Determination of membrane potentials in human and Amphiuma red blood cells by means of fluorescent probe

Abstract

1. Changes in the fluorescent intensity of the dye, 3,3'-dihexyl-2,2'-oxacarbocyanine, added to suspensions of human and Amphiuma red blood cells were measured in parallel with changes in the membrane potentials of these cells. In these studies the membrane potential was altered in three different ways: by the addition of valinomycin to alter the ratio, P(K)/P(Cl), by a change in the pH of the medium to alter the ratio, Cl(c)/Cl(0), and by the substitution of impermeant anions for Cl(0) again to alter the ratio, Cl(c)/Cl(0). In each case hyperpolarization led to a decrease and depolarization to an increase in fluorescent intensity.2. The change in fluorescence with the addition of valinomycin was dependent on the concentration of K in both the cells and the medium. Changes in fluorescence were not observed when valinomycin was added to suspensions of frozen-thawed, haemoglobin-free ghosts with dye in KCl or NaCl solutions. Such changes were observed with reconstituted ghosts provided that there was a K concentration gradient across the membrane.3. From values of cellular K and the corresponding external K concentrations for which there were no changes in fluorescence with valinomycin, estimations of membrane potentials were made. The potential was -5 to -8 mV for the human red cell and -19 mV for Amphiuma. These values are in good agreement with the potentials estimated from the Cl ratios (-9 mV for human and -17 to -20 mV for Amphiuma) and from those obtained by direct electrical measurements (-15 mV for Amphiuma).4. Fluorescent intensity of the dye in suspensions of human red cells was shown to be a linear function of the log Cl(c)/Cl(0).5. The dye (2.9 x 10(-6)M) increased the inward rate constants for (24)Na (3-4-fold) and (42)K (0.5-2-fold) for human red cells. In addition, the dye was found to be haemolytic (5-6% in 1 hr) at 22 degrees C.6. In contrast, the dye did not alter the rate of (35)SO(4) efflux at 37 degrees C from human red cells previously equilibrated with a Cl-free SO(4) medium.7. The dye was also seen to interact with certain impermeant anions and other compounds, e.g. inhibitors of anion permeability, of interest. These interactions and other limitations of the use of this dye are discussed.