title: The voltage-sensitive dye RH421 detects a Na+,K+-ATPase conformational change at the membrane surface.
abstract: RH421 is a voltage-sensitive fluorescent styrylpyridinium dye which has often been used to probe the kinetics of Na+,K+-ATPase partial reactions. The origin of the dye's response has up to now been unclear. Here we show that RH421 responds to phosphorylation of the Na+,K+-ATPase by inorganic phosphate with a fluorescence increase. Analysis of the kinetics of the fluorescence response indicates that the probe is not detecting phosphorylation itself but rather a shift in the protein's E1/E2 conformational equilibrium induced by preferential phosphate binding to and phosphorylation of enzyme in the E2 conformation. Molecular dynamics simulations of crystal structures in lipid bilayers indicate some change in the protein's hydrophobic thickness during the E1-E2 transition, which may influence the dye response. However, the transition is known to involve significant rearrangement of the protein's highly charged lysine-rich cytoplasmic N-terminal sequence. Using poly-l-lysine as a model of the N-terminus, we show that an analogous response of RH421 to the E1→E2P conformational change is produced by poly-l-lysine binding to the surface of the Na+,K+-ATPase-containing membrane fragments. Thus, it seems that the prime origin of the RH421 fluorescence response is a change in the interaction of the protein's N-terminus with the surrounding membrane. Quantum mechanical calculations of the dye's visible absorption spectrum give further support to this conclusion. The results obtained indicate that membrane binding and release of the N-terminus of the Na+,K+-ATPase α-subunit are intimately involved in the protein's catalytic cycle and could represent an effective site of regulation.
authors.affiliation: School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Garcia
authors.affiliation: Department of Physics and Astronomy, University of North Carolina at Greensboro, Greensboro, NC 27402-6170, USA.
authors.forename: Promod R
authors.initials: PR
authors.lastname: Pratap
authors.affiliation: Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, D-60438 Frankfurt am Main, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Lüpfert
authors.affiliation: Department of Biomedicine, University of Aarhus, DK-8000, Aarhus C, Denmark.
authors.forename: Flemming
authors.initials: F
authors.lastname: Cornelius
authors.affiliation: CEISAM Lab, UMR CNRS 6230, Science and Technology Faculty, BP92208, University of Nantes, 44322 Nantes Cedex 3, France; Institut Universitaire de France, 1, Rue Descartes, 75231, Paris, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Jacquemin
authors.affiliation: School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne, Australia.
authors.forename: Bogdan
authors.initials: B
authors.lastname: Lev
authors.affiliation: School of Applied Science and Health Innovations Research Institute, RMIT University, Melbourne, Australia; Department of Chemistry, University of California, Davis, CA 95616, USA.
authors.forename: Toby W
authors.initials: TW
authors.lastname: Allen
authors.affiliation: School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia. Electronic address: ronald.clarke@sydney.edu.au.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Clarke
mesh_terms: D005456:Fluorescent Dyes; D008051:Lipid Bilayers / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D010766:Phosphorylation; D011107:Polylysine / Q000737:chemistry; D011487:Protein Conformation; D020539:Sequence Analysis, Protein; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D011107:Polylysine; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Molecular dynamics; N-terminus; Na(+),K(+)-ATPase; Phosphorylation; RH421 fluorescence; Stopped-flow kinetics