Potential difference and the distribution of ions across the human red blood cell membrane; a study of the mechanism by which the fluorescent cation, diS‐C3‐(5) reports membrane potential.

Abstract

The dye seems to bind both to cell membranes and to cell contents. The component of the cytoplasm which binds the dye is non-dialyzable, presumably Hb. Dye added to a suspension of intact cells shows a strong absorption at 590 nm, indicating that the dye has bound to the cell contents and that the membrane is permeable to the dye. The amount of dye which partitions into (and on to) the cells can be determined from the fluorescence of the dye remaining in the supernatant after the cells are centrifuged to the bottom of the suspension. In most conditions the proportion of the cell associated dye which is either free inside the cell or bound to the outside face of the membrane is negligible compared to the proportion bound to the cell contents. On the assumption that the dye is not actively transported, the ratio of the equilibrium dye activities inside and outside the cell, .alpha.i/.alpha.o, is determined by the membrane potential according to the Nernst relation. An empirical relation between cell associated dye and internal activity was determined by measuring the dye partition between cells and medium at different eternal activities. Using this empirical relation, and providing that any changes in cell composition do not affect the dye binding, the internal activity at any potential can be calculated from the measured amount of cell associated dye. The external activity was estimated fluorimetrically. The membrane potential is then calculated from the activity ratio. The membrane potential of cells was altered by adding valinomycin in the presence of different K gradients. Dye binding to Hb is influenced by pH and dye partitioning into cells apparently changes with intracellular pH. When large potentials are produced with valinomycin, there is no change in intracellular pH. This result indicates that in red blood cells intracellular pH is determined by the external pH and Cl concentration ratio and not by the membrane potential. DiS-C3-(5) can be used to estimate potentials across resealed ghost membranes. For KCl ghosts, the constant field permeability for K in the presence of valinomycin is about 20-25 times that of Cl. The similarity between this value and that for cells suggests that the Cl conductance of the membrane is not greatly altered by the ghosting and resealing procedures. Because Hb is negatively charged and present at high concentration, the electrical potential inside the cell varies locally within the cell. This variation in potential tends to increase the apparent activity coefficient for Cl and reduce that for K. The partition of the dye will accurately predict partition of other univalent ions but not of divalent ions.