The measurement of membrane potential using optical indicators

Abstract

Conclusions Optical methods have become established as a major experimental protocol for following membrane potential. They can provide a rapid, continuous record of the potential and have a very wide applicability. However, when used to make quantitative assertions about membrane potential, optical methods have a number of weaknesses. Even the most reliable calibration procedures depend on accurate evaluation of a small number, namely the internal ion concentration, in a large background, that is total ion levels. However, a consensus seems to be emerging that the plasma membrane potential of non-excitable cells nevertheless has considerable magnitude: typical values are −60 mV for lymphocytes (Rink et al., 1980), −20 to −100 mV, depending on metabolic load, for Ehrlich ascites tumour cells (Philo & Eddy, 1978; but see also Smith & Robinson, 1980), and −66 to −86 mV for neutrophils (Tatham et al., 1980). In our own experiments using monolayer cultures of cells grown to confluence (Bashford et al., 1981) the potential across the plasma membrane is of the order of −100 mV (see Fig. 2). Membrane potentials of similar magnitude have been found using ion-distribution methods and microelectrodes in neuroblastoma cells and lymphocytes (Deutsch et al., 1979a,b). In the latter studies ions of different charge were used to provide upper and lower estimates of the potential, the presumed effects of binding being very different for anions and cations. A similar approach, in this case the use of optical indicators of different charge, has been taken by Rink et al. (1980), and this would seem to be one way in which to diminish the uncertainties involved in dye calibration. Unfortunately many anions, particularly oxonols, form complexes with valinomycin (Lavie & Sonenberg, 1980; Rink et al., 1980), although we have found no evidence for such a complex with bis isoxazolone oxonols (J.C. Smith and C.L. Bashford, unpublished observations). It is apparent that calibration procedures not dependent on valinomycin should be sought in order to establish optical methods as a quantitative approach to the study of membrane potential.