Cell Biology
Dynamic Regulation of the Mitochondrial Proton Gradient during Cytosolic Calcium Elevations*

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Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨm) and pH gradient (ΔpHm) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpHm, whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpHm 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 ΔpHm (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpHm decreased during cytosolic Ca2+ elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca2+-ATPases and transmitted to mitochondria by Pi/H+ symport and K+/H+ exchange, whereas the decrease in ΔpHm 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 Ca2+ and pH, mitochondria alkalinized and ΔpHm increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca2+ caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpHm are regulated by opposing Ca2+-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.

Bioenergetics
Cell Metabolism
Cell pH
Mitochondrial Metabolism
Proton Transport
pHluorin

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*

This work was supported in part by Swiss National Foundation Grant 31-068317 (to N. D.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6.

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Supported in part by the Canadian Natural Sciences and Engineering Research Council.