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

The Na, K-ATPase alpha(2) subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas alpha(1) has a more conventional role of maintaining ion homeostasis. The beta subunit differentially regulates maturation, trafficking, and activity of alpha-beta heterodimers. It is not known whether the distinct role of alpha(2) in the heart is related to selective assembly with a particular one of the three beta isoforms. We show here by immunofluorescence and co-immunoprecipitation that alpha(2) is preferentially expressed with beta(2) in T-tubules of cardiac myocytes, forming alpha(2)beta(2) heterodimers. We have expressed human alpha(1)beta(1), alpha(2)beta(1), alpha(2)beta(2), and alpha(2)beta(3) in Pichia pastoris, purified the complexes, and compared their functional properties. alpha(2)beta(2) and alpha(2)beta(3) differ significantly from both alpha(2)beta(1) and alpha(1)beta(1) in having a higher K0.5K+ and lower K0.5Na+ for activating Na, K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of alpha(2)beta(2) and alpha(2)beta(3) over alpha(1)beta(1) (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 alpha(2)beta(2) could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of alpha(2)beta(2) for cardiac muscle contractility. The high sensitivity of alpha(2)beta(2) to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of alpha(2)beta(2)-selective digoxin derivatives for reducing cardiotoxicity.