels. Treatment with angiotensin II or increased extracellular potassium levels further stimulated aldosterone production in both CACNA1H(WT)- and CACNA1H(M1549V)-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 CACNA1H(WT) and CACNA1H(M1549V) on CYP11B2 expression. These results directly link CACNA1H(M1549V) 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.
我们最近证明，T型钙通道CACNA1H（M1549V）中的复发性功能获得性突变会导致一种新型孟德尔疾病，其特征为早发性原发性醛固酮增多症和高血压。该变体在五个家庭中独立发现。CACNA1H（M1549V）导致通道失活受损，并在更多超极化电位下激活，推测会导致钙内流增加。我们在此旨在研究该变体对醛固酮产生的影响。我们在产生醛固酮的肾上腺皮质癌细胞系H295R及其亚克隆HAC15中异源表达空载体、CACNA1H（野生型）和CACNA1H（M1549V）。CACNA1H（野生型）和CACNA1H（M1549V）之间通道的转染率、表达水平和亚细胞分布相似。我们通过酶联免疫吸附测定法测量醛固酮产生，并通过实时聚合酶链反应测量CYP11B2（醛固酮合成酶）表达。在未刺激的细胞中，与载体转染的细胞相比，CACNA1H（野生型）的转染导致醛固酮水平增加2倍。CACNA1H（M1549V）的表达导致醛固酮水平增加7倍。用血管紧张素II处理或增加细胞外钾水平进一步刺激了CACNA1H（野生型）和CACNA1H（M1549V）转染细胞中的醛固酮产生。CYP11B2表达也获得了类似结果。用T型钙通道阻滞剂米贝地尔抑制CACNA1H通道完全消除了CACNA1H（野生型）和CACNA1H（M1549V）对CYP11B2表达的影响。这些结果直接将CACNA1H（M1549V）与醛固酮产生增加联系起来。它们表明钙通道阻滞剂可能对治疗一部分原发性醛固酮增多症患者有益。此类阻滞剂可以靶向CACNA1H或同时靶向CACNA1H和也在肾上腺中表达且在原发性醛固酮增多症患者中发生突变的L型钙通道CACNA1D。
