Two mechanisms by which fluorescent oxonols indicate membrane potential in human red blood cells

Optical potentiometric indicators have been used to monitor the transmembrane electrical potential (Em) of many cells and organelles. A better understanding of the mechanisms of dye response is needed for the design of dyes with improved responses and for unambiguous interpretation of experimental results. This paper describes the responses to delta Em of 20 impermeant oxonols in human red blood cells. Most of the oxonols interacted with valinomycin, but not with gramicidin. The fluorescence of 15 oxonols decreased with hyperpolarization, consistent with an "on-off" mechanism, whereas five oxonols unexpectedly showed potential-dependent increases in fluorescence at less than 2 microM [dye]. Binding curves were determined for two dyes (WW781, negative response and RGA451, positive response) at 1 mM [K]o (membrane hyperpolarized with gramicidin) and at 90 mM [K]o (delta Em = 0 with gramicidin). Both dyes showed potential-dependent decreases in binding. Changes in the fluorescence of cell suspensions correlated with changes in [dye]bound for WW781, in accordance with the "on-off" mechanism, but not for RGA451. Large positive fluorescence changes (greater than 30%) dependent on Em were observed between 0.1 and 1.0 microM RGA451. A model is suggested in which RGA451 moves between two states of different quantum efficiencies within the membrane.