Dynamic Regulation of the Mitochondrial Proton Gradient during Cytosolic Calcium Elevations

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential ( m ) and pH gradient (pH m ) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by pH m , whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of pH m in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (7.6) and pH m (0.45) of HeLa cells at 37 C were lower than previously reported. Unexpectedly, mitochondrial pH and pH m decreased during cytosolic Ca 2 elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca 2 -ATPases and transmitted to mitochondria by P i /H symport and K /H exchange, whereas the decrease in pH m reflected the low H -buffering power of mitochondria (5 mM, pH 7.8) compared with the cytosol (20 mM, pH 7.4). Upon agonist washout and restoration of cytosolic Ca 2 and pH, mitochondria alkalinized and pH m increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 M Ca 2 caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and pH m are regulated by opposing Ca 2 -dependent processes of stimulated mitochondrial respiration and cytosolic acidification.