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

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 alpha(1) and alpha(2) isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (alpha(S/S)(1)alpha(R/R)(2) and sk alpha(-/-)(2)). 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 alpha(2) isozymes for K+ is significantly lower than alpha(1). When measured in intact fibers of WT and alpha(S/S)(1)alpha(R/R)(2) mice in the presence of 10 mu M ouabain; the K-1/2,K-K of alpha(1) and alpha(2) 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.