Imaging of mitochondrial matrix pH dynamics reveals a functional interaction between the ADP/ATP carrier and ATP synthase to regulate H(+) distribution.

In mitochondria, the energy derived from the proton gradient across the mitochondrial inner membrane (IMM) is converted into ATP and heat. For these conversions to occur, H(+) is pumped out of the matrix via the electron transport chain (ETC) and then re-enters either via the ATP synthase to produce ATP or via the ADP/ATP carrier (AAC) to release heat. Due to its dual functions of ADP/ATP exchange and H(+) transport, AAC may be considered a major regulator of the energy distribution of mitochondria between ATP synthesis and thermogenesis. Using real-time imaging of pH with a fluorescent pH probe targeted to the mitochondrial matrix, we investigated in a myoblast cell model how H(+) fluxes across the IMM are regulated by AAC and the ATP synthase. Our data show that activation of AAC-dependent H(+) transport by the mitochondrial uncoupler BAM15 causes an acidification of the matrix followed by a re-alkalization phase due to the reversed activity of the ATP synthase. Similar re-alkalization and reversal of ATP synthase activity were observed after acidification caused by inhibition of the electron transport chain. Lastly, the discovery that strong protonophoric activity independent of AAC suppresses the re-alkalization phase and consequently the reverse action of the ATP synthase, suggests the need for strict control of the H(+) flux through the IMM by AAC. Thus, real-time imaging of matrix pH reveals a functional interaction between AAC and the ATP synthase for the control of H(+) fluxes across the IMM.