title: Identification of electric-field-dependent steps in the Na(+),K(+)-pump cycle.
abstract: The charge-transporting activity of the Na(+),K(+)-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme's reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na(+),K(+)-ATPase's transport sites in competition with Na(+) and K(+), but is not occluded within the protein. We find that only the occludable ions Na(+), K(+), Rb(+), and Cs(+) cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na(+) or K(+) binding in a high field access channel is a major electrogenic reaction of the Na(+),K(+)-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.
authors.affiliation: School of Chemistry, University of Sydney, Sydney, Australia.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Mares
authors.affiliation: Department of Cardiology, Royal North Shore Hospital, Sydney, Australia; Kolling Institute, University of Sydney, Sydney, Australia.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Garcia
authors.affiliation: Department of Cardiology, Royal North Shore Hospital, Sydney, Australia; Kolling Institute, University of Sydney, Sydney, Australia.
authors.forename: Helge H
authors.initials: HH
authors.lastname: Rasmussen
authors.affiliation: Department of Biomedicine, University of Aarhus, Aarhus, Denmark.
authors.forename: Flemming
authors.initials: F
authors.lastname: Cornelius
authors.affiliation: Department of Biomedicine, University of Aarhus, Aarhus, Denmark.
authors.forename: Yasser A
authors.initials: YA
authors.lastname: Mahmmoud
authors.affiliation: Department of Pharmacology and Physiology, Rutgers University, Newark, New Jersey.
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Berlin
authors.affiliation: School of Applied Science and Health Innovations Research Institute, REMIT University, Melbourne, Australia.
authors.forename: Bogdan
authors.initials: B
authors.lastname: Lev
authors.affiliation: School of Applied Science and Health Innovations Research Institute, REMIT University, Melbourne, Australia.
authors.forename: Toby W
authors.initials: TW
authors.lastname: Allen
authors.affiliation: School of Chemistry, University of Sydney, Sydney, Australia. Electronic address: ronald.clarke@sydney.edu.au.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Clarke
mesh_terms: D000818:Animals; D055724:Electrophysiological Phenomena / Q000187:drug effects; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D053835:Unilamellar Liposomes / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D000644:Quaternary Ammonium Compounds; D053835:Unilamellar Liposomes; D000254:Sodium-Potassium-Exchanging ATPase; C556155:benzyltriethylammonium