Functional consequences of a posttransfection mutation in the H2-H3 cytoplasmic loop of the alpha subunit of Na,K-ATPase

During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G(950) to, A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and alpha 1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and alpha 1 have similar apparent affinities for cytoplasmic Na+ and extracellular K+. In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K+ deocclusion pathway of the reaction is rate-limiting. At 1 mu M ATP K+ inhibits Na+-ATPase of alpha 1, but activates Na+-ATPase of E233K, This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E(1)) from K+-occluded enzyme (E(2)(K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of V-max to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than alpha 1. These distinct kinetics of E233K suggest a shift in its E(1)/E(2) conformational equilibrium toward E(1). Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu(233) --> LyS mutation.