Modulation of Calcium Signaling on Demand to Decipher the Molecular Mechanisms of Primary Aldosteronism

Abstract

Background: Primary aldosteronism is the most common form of secondary hypertension. The most frequent genetic cause of aldosterone-producing adenomas is somatic mutations in the potassium channel KCNJ5. They affect the ion selectivity of the channel, with sodium influx leading to cell membrane depolarization and activation of calcium signaling, the major trigger for aldosterone biosynthesis.

Methods: To investigate how KCNJ5 mutations lead to the development of aldosterone-producing adenomas, we established an adrenocortical cell model in which sodium entry into the cells can be modulated on demand using chemogenetic tools [H295R-S2 α7-5HT3-R (α7-5HT3 receptor) cells]. We investigated their functional and molecular characteristics with regard to aldosterone biosynthesis and cell proliferation.

Results: A clonal cell line with stable expression of the chimeric α7-5HT3-R in H295R-S2 (human adrenocortical carcinoma cell line, Strain 2) cells was obtained. Increased sodium entry through α7-5HT3-R upon stimulation with uPSEM-817 (uPharmacologically Selective Effector Molecule-817) led to cell membrane depolarization, opening of voltage-gated Ca²⁺ channels, and increased intracellular Ca²⁺ concentrations, resulting in the stimulation of CYP11B2 expression and increased aldosterone biosynthesis. Increased intracellular sodium influx did not increase proliferation but rather induced apoptosis. RNA sequencing and steroidome analyses revealed unique profiles associated with Na⁺ entry, with only partial overlap with Ang II (angiotensin II) or potassium-induced changes.

Conclusions: H295R-S2 α7-5HT3-R cells are a new model reproducing the major features of cells harboring KCNJ5 mutations. Increased expression of CYP11B2 and stimulation of the mineralocorticoid biosynthesis pathway are associated with a decrease of cell proliferation and an increase of apoptosis, indicating that additional events may be required for the development of aldosterone-producing adenomas.

Keywords: adrenal cortex; calcium signaling; cell proliferation; mutation; potassium channels; sodium.