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 spo ntaneous 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(9 50) to, A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and alpha 1 cDNAs were transfecte d into HeLa cells and their kinetic behavior was compared. Transport s tudies carried out under physiological conditions with intact cells in dicate that the E233K mutant and alpha 1 have similar apparent affinit ies for cytoplasmic Na+ and extracellular K+. In contrast, distinct ki netic properties are observed when ATPase activity is assayed under co nditions (low ATP concentration) in which the K+ deocclusion pathway o f 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 behavi or 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 aff inity 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 lowe r catalytic turnover than alpha 1. These distinct kinetics of E233K su ggest a shift in its E(1)/E(2) conformational equilibrium toward E(1). Furthermore, the importance of the H2-H3 loop in coupling conformatio nal changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu(233) --> LyS mutation.