Selective assembly of Na,K-ATPase α₂ β₂ Heterodimers in the Heart: Distinct functional properties and isoform-selective inhibitors

The Na,K-ATPase α₂ subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca ²⁺ , whereas α₁ 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 α₂ 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 α₂ is preferentially expressed with β₂ in T-tubules of cardiac myocytes, forming α₂ β₂ heterodimers. We have expressed human α₁ β₁ , α₂ β₁ , α₂ β₂ , and α₂ β₃ in Pichia pastoris, purified the complexes, and compared their functional properties. α₂ β₂ and α₂ β₃ differ significantly from both α₂ β₁ and α₁ β₁ in having a higher K_0.5 K⁺ and lower K_0.5 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 E₁ P-E₂ P conformational equilibrium toward E1 P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α₂ β₂ and α₂ β₃ over α₁ β₁ (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 α₂ β₂ could allow an acute response to raised ambient K⁺ concentrations in physiological conditions and explain the importance of α₂ β₂ for cardiac muscle contractility. The high sensitivity of α₂ β₂ to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α₂ β₂ -selective digoxin derivatives for reducing cardiotoxicity.