Functional Analysis of Plasma Membrane Ca2+ATPase 3 and Its Primary Aldosteronism-Associated Mutations

Primary aldosteronism (PA) is the most common form of secondary hypertension and can lead to higher rates of cardiovascular complications and death than essential hypertension. Aldosterone-producing adenomas (APAs) are the most frequent cause of PA. Most APAs have mutations in plasma membrane ion-transport proteins with 0.6%-9% carrying a mutation in the Plasma Membrane Ca2+ ATPase 3 (PMCA3). We have developed a Xenopus oocyte expression system to measure the function of PMCA3 wildtype (PMCA3WT) and an APA associated deletion mutant (PMCA3L425_V426del.) with minimal endogenous contamination. We measured ATPase activity at 37 °C in plasma membrane preparations from oocytes expressing PMCA3. Ca2+-dependent ATPase was observed in preparations from oocytes expressing PMCA3WT, with a [Ca2+] dependence well described with a rectangular hyperbola (K0.5 = 25.6 ± 5.1 μM) (triplicates from three independent membrane preparations). In contrast, membrane preparations from PMCA3L425_V426del. expressing oocytes lacked Ca2+-dependent ATPase activity, indicating loss of enzymatic activity in the mutant. We used two-electrode voltage clamp electrophysiology to evaluate the presence of PMCA3-variant mediated currents. At resting intracellular [Ca2+], PMCA3WT-injected oocytes had currents indistinguishable from those in uninjected oocytes, at all voltages. When bathed by extracellular 125 mM Na+, PMCA3L425_V426del.-injected oocytes presented inward currents at negative membrane potentials and outward currents at positive ones, consistent with induction of an aberrant channel-like current. The currents were outwardly directed upon substitution of external Na+ with N-methyl D-glucamine+ and were insensitive to changes in extracellular [Ca2+]. These results indicate that PMCA3L425_V426del. causes hyperaldosteronism due to the concomitant loss of active Ca2+ transport and induction of a depolarizing Na+-mediated current. Current experiments are evaluating the ATPase and physiological characteristics of other PA-associated PMCA3 mutations. Funded by NSF-MCB 2003251.