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Int. J. Mol. Sci. 2018, 19(9), 2725; https://doi.org/10.3390/ijms19092725

K+ and Rb+ Affinities of the Na,K-ATPase α1 and α2 Isozymes: An Application of ICP-MS for Quantification of Na+ Pump Kinetics in Myofibers

1
Department of Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, OH 45229, USA
2
Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA
3
Agilent Technologies Metallomics Center of the Americas, University of Cincinnati, Cincinnati, OH 45221, USA
4
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, OH 45229, USA
*
Author to whom correspondence should be addressed.
Received: 23 August 2018 / Revised: 10 September 2018 / Accepted: 10 September 2018 / Published: 12 September 2018
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Abstract

The potassium affinities of Na,K-ATPase isozymes are important determinants of their physiological roles in skeletal muscle. This study measured the apparent K+ and Rb+ affinities of the Na,K-ATPase α1 and α2 isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (α1S/Sα2R/R and skα2−/−). It also validates a new method to quantify cations in intact, dissociated myofibers, using inductively coupled plasma mass spectrometry (ICP-MS). Our findings were that: (1) The extracellular substrate sites of Na,K-ATPase bind Rb+ and K+ with comparable apparent affinities; however; turnover rate is reduced when Rb+ is the transported ion; (2) The rate of Rb+ uptake by the Na,K-ATPase is not constant but declines with a half-time of approximately 1.5 min; (3) The apparent K+ affinity of the α2 isozymes for K+ is significantly lower than α1. When measured in intact fibers of WT and α1S/Sα2R/R mice in the presence of 10 µM ouabain; the K1/2,K of α1 and α2 isozymes are 1.3 and 4 mM, respectively. Collectively, these results validate the single fiber model for studies of Na,K-ATPase transport and kinetic constants, and they imply the existence of mechanisms that dynamically limit pump activity during periods of active transport. View Full-Text
Keywords: Na,K-ATPase; isozymes; skeletal muscle; potassium; rubidium; myofiber; affinity; ICP-MS Na,K-ATPase; isozymes; skeletal muscle; potassium; rubidium; myofiber; affinity; ICP-MS
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Hakimjavadi, H.; Stiner, C.A.; Radzyukevich, T.L.; Lingrel, J.B.; Norman, N.; Landero Figueroa, J.A.; Heiny, J.A. K+ and Rb+ Affinities of the Na,K-ATPase α1 and α2 Isozymes: An Application of ICP-MS for Quantification of Na+ Pump Kinetics in Myofibers. Int. J. Mol. Sci. 2018, 19, 2725.

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