title: Rescue of Na+ affinity in aspartate 928 mutants of Na+,K+-ATPase by secondary mutation of glutamate 314.
abstract: The Na(+),K(+)-ATPase binds Na(+) at three transport sites denoted I, II, and III, of which site III is Na(+)-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na(+) affinity in the α1-, α2-, and α3-isoforms of Na(+),K(+)-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na(+)-coordinating residues in site III. Remarkably, the Na(+) affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na(+) binding and does not affect the E1-E2 conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na(+) affinity is likely intrinsic to the Na(+) binding pocket, and the underlying mechanism could be a tightening of Na(+) binding at Na(+) site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na(+),K(+) pump function in intact cells. Rescue of Na(+) affinity and Na(+) and K(+) transport by second-site mutation is unique in the history of Na(+),K(+)-ATPase and points to new possibilities for treatment of neurological patients carrying Na(+),K(+)-ATPase mutations.
authors.affiliation: From the Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Building 1160, DK-8000 Aarhus C, Denmark.
authors.forename: Rikke
authors.initials: R
authors.lastname: Holm
authors.affiliation: From the Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Building 1160, DK-8000 Aarhus C, Denmark.
authors.forename: Anja P
authors.initials: AP
authors.lastname: Einholm
authors.affiliation: From the Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Building 1160, DK-8000 Aarhus C, Denmark.
authors.forename: Jens P
authors.initials: JP
authors.lastname: Andersen
authors.affiliation: From the Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Building 1160, DK-8000 Aarhus C, Denmark bv@biomed.au.dk.
authors.forename: Bente
authors.initials: B
authors.lastname: Vilsen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D001692:Biological Transport / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013489:Suppression, Genetic
chemical_list: D007527:Isoenzymes; D001224:Aspartic Acid; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium
keywords: Alternating Hemiplegia of Childhood; Membrane Transport; Na+,K+ Pump; Na+/K+-ATPase; Neurological Disease; P-type ATPase; Rapid-onset Dystonia Parkinsonism; Second-site Revertant; Site-directed Mutagenesis; Sodium Transport