TY - JOUR
T1 - Selective assembly of Na,K-ATPase α2 β2 Heterodimers in the Heart
T2 - Distinct functional properties and isoform-selective inhibitors
AU - Habeck, Michael
AU - Tokhtaeva, Elmira
AU - Nadav, Yotam
AU - Zeev, Efrat Ben
AU - Ferris, Sean P.
AU - Kaufman, Randal J.
AU - Bab-Dinitz, Elizabeta
AU - Kaplan, Jack H.
AU - Dada, Laura A.
AU - Farfel, Zvi
AU - Tal, Daniel M.
AU - Katz, Adriana
AU - Sachs, George
AU - Vagin, Olga
AU - Karlish, Steven J.D.
N1 - Funding Information:
This work was supported by Israel Science Foundation (789/12) and US-Israel Binational Science Foundation (711993, to S. J. D. K.) and, in part, by National Institutes of Health Grants R37-HL48129 (to L. A. D.), RO1HL113350 (to L. A. D. and O. V.), USVA 2I01BX001006 (to G. S.), and1RO1DK105156-01 (to G. S.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
PY - 2016/10/28
Y1 - 2016/10/28
N2 - 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 K0.5 K+ and lower K0.5 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.
AB - 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 K0.5 K+ and lower K0.5 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.
UR - http://www.scopus.com/inward/record.url?scp=84993949986&partnerID=8YFLogxK
U2 - 10.1074/jbc.M116.751735
DO - 10.1074/jbc.M116.751735
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AN - SCOPUS:84993949986
SN - 0021-9258
VL - 291
SP - 23159
EP - 23174
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 44
ER -