Mitochondrial Inner Membrane Electrophysiology Assessed by Rhodamine-123 Transport and Fluorescence

Abstract

Membrane potential sensitive dyes such as rhodamine-123 (R123) are widely used to make noninvasive dynamic measurements of mitochondrial membrane potential both in vitro and in situ. Yet interpretation of measurements is difficult due to a lack of understanding of how membrane potential and measured fluorescence are related. To develop a means to quantitatively assay the kinetics of the mitochondrial inner membrane potential, a model for dye transport, including electrogenic transport across the mitochondrial inner membrane and partition into the membrane was developed. The model accounts for dye self-quenching experimentally measured in our lab and was integrated into a previously developed model of mitochondrial electrophysiology in order to estimate transients in mitochondrial membrane potential from kinetic measurements of fluorescence intensity. Our analysis indicates that (i.) the intensity of R123 fluorescence (at excitation and emission wavelengths of 503 nm and 527 nm, respectively) has a peak near concentration of 50 mM and decreases to zero at higher concentrations due to self-quenching; (ii.) measured fluorescence intensity and membrane potential are related by a non-linear calibration curve sensitive to certain experimental details, including total concentration of dye and mitochondria in suspensions; and (iii.) the time courses of membrane potential and electron transport fluxes following a perturbation (i.e. addition of ADP) significantly differ from observed transients in fluorescence intensity. These findings reveal that mitochondria display a characteristic time of response to changes in substrate concentration of less than 0.1 second, corresponding to the time scale over which the rate of ATP synthesis changes to meet changes in ATP demand.