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

The Na,K-ATPase α ₂ subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca ²⁺ , whereas α ₁ 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 ₂ 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 ₂ is preferentially expressed with β ₂ in T-tubules of cardiac myocytes, forming α ₂ β ₂ heterodimers. We have expressed human α ₁ β ₁ , α ₂ β ₁ , α ₂ β ₂ , and α ₂ β ₃ in Pichia pastoris, purified the complexes, and compared their functional properties. α ₂ β ₂ and α ₂ β ₃ differ significantly from both α ₂ β ₁ and α ₁ β ₁ 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 E ₁ P-E ₂ P conformational equilibrium toward E ₁ 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 ₂ β ₂ and α ₂ β ₃ over α ₁ β 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 α ₂ β ₂ could allow an acute response to raised ambient K ⁺ concentrations in physiological conditions and explain the importance of α ₂ β ₂ for cardiac muscle contractility. The high sensitivity of α ₂ β ₂ to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α ₂ β ₂ -selective digoxin derivatives for reducing cardiotoxicity.
(© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)