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

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/S α2R/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/S α2R/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.