Selective Assembly of Na,K-ATPase ?2?2 Heterodimers in the Heart: DISTINCT FUNCTIONAL PROPERTIES AND ISOFORM-SELECTIVE INHIBITORS.

ABSTRACT: The Na,K-ATPase ?2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas ?1 has a more conventional role of maintaining ion homeostasis. The ? subunit differentially regulates maturation, trafficking, and activity of ?-? heterodimers. It is not known whether the distinct role of ?2 in the heart is related to selective assembly with a particular one of the three ? isoforms. We show here by immunofluorescence and co-immunoprecipitation that ?2 is preferentially expressed with ?2 in T-tubules of cardiac myocytes, forming ?2?2 heterodimers. We have expressed human ?1?1, ?2?1, ?2?2, and ?2?3 in Pichia pastoris, purified the complexes, and compared their functional properties. ?2?2 and ?2?3 differ significantly from both ?2?1 and ?1?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 ?2?2 and ?2?3 over ?1?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 ?2?2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of ?2?2 for cardiac muscle contractility. The high sensitivity of ?2?2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of ?2?2-selective digoxin derivatives for reducing cardiotoxicity.

SUBMITTER: Habeck M 

PROVIDER: S-EPMC5087734 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature