Phospholemman (FXYD1) Raises the Affinity of the Human α1β1 Isoform of Na,K-ATPase for Na Ions

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2011
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Katz, Adriana
Mishra, Neeraj Kumar
Belogus, Talya
Lifshitz, Yael
Garty, Haim
Karlish, Steven J. D.
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Biochemistry ; 50 (2011), 18. - pp. 3736-3748. - ISSN 0006-2960. - eISSN 1520-4995
Abstract
The human α1/His10-β1 isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-β-maltoside and purified by metal chelate chromatography. The α1β1 complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α1/His10-β1 complex and stabilizes it in an active mode. The functional properties of the α1/His10-β1 and α1/His10-β1/FXYD1 complexes have been investigated by fluorescence methods. The electrochromic dye RH421 which monitors binding to and release of ions from the binding sites has been applied in equilibrium titration experiments to determine ion binding affinities and revealed that FXYD1 induces an ~30% increase of the Na+-binding affinity in both the E1 and P-E2 conformations. By contrast, it does not affect the affinities for K+ and Rb+ ions. Phosphorylation induced partial reactions of the enzyme have been studied as backdoor phosphorylation by inorganic phosphate and in kinetic experiments with caged ATP in order to evaluate the ATP-binding affinity and the time constant of the conformational transition, Na3E1-P → P-E2Na3. No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb2E1→ E2(Rb2) and E2(Rb2) → Na3E1, investigated with fluorescein-labeled Na,K-ATPase showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiment is that FXYD1 raises the binding affinity of α1β1 for Na ions, presumably at the third Na-selective binding site. In whole cell expression studies FXYD1 reduces the apparent affinity for Na ions. Possible reasons for the difference from this study using the purified recombinant Na,K-ATPase are discussed.
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ISO 690CIRRI, Erica, Adriana KATZ, Neeraj Kumar MISHRA, Talya BELOGUS, Yael LIFSHITZ, Haim GARTY, Steven J. D. KARLISH, Hans-Jürgen APELL, 2011. Phospholemman (FXYD1) Raises the Affinity of the Human α1β1 Isoform of Na,K-ATPase for Na Ions. In: Biochemistry. 50(18), pp. 3736-3748. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi2001714
BibTex
@article{Cirri2011-05-10Phosp-14178,
  year={2011},
  doi={10.1021/bi2001714},
  title={Phospholemman (FXYD1) Raises the Affinity of the Human α1β1 Isoform of Na,K-ATPase for Na Ions},
  number={18},
  volume={50},
  issn={0006-2960},
  journal={Biochemistry},
  pages={3736--3748},
  author={Cirri, Erica and Katz, Adriana and Mishra, Neeraj Kumar and Belogus, Talya and Lifshitz, Yael and Garty, Haim and Karlish, Steven J. D. and Apell, Hans-Jürgen}
}
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