Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart: DISTINCT FUNCTIONAL PROPERTIES AND ISOFORM-SELECTIVE INHIBITORS

The Na,K-ATPase α subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca , whereas α has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α is preferentially expressed with β in T-tubules of cardiac myocytes, forming α β heterodimers. We have expressed human α β , α β , α β , and α β in Pichia pastoris, purified the complexes, and compared their functional properties. α β and α β differ significantly from both α β and α β in having a higher K K and lower K Na for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K and shift of the E P-E P conformational equilibrium toward E P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α β and α β over α β (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K affinity of α β could allow an acute response to raised ambient K concentrations in physiological conditions and explain the importance of α β for cardiac muscle contractility. The high sensitivity of α β to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α β -selective digoxin derivatives for reducing cardiotoxicity.