The generation of a proton gradient across the inner mitochondrial membrane (IMM) is an essential energy conservation event that couples the oxidation of carbohydrates and fat to the synthesis of ATP. Studies in isolated mitochondria have established that the chemical gradient for protons (ΔpHm) and the mitochondrial membrane potential (ΔΨm) contribute independently to the proton-motive force (Δp) that drives the synthesis of ATP. Because ΔΨm contributes most of the Δp and can be easily measured in intact cells with fluorescent dyes, most studies ignore the contribution of ΔpHm and only record changes in ΔΨm to track the metabolic state of mitochondria. ΔpHm, however, drives the fluxes of metabolic substrates required for mitochondrial respiration and the activity of electroneutral ion exchangers that maintain mitochondria osmolarity and volume, and recent studies indicate that the mitochondrial pH (pHmito) plays an important and underappreciated role in physiological and pathological situations such as apoptosis, neurotransmission, and insulin secretion. In this Perspective, we discuss the putative roles of the pHmito and review the different techniques used to measure pHmito and ΔpHm in isolated mitochondria and in intact cells, focusing on our recent results obtained with genetically encoded pH-sensitive indicators. These measurements have revealed that the pHmito is in dynamic equilibrium with the cytosolic pH and that spontaneous pHmito elevations coinciding with ΔΨm drops occur in single mitochondria. Unlike the “superoxide flashes” reported with a pH-sensitive circularly permuted YFP (cpYFP), these “pH flashes” preserve the Δp during spontaneous fluctuations in ΔΨm; therefore, we propose that the flashes are energy conservation events that reflect the intrinsic properties of the mitochondrial proton circuit.
