title: Oxonol VI as an optical indicator for membrane potentials in lipid vesicles.
abstract: Experiments with large unilamellar dioleoylphosphatidylcholine vesicles were carried out in order to study the effect of membrane potential on the fluorescence of Oxonol VI. A partition equilibrium of dye between membrane and water was found to exist with a partition coefficient gamma identical to c lipid/c water of about 19,000 (at zero voltage). In the presence of an inside-positive membrane potential, the negatively charged dye accumulates in the intravesicular aqueous space according to a Nernst equilibrium. This leads to an increased adsorption of dye to the inner lipid monolayer and to a concomitant increase of fluorescence. The fluorescence change can be calibrated as a function of transmembrane voltage by generating a potassium diffusion potential in the presence of valinomycin. The intrinsic fluorescence of the membrane-bound dye is not affected by voltage; the whole influence of voltage on the fluorescence results from voltage-dependent partitioning of the dye between water and membrane. The voltage dependence of the apparent partition coefficient can be quantitatively described by a three-capacitor model in which the dye is assumed to bind to adsorption planes located on the hydrocarbon side of the membrane/solution interface. Oxonol VI was found to be suitable for detecting changes of membrane potential associated with the activity of the (Na+ + K+)-ATPase in reconstituted vesicles. When ATP is added to the external medium, pump molecules with the ATP-binding side facing outward become activated; this results in a translocation of net positive charge towards the vesicle interior. Under this condition, fluorescence changes corresponding to (inside-positive) potentials of up to 150-200 mV are observed. After the build-up of the membrane potential, a quasi-stationary state is reached in which the pump current is compensated by a back-flow of charge through passive conductance pathways.
mesh_terms: D000327:Adsorption; D004058:Diffusion; D005453:Fluorescence; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007473:Ion Channels / Q000378:metabolism; D007555:Isoxazoles; D008081:Liposomes / Q000378:metabolism; D008564:Membrane Potentials; D010080:Oxazoles; D010713:Phosphatidylcholines; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007473:Ion Channels; D007555:Isoxazoles; D008081:Liposomes; D010080:Oxazoles; D010713:Phosphatidylcholines; D012996:Solutions; D014634:Valinomycin; C031184:oxonol VI; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; C017251:1,2-oleoylphosphatidylcholine; D011188:Potassium