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  • Title: Oxonol VI as an optical indicator for membrane potentials in lipid vesicles.
    Author: Apell HJ, Bersch B.
    Journal: Biochim Biophys Acta; 1987 Oct 16; 903(3):480-94. PubMed ID: 2444259.
    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.
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