Linear equation for calibration of the mitochondrial membrane potential

Abstract

Membrane potential measurements of small cells or vesicles which are too small to use microelectrodes are performed with the use of a lipophilic ion as a probe. Although the principle of this method is simple and clear, adsorption of probes to membranes and/or intracellular constituents causes errors. On the basis of the binding model proposed previously and with the use of data on the binding to intact mitochondria, the relation between the true and the observed membrane potentially (called the uncorrected value) is calculated and a linear calibration equation, (true membrane potential) = A × (uncorrected value) + B, is found to hold for mitochondria in the range from −100 to −200 mV (determination coefficient > 0.99 for all cases). Here, membrane potentials are defined with respect to the outside. The values of A and B depend on the initial probe concentration, c o. But for TPMP + (triphenylmethylphosphonium), the dependence on c o is small, and this fact has been shown experimentally. On the other hand, for TPP + (tetraphenylphosphonium) the dependence is evident. For TPMP +, A and B do not depend on the protein concentration while for TPP +, they do depend on it. The values of the inner volume change A and B. Fortunately, actual calculation of the membrane potential is not affected very much by the value of volume employed, especially when dilute TPP + is used. This type of calibration equation has been obtained experimentally by other authors, and is very useful from a practical point of view. The equation obtained in this study agrees well with that obtained experimentally by other authors for mitochondria suspended in a medium of high ionic strength, although the medium was not strictly identical. They considered the value obtained with valinomycin + Rb + to be the true membrane potential.