Quantitative measurement of cationic fluxes, selectivity and membrane potential using liposomes multilabelled with fluorescent probes.

Liposomes of egg PC/PG (8:2, mol/mol) were multilabelled with PBFI, pyranine and oxonol VI, fluorescent probes for, respectively, K+, H+ and membrane potential. Monitoring fluorescence with a multichannel photoncounting spectrofluorometer during K+ filling experiments allowed to measure K+ influx, the associated H+ efflux and the membrane potential, continuously and simultaneously. The proton net efflux quantitatively mirrored the K+ net influx. The rate of the K+/H+ exchange diminished progressively as a quasi-equilibrium was reached for both K+ and H+. In the presence of valinomycin, the measured membrane potential during the K+ filling actually corresponded to the Nernst potential calculated from the observed K+ gradient. In the absence of valinomycin, it corresponded to the Nernst potential calculated from the observed H+ gradient. In the latter case, the permeability coefficient of liposomes to K+, calculated from the Goldman-Hodgkin-Katz relation, was 6.10(-13) m s-1. The selectivity sequence for alkali cations of liposomes was determined from the measured H+ efflux associated to the influx of the different cations. The selectivity sequence corresponded to the series VI of Eisenman, suggesting interaction of the cation with an anionic field of intermediate strength.