A novel fluorescent technique for direct assessment of membrane potential was tested on suspensions of large unilamellar vesicles (LUV). The method is based on monitoring shifts in the fluorescence maximum, lambda(max), of the redistribution dye diS-C3(3) caused by dye binding to the LUV membrane. A theory describing the behavior of this dye in LUV suspensions was elaborated and tested. The diffusion potentials across the LUV membrane were adjusted by ion gradients in the absence of valinomycin. When using KCl and choline chloride without valinomycin the potential can be set as high as -70 mV. These potentials exhibited long-term stability and the theory allowed to determine the upper limits of membrane permeabilities for Cl-, choline cations, protons and hydroxyls relative to the K+ permeability. The absolute values of membrane potential set by ion gradients were calibrated using valinomycin. The monitoring of the lambda(max) shift permitted us to show real-time changes in membrane potential, since addition of valinomycin to the LUV was followed by an immediate depolarization. The setting of the potential and the dye re-equilibration after valinomycin addition took place within a second.

Department of Biophysics Institute of Physics Charles University Prague Czech Republic

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