Overview
The mitochondrion maintains a membrane potential at all possible times as a driving force along with the pH gradient for ATP synthesis, uptake of substrates and import of proteins into the organelle. In the absence of oxidative phosphorylation, for example when cells are living of glycolysis, the membrane potential is generated by reversal of the adenine nucleotide transporter (ANT) to bring ATP into the organelle from the cytosol in exchange for ADP moved out.

Compounds that are weak, hydrophobic acids can enter the mitochondrion, driven by the membrane potential, and uncouple electron transfer from ATP synthesis, dissipate the membrane potential, and accumulate in mitochondria to high concentrations.

Other compounds that are not weak acids can also alter membrane potential by direct effect on ANT and other inner membrane solute carriers, and as yet undefined other mechanisms.

Assay Details
The assay uses a widely accepted monitor of membrane potential, the cationic dye, tetraethylbenzimidazolylcarbocyanine iodide (JC1). JC1 accumulates in mitochondria, and becomes concentrated mostly in the inner membrane. When present at low concentrations it is predominantly a monomer, which yields green fluorescence. At higher concentrations, i.e at high membrane potentials, the dye aggregates to give a reddish colored emission with a maximum at ~590 nm. Our assay is set up as a 96 well plate screen with the data monitored as a ratio of aggregate to monomer form. A typical result is shown in figure 1 which is a titration of the effect of the classic uncoupler CCCP on the membrane potential of HepG2 cells grown in glucose.

Figure 1. IC₅₀ (8.7µM) determination of the effect of CCCP on the mitochondrial membrane potential in HepG2 cells.

Frequently Asked Questions

Q1. I have heard that JC1 is a poorly understood dye in terms of the mechanism of its action. Is there something better with which to measure membrane potential
A1. Yes and No. There are other dyes that can monitor membrane potential equally well but we choose JC1 because of the strong color change, the dye does not itself alter cellular properties, and there is a vast literature using the dye for comparison.

Q2. What interpretations can I make from the membrane potential measurements?
A1. From the foregoing, it should be evident that a compound that dissipates the mitochondrial membrane potential will greatly diminish energy metabolism via oxidative phosphorylation. Additionally, if the compound dissipates the gradient, in most cases it will be doing this by moving across the mitochondrial inner membrane. The result is that there will be accumulation of the compound in the organelle to the point that the concentration inside and at a potential target for toxicity can be many times the cell concentration. This is important to realize when considering an IC₅₀ for toxicity against the Cmax for a drug.