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 αβ heterodimers. We have expressed human αβ, αβ, αβ, and αβ in Pichia pastoris, purified the complexes, and compared their functional properties. αβ and αβ differ significantly from both αβ and αβ in having a higher KK and lower KNa for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K and shift of the EP-EP conformational equilibrium toward EP. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of αβ and αβ over αβ (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 αβ could allow an acute response to raised ambient K concentrations in physiological conditions and explain the importance of αβ for cardiac muscle contractility. The high sensitivity of αβ to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of αβ-selective digoxin derivatives for reducing cardiotoxicity.