title: Substitution of glutamic 779 with alanine in the Na,K-ATPase alpha subunit removes voltage dependence of ion transport.
abstract: The effects of changing Glu-779, located in the fifth transmembrane segment of the Na,K-ATPase alpha subunit, on the phosphorylation characteristics and ion transport properties of the enzyme were investigated. HeLa cells were transfected with cDNA coding the E779A substitution in an ouabain-resistant sheep alpha1 subunit (RD). Steady state phosphorylation stimulated by Na+ concentrations less than 20 mM or by imidazole were similar for RD and E779A enzymes, an indication that phosphorylation and Na+ occlusion were not altered by this mutation. With E779A enzyme, higher Na+ concentrations reduced the level of phosphoenzyme and stimulated Na-ATPase activity in the absence of K+. These effects were a consequence of Na+ increasing the rate of protein dephosphorylation. In voltage-clamped HeLa cells expressing E779A enzyme, a prominent electrogenic Na+-Na+ exchange was observed in the absence of extracellular K+. Thus, increased Na-ATPase activity and Na+-dependent dephosphorylation result from Na+ acting as a K+ congener with low affinity at extracellular binding sites. These data suggest that E779A does not directly participate in ion binding but does affect the connection between extracellular ion binding and intracellular enzyme dephosphorylation. In cells expressing control RD enzyme, Na,K-pump current was dependent on membrane potential and extracellular K+ concentration. However, Na,K-pump current in cells expressing E779A enzyme was voltage independent at all extracellular K+ tested. These results indicate that Glu-779 may be part of the access channel determining the voltage dependence of ion transport by the Na, K-ATPase.
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0524, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Argüello
authors.forename: R D
authors.initials: RD
authors.lastname: Peluffo
authors.forename: J
authors.initials: J
authors.lastname: Feng
authors.forename: J B
authors.initials: JB
authors.lastname: Lingrel
authors.forename: J R
authors.initials: JR
authors.lastname: Berlin
mesh_terms: D000409:Alanine / Q000235:genetics*; D000818:Animals; D018698:Glutamic Acid / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating; D017136:Ion Transport; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D018698:Glutamic Acid; D000254:Sodium-Potassium-Exchanging ATPase; D000409:Alanine