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 α&sub1; and α&sub2; isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (α&sub1;S/Sα&sub2;R/R and skα&sub2;-/-). 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&sub2; isozymes for K⁺ is significantly lower than α&sub1;. When measured in intact fibers of WT and α&sub1;S/Sα&sub2;R/R mice in the presence of 10 µM ouabain; the K1/2,K of α&sub1; and α&sub2; 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.