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Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase

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MISHRA, Neeraj Kumar, Michael HABECK, Corinna KIRCHNER, Haim HAVIV, Yoav PELEG, Miriam EISENSTEIN, Hans Jürgen APELL, Steven J. D. KARLISH, 2015. Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase. In: The Journal of Biological Chemistry. 290(48), pp. 28746-28759. ISSN 0021-9258. eISSN 1083-351X

@article{Mishra2015Molec-33249, title={Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase}, year={2015}, doi={10.1074/jbc.M115.687913}, number={48}, volume={290}, issn={0021-9258}, journal={The Journal of Biological Chemistry}, pages={28746--28759}, author={Mishra, Neeraj Kumar and Habeck, Michael and Kirchner, Corinna and Haviv, Haim and Peleg, Yoav and Eisenstein, Miriam and Apell, Hans Jürgen and Karlish, Steven J. D.} }

Mishra, Neeraj Kumar Karlish, Steven J. D. Kirchner, Corinna Mishra, Neeraj Kumar Apell, Hans Jürgen Haviv, Haim Habeck, Michael eng 2015 Apell, Hans Jürgen 2016-03-07T12:16:19Z Eisenstein, Miriam Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants S63E, S68E, and S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control α1β1, FXYD1 reduces Vmax and turnover rate and raises K<sub>0.5</sub>Na. The phosphomimetic mutants reverse these effects and reduce K<sub>0.5</sub>Na below control K<sub>0.5</sub>Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K<sub>0.5</sub>Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E<sub>2</sub>(2K)ATP → E<sub>1</sub>(3Na)ATP but does not affect 3NaE<sub>1</sub>P → E<sub>2</sub>P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60-70) docks between the αN and αP domains in the E<sub>2</sub> conformation, but docking is weaker in E<sub>1</sub> (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na(+)-selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies. Karlish, Steven J. D. Haviv, Haim Peleg, Yoav Habeck, Michael Kirchner, Corinna Peleg, Yoav Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase 2016-03-07T12:16:19Z Eisenstein, Miriam

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