The role of Na,K-ATPase alpha subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties of the Na,K-pump.
The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K-ATPase alpha 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 alpha1 subunit of sheep Na,K-ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+ (37 degrees 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 Glu779 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.
Peluffo
R D
RD
Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
Argüello
J M
JM
Berlin
J R
JR
D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D017354:Point Mutation / Q000502:physiology*; D011188:Potassium / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*
D018698:Glutamic Acid; D012694:Serine; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium