Cellular Pathophysiology of an Adrenal Adenoma-Associated Mutant of the Plasma Membrane Ca2+-ATPase ATP2B3

Abstract

Adrenal aldosterone-producing adenomas (APAs) are a main cause for primary aldosteronism leading to arterial hypertension. Physiologically, aldosterone production in the adrenal gland is stimulated by angiotensin II and high extracellular potassium. These stimuli lead to a depolarization of the plasma membrane and, as a consequence, an increase of intracellular Ca(2+). Mutations of the plasma membrane Ca(2+)-ATPase ATP2B3 have been found in APAs with a prevalence of 0.6%-3.1%. Here, we investigated the effects of the APA-associated ATP2B3(Leu425_Val426del) mutation in adrenocortical NCI-H295R and human embryonic kidney (HEK-293) cells. Ca(2+) measurements revealed a higher basal Ca(2+) level in cells expressing the mutant ATP2B3. This rise in intracellular Ca(2+) was even more pronounced under conditions with high extracellular Ca(2+) pointing to an increased Ca(2+) influx associated with the mutated protein. Furthermore, cells with the mutant ATP2B3 appeared to have a reduced capacity to export Ca(2+) suggesting a loss of the physiological pump function. Surprisingly, expression of the mutant ATP2B3 caused a Na(+)-dependent inward current that strongly depolarized the plasma membrane and compromised the cytosolic cation composition. In parallel to these findings, mRNA expression of the cytochrome P450, family 11, subfamily B, polypeptide 2 (aldosterone synthase) was substantially increased and aldosterone production was enhanced in cells overexpressing mutant ATP2B3. In summary, the APA-associated ATP2B3(Leu425_Val426del) mutant promotes aldosterone production by at least 2 different mechanisms: 1) a reduced Ca(2+) export due to the loss of the physiological pump function; and 2) an increased Ca(2+) influx due to opening of depolarization-activated Ca(2+) channels as well as a possible Ca(2+) leak through the mutated pump.

Of channels and pumps: different ways to boost the aldosterone?

“…In the human adrenal gland, ATP2B1 and ATP2B3 are moderately expressed, whereas ATP2B4 shows the highest expression level, and ATP2B2 is only weakly expressed under control conditions [http://www.humanproteomemap.org, (Tauber et al . )]. Mice adrenal glands exhibit a similar expression pattern, but show the highest expression of Atp2b3.…”

“…, Tauber et al . ). With respect to their role in Ca 2+ extrusion and their high adrenal expression level, there is no doubt about an important function of PMCAs for the Ca 2+ signalling in glomerulosa cells.…”

The Plasma Membrane Calcium ATPasesand Their Role as Major New Players in Human Disease

“…Clinically APAs containing ATP2B3 mutations are associated with higher levels of aldosterone secretion when compared with wild-type APAs (27,186,397), most likely as a result of an observed increase in aldosterone synthase CYP11B2 expression (250,254,396). Recently these associations have been confirmed through the transfection of ATP2B3 carrying the common Leu425_Val426 deletion into adrenocortical NCI-H295R cells, resulting in impaired Ca 2ϩ clearance accompanied by elevated basal Ca 2ϩ , CYP11B2 expression, and aldosterone secretion (370). This highlights a highly specialized function for this isoform in nonneuronal tissue.…”

Somatic and inherited mutations in primary aldosteronism

“…All the mutations identified in ATP2B3 are located in the M4 domain, deletions of at least two amino acids in a specific region between amino acids 425 and 433 , Dutta et al 2014, FernandesRosa et al 2014, Akerstrom et al 2015, Scholl et al 2015a, Zheng et al 2015, a region crucial for the binding of Ca 2+ ions but also close to the catalytic core of the pump (Di Leva et al 2008). Electrophysiological studies have shown that the ATP2B3 p.Leu425_Val426del mutation leads to increased intracellular Ca 2+ concentration in transfected human embryonic kidney (HEK-293) cells due to an increased Ca 2+ influx and reduced capacity to export Ca 2+ , suggesting a loss of the physiological pump function (Tauber et al 2016). In addition to the alteration of Ca 2+ equilibrium, ATP2B3 mutations, like KCNJ5 but to a less extent, lead to Na + and possibly Ca 2+ influx into the cells, contributing to increased intracellular Ca 2+ concentration also through depolarization of the cell membrane and opening of voltage-gated Ca 2+ channel.…”

“…In addition to the alteration of Ca 2+ equilibrium, ATP2B3 mutations, like KCNJ5 but to a less extent, lead to Na + and possibly Ca 2+ influx into the cells, contributing to increased intracellular Ca 2+ concentration also through depolarization of the cell membrane and opening of voltage-gated Ca 2+ channel. These alterations are associated to increased aldosterone production and CYP11B2 expression in adrenocortical cells (Tauber et al 2016).…”