Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations

Abstract

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ(m)) and pH gradient (ΔpH(m)) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH(m), whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH(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 ΔpH(m) (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH(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 ΔpH(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 ΔpH(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 μm Ca(2+) caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH(m) are regulated by opposing Ca(2+)-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.

FIGURE 1.

SypHer characterization. A, changes in HyPer (top) and SypHer ratio (bottom) evoked by an alkaline load (30 mm NH₄Cl) and by H₂O₂ (200 μm). B, confocal images of mitoSypHer (green) in fixed cells stained for TOM20 (red; top) or live cells labeled with MitoTracker Red CMXRos (red; bottom). Merged images (right) show clear mitochondrial targeting of mitoSypHer. Scale bars, 5 μm (top) and 10 μm (bottom). C, excitation spectra of mitoSypHer in situ, at 37 °C using a 505-nm dichroic and 535/25 emission filter. D, in situ calibration of mitoSypHer. Inset, dynamic range at physiological pH. E, average resting pH_cyto and pH_mito measured by single-cell (left box) and high throughput fluorescence imaging. Boxes show quartiles with circles centered on means; error bars show S.E. for the individual cells and 95% intervals for high throughput data.

FIGURE 2.

Mitochondria acidify during [Ca²⁺]_cyt elevations. A, [Ca²⁺]_cyt responses to histamine measured with fura-2 (green; raw ratio) in cells expressing mitoSypHer (black; raw ratio). Traces show the mean response of seven cells in a single field of view with an inset of simultaneous responses from a single cell. B, parallel pH_mito (black, mitoSypHer; mean of 11 cells) and pH_cyto (blue, cytoSypHer; mean of four cells) acidification following a 30 μm histamine addition. Scale bars, 10 μm.

FIGURE 3.

ΔpH_m changes during and after [Ca²⁺]_cyt elevations. A, simultaneous pH_mito (black, mitoSypHer) and pH_cyto (blue, SNARF) measurements in cells repeatedly stimulated with histamine (30 μm). For each cell, ΔpH_m was estimated as pH_mito − pH_cyto (red). Diamonds and circles, mean ± S.D. from 74 cells. B, typical mitoSypHer (top, λ_ex 480 nm) and SNARF fluorescence images (bottom, λ_em 580 nm). C, average changes in pH_cyto (i), pH_mito (ii), and ΔpH_m (iii) during each histamine addition and in ΔpH_m (iv) after histamine washout (mean ± S.E. (error bars) of 74 cells). *, p < 0.05 for repeated measures general linear model analysis of variance.

FIGURE 4.

Ca²⁺ dependence of the decreases in mitochondrial and cytosolic pH. A, simultaneous [Ca²⁺]_cyto (i; fura) and pH_mito (ii; mitoSypHer) recordings illustrate that the change in mitoSypHer ratio (iii; black) closely match the instantaneous integral of the fura-2 ratio (iii; green). Traces are taken from Fig. 2A. B, effect of extracellular Ca²⁺ removal and restitution on [Ca²⁺]_cyt (YC3.6 cameleon, 4 cells) (i), pH_mito (11 cells) (ii), and pH_cyto (nine cells) (iii) responses evoked by histamine (30 μm) and thapsigargin (1 μm). Insets show kinetics of SERCA-independent Ca²⁺_cyto clearance (i) and pH_mito recovery (ii).

FIGURE 5.

PMCA mediates the Ca²⁺-dependent decreases in pH_cyto and pH_mito. A, La³⁺ (5 mm) blocks clearance of a [Ca²⁺]_cyt elevation (i) and decrease in pH_mito (ii) evoked by histamine (30 μm) when SERCA is blocked with thapsigargin (Tg; 1 μm). B, effect of La³⁺ (5 mm) on the clearance of [Ca²⁺]_cyt elevations (i) and on decreases in pH_mito (ii) evoked by Ca²⁺ readdition to cells treated with histamine and thapsigargin in low extracellular Na⁺ to block Ca²⁺ clearance by the Na⁺/Ca²⁺-exchanger. C, effect of alkaline pH_o (8.8) on [Ca²⁺]_cyt (YC3.6 cameleon, four cells) recovery (i) after histamine removal and the decrease in pH_cyto (SNARF) and pH_mito (mitoSypHer) (ii) evoked by histamine. La³⁺ and alkaline pH_o both prevent Ca²⁺ clearance and prevent the decreases in cytosolic and mitochondrial pH.

FIGURE 6.

Low mitochondrial buffering power at alkaline pH_mito underlies the loss of ΔpH_m during Ca²⁺ elevations. A, i, protocol used to calculate H⁺-buffering power (β) by the stepwise addition/removal of permeant weak acid. Black traces, mitoSypHer; blue traces, SNARF. Amiloride (100 μm), rotenone (5 μm), antimycin (5 μm), and oligomycin (5 μg/ml) were added to prevent membrane H⁺ transport. ii, pH dependence of intrinsic H⁺-buffering power (β_cyto and β_mito). β values were binned every 0.1 pH units (mean ± S.E.). Dotted lines, β at mean resting pH_cyto and pH_mito. Solid lines, fitted sigmoid functions. B, calculation of J_H⁺ for a given histamine-mediated pH change, where β is a function of pH. C, i, changes in pH_mito as a function of the changes in pH_cyto in the same cell during histamine responses. Regression of δpH_mito against δpH_cyto has a slope of >1, reflecting the loss of ΔpH_m during stimulation. ii, mitochondrial versus cytosolic proton fluxes. Regression of the calculated J_H⁺mito versus J_H⁺cyto has a slope of <1, indicating that mitochondria resist cytosolic H⁺ fluxes. Regressions were constrained to pass through the origin.

FIGURE 7.

Opposite effects of Ca²⁺ and H⁺ on mitochondrial matrix pH. A, pH_mito responses evoked by the addition of H⁺ and Ca²⁺ to permeabilized cells. Bath pH was transiently decreased from 7.4 to 7.2, and then 10 μm Ca²⁺ was added at pH 7.4 or during the pH switch to mimic the changes occurring in intact cells. B, averaged pH_mito responses evoked in permeabilized cells by a decrease in environmental pH to 7.2, by the addition of 10 μm Ca²⁺ at pH 7.4, or by the addition of 10 μm Ca²⁺ during a switch to pH 7.2.

FIGURE 8.

Contributions of mitochondrial transporters to mitochondrial proton fluxes. A, effects of inhibitors of the ETC (5 μm rotenone, 5 μm antimycin with or without 5 μg/ml oligomycin) and of the mitochondrial NCX (10 μm CGP-37157) on histamine-mediated pH_mito (black, mitoSypHer) and pH_cyto (blue, SNARF) responses in intact cells. Bar graphs show mean ± S.E. drug effects for pH_cyto (blue) and pH_mito (gray) acidification and loss of ΔpH_m (pink). n is displayed in the bars and p values are shown for t tests. B, effects of inhibitors of the ETC and of the mitochondrial NCX (as above) on the pH_mito responses evoked by the addition of H⁺ to permeabilized cells. Bath pH was transiently decreased from 7.4 to 7.2 before and after the addition of the inhibitors. Bar graphs show mean ± S.E. decrease in pH_mito evoked by the pH switch from 7.4 to 7.2. C, average effect of the inhibitors on the J_H⁺mito/J_H⁺cyto flux ratio measured in intact cells during the third histamine response, using the second histamine response as internal control. Bars, mean ± S.E. (error bars) from n cells (number in bars) from ≥6 coverslips. *₁, significantly different from control by Dunnett's test with groupwise α = 0.05. D, average effect of the inhibitors on the J_H⁺mito flux measured in permeabilized cells during a pH switch from 7.4 to 7.2. *₂, significantly different (p < 0.05) by one-sample t test from internal control in the absence of inhibitor.