The role of Na,K-ATPase α subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties of the Na,K-pump

Abstract

The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K-ATPase α subunit, in determining the voltage and extracellular K+ (K(o)+) dependence of enzyme-mediated ion transport, were examined in this study. HeLa cells expressing the αl subunit of sheep Na,K-ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+ (37°C). Na,K-pump current produced by the ouabain-resistant control enzyme (RD), containing amino acid substitutions Gln111Arg and Asn122Asp, displayed a membrane potential and K(o)+ dependence 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 GluT79 produced 155-and 15-fold increases, respectively, in the K(o)+ concentration that half-maximally activated Na,K-pump current at 0 mV in extracellular Na+-free solutions. However, the voltage dependence of Na,K- pump current was unchanged in RD and alanine-substituted enzymes. Thus, large changes in apparent K(o)+ affinity could be produced by mutations in the fifth transmembrane segment of the Na,K-ATPase with little effect on voltage- dependent 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 responsible for the voltage dependence of K(o)+ binding to the Na,K-ATPase.