K(+) and Rb(+) Affinities of the Na,K-ATPase α(1) and α(2) 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 α(1) and α(2) isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (α(1)(S/S)α(2)(R/R) and skα(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 α(2) isozymes for K(+) is significantly lower than α(1). When measured in intact fibers of WT and α(1)(S/S)α(2)(R/R) mice in the presence of 10 µM ouabain; the K(1/2,K) of α(1) and α(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.