Modulation of calcium signalling on demand to decipher the molecular mechanisms of primary aldosteronism

Background. Primary aldosteronism is the most common form of secondary hypertension. The most frequent genetic cause of aldosterone producing adenoma (APA) are 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 signalling, the major trigger for aldosterone biosynthesis. Methods. To investigate how KCNJ5 mutations lead to the development of APA, 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 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 receptor in H295R-S2 cells was obtained. Increased sodium entry through the α7-5HT3 receptor upon stimulation with uPSEM-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 angiotensin II or potassium induced changes. Conclusion. H295R-S2 α7-5HT3 cells are a new model reproducing the major features of cells harbouring 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 APA. To investigate the effect of modulating intracellular Na+ concentration in an adrenocrotical cell model (H295R-S2 cells) expressing the chimeric α7-5HT3 receptor, which allow Na+ entry into the cells when treated with a synthetic molecule called PSAM. The cells were treated 8h or 24h with 10-7M of PSEM and we perfroemd gene expression profiling by RNA-sequencing.