Inward currents through somatic Na/K‐ATPase α1‐subunit mutants are not required for hyperaldosteronism

Hyperaldosteronism (HA), the most common cause of secondary hypertension, is often initiated by an aldosterone‐producing adenoma within the zona glomerulosa of the adrenal cortex. Somatic mutations to several genes were identified in these adenomas, including to the Na/K‐ATPase (NKA) α1‐subunit. The membrane potential (V m ) of cells harboring these NKA‐α1 mutations is depolarized, which is believed to underlie the pathophysiological aspects of HA. At physiological Na + and K + concentrations, wild type NKA activity generates outward current due to the export of 3 Na + and import of 2 K + per cycle against their gradients. Under similar conditions, all HA‐associated NKA mutants evaluated thus far have dramatically reduced activity and an inward current, mediated by Na + or H + , proposed to be a ‘gain‐of‐function’ required for V m depolarization. The outward current of wild type NKA typically contributes minimally to V m . Thus, for the inward current through HA‐associated NKA mutants to significantly depolarize V m , its amplitude is expected to be significantly larger than the amplitude of wild type NKA outward current. Assuming similar expression levels, the transport rate of inward current through NKA mutant pumps should be larger than the pumping turnover rate of a single wild type NKA. To calculate the turnover rate of inward current through human NKA mutants, we utilized two‐electrode voltage clamp (TEVC) of Xenopus oocytes heterologously expressing HA‐associated mutants to measure the ouabain‐sensitive inward current amplitude in the presence of Na + , and 3 H‐ouabain binding assays on the same oocyte. As ouabain binds to NKA at a 1:1 ratio, its quantity is equivalent to the number of pumps in the oocyte's plasma membrane. The ratio of current at −50 mV (a physiologically‐relevant voltage) to the moles of ouabain bound to the oocyte yielded inward current turnover rates of 22.0 ± 1.8 s −1 for L104R, 417.3 ± 29.4 s −1 for delF100‐L104, 60.7 ± 5.8 s −1 for V332G, and 19.1 ± 1.4 s −1 for EETA963S, while turnover rate of wild type K + ‐induced pump current was 32.0 ± 1.5 s −1 . This indicates that the inward current of several NKA mutants may not suffice to significantly depolarize V m , and that the pathophysiological effects may simply reflect lack of function. Furthermore, oocytes expressing G99R, a mutant recently associated with HA, lacked inward current under physiological conditions, instead presenting K + ‐induced outward pump current. TEVC measurements of extracellular K + ‐induced current in G99R revealed a ~3‐fold reduction of K + apparent affinity compared to wild type, while measurements of intracellular Na + ‐dependent, ATP‐activated currents in giant inside‐out patches showed a ~3‐fold reduction of Na + apparent affinity, demonstrating impaired function under physiological conditions. These results suggest that lack of function is the main mechanism leading to HA in all mutants. Support or Funding Information Supported by NSF MCB‐1515434