Dataset Information

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

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Publications

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

Habeck Michael M Tokhtaeva Elmira E Nadav Yotam Y Ben Zeev Efrat E Ferris Sean P SP Kaufman Randal J RJ Bab-Dinitz Elizabeta E Kaplan Jack H JH Dada Laura A LA Farfel Zvi Z Tal Daniel M DM Katz Adriana A Sachs George G Vagin Olga O Karlish Steven J D SJ

The Journal of biological chemistry 20160913 44

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 an ...[more]

PMID: 27624940

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Project description:Due to the critical role of cochlear ion channels for hearing, the focus of the present study was to examine age-related changes of Na, K-ATPase (NKA) subunits in the lateral wall of mouse cochlea. We combined qRT-PCR, western blot and immunocytochemistry methodologies in order to determine gene and protein expression levels in the lateral wall of young and aged CBA/CaJ mice. Of the seven NKA subunits, only the mRNA expressions of α1, β1 and β2 subunit isoforms were detected in the lateral wall of CBA/CaJ mice. Aging was accompanied by dys-regulation of gene and protein expression of all three subunits detected. Hematoxylin and eosin (H&E) staining revealed atrophy of the cochlear stria vascularis (SV). The SV atrophy rate (20%) was much less than the ∼80% decline in expression of all three NKA isoforms, indicating lateral wall atrophy and NKA dys-regulation are independent factors and that there is a combination of changes involving the morphology of SV and NKA expression in the aging cochlea which may concomitantly affect cochlear function. Immunoprecipitation assays showed that the α1-β1 heterodimer is the selective preferential heterodimer over the α1-β2 heterodimer in cochlea lateral wall. Interestingly, in vitro pathway experiments utilizing cultured mouse cochlear marginal cells from the SV (SV-K1 cells) indicated that decreased mRNA and protein expressions of α1, β1 and β2 subunit isoforms are not associated with reduction of NKA activity following in vitro application of ouabain, but ouabain did disrupt the α1-β1 heterodimer interaction. Lastly, the association between the α1 and β1 subunit isoforms was present in the cochlear lateral wall of young adult mice, but this interaction could not be detected in old mice. Taken together, these data suggest that in the young adult mouse there is a specific, functional selection and assembly of NKA subunit isoforms in the SV lateral wall, which is disrupted and dys-regulated with age. Interventions for this age-linked ion channel disruption may have the potential to help diagnose, prevent, or treat age-related hearing loss.

Dataset Information

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

Publications

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

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