TRANSMEMBRANE POTENTIALS IN CELLS - A DIS-C-3(3) ASSAY FOR RELATIVE POTENTIALS AS AN INDICATOR OF REAL CHANGES

The mechanism by which the fluorescent cationic dye diS-C-3(3) reports on cellular transmembrane potential has been investigated in murine h aemopoietic cells. Due to the large molar absorbance of diS-C-3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5.10(-8) to 2.10(-7) M). In con trast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescenc e intensity of diS-C-3(3) increases when the dye accumulates in the ce lls. The method of synchronous emission spectroscopy was used to resol ve the intracellular and extracellular components of the diS-C-3(3) fl uorescence of suspensions of labelled cells. In comparing changes in t hese signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the pr esence of valinomycin, the logarithm of the ratio of intensities of th ese two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric an alysis, the logarithm of the mean population signal similarly being fo und to provide a good linear measure of the transmembrane potential. T he conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and th eir validity with respect to the possibly contentious role of mitochon dria in such measurements examined in particular. The use of the metho d in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane p otential between different physiological states, the possible extensio n to determinations of absolute differences in potential between diffe rent cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these p artially complementary measurements, are discussed.