The role of Na,K-ATPase alpha subunit Serine 775 and Glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties ofthe Na,K-pump

The roles of Ser775 and Glu779, two amino acids in the putative fifth trans membrane segment of the Na,K-ATPase alpha subunit, in determining the volta ge and extracellular K+ (K-o(+)) dependence of enzyme-mediated ion transpor t, were examined in this study. HeLa cells expressing the alpha 1 subunit o f sheep Na,K-ATPase were voltage clamped via patch electrodes containing so lutions with 115 mM Na+ (37 degrees C). Na,K-pump current produced by the o uabain-resistant control enzyme (RD), containing amino acid substitutions G ln111Arg and Asn122Asp, displayed a membrane potential and K-o(+) dependenc e similar to wild-type Na,K-ATPase during superfusion with 0 and 148 mM Na-containing salt solutions. Additional substitution of alanine at Ser775 or Glu779 produced 155- and 15-fold increases, respectively, in the K-o(+) co ncentration that half-maximally activated Na,K-pump current at 0 mV in extr acellular Na+-free solutions. However, the voltage dependence of Na,K-pump current was unchanged in RD and alanine-substituted enzymes. Thus, large ch anges in apparent K-o(+) affinity could be produced by mutations in the fif th transmembrane segment of the Na,K-ATPase with little effect on voltage-d ependent properties of K+ transport. One interpretation of these results is that protein structures responsible for the kinetics of K-o(+) binding and /or occlusion may be distinct, at least in part, from those that are respon sible for the voltage dependence of K-o(+) binding to the Na,K-ATPase.