Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria

Key points Within cells, mitochondria oxidize carbohydrates, and fatty and amino acids to use the released energy to form ATP, and in the process, they also generate reactive oxygen species. Their maximal rates are linked to the magnitude of the mitochondrial membrane potential. Here we derive a model of fluorescent potentiometric probe dynamics, and on these principles we introduce an absolute quantitative method for assaying mitochondrial membrane potential in millivolts in individual cultured cells. This is the first micro-scale method to enable measurement of differences in mitochondrial membrane potential between cells with different properties, e.g. size, mitochondrial density and plasma membrane potential, including cases when plasma membrane potential fluctuates. Mitochondrial membrane potential in cultured rat cortical neurons is -139 mV at rest. In response to electrical stimulation of the cells, it is regulated between -108 mV and -158 mV by concerted increases in energy demand and metabolic activation.