Abstract

The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, 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 α2β2 heterodimers. We have expressed human α1β1, α2β1, α2β2, and α2β3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2β2 and α2β3 differ significantly from both α2β1 and α1β1 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 α2β2 and α2β3 over α1β1 (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 α2β2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2β2 for cardiac muscle contractility. The high sensitivity of α2β2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2β2-selective digoxin derivatives for reducing cardiotoxicity.

Highlights

  • IntroductionSubunit differentially regulates maturation, trafficking, and activity of ␣-␤ heterodimers

  • From the ‡Department of Biomolecular Sciences and ¶Israel National Centre for Personalized Medicine, Weizmann Institute of Science, Rehovoth 7610001, Israel, the §§School of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel, the §Department of Physiology, School of Medicine, UCLA and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073, the ʈDepartment of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109, the **Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607, and the ‡‡Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

  • 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

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Summary

Introduction

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. The Na,K-ATPase plays a key role in cardiac muscle contractility by regulating the cytosolic Ca2ϩ concentration, via the Naϩ/Ca2ϩ exchanger, and the excitation-contraction coupling in cardiac myocytes [1]. Measurements of the Na,K-ATPase function of isoforms in cardiac myocytes suggest that the ␣2 subunit is more concentrated in T-tubular membranes than in the external sarcolemma, whereas the ␣1 subunit is distributed in the plasma membrane [11, 15, 16].

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