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

Abstract

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.

Keywords: ADP/ATP carrier; ATP synthase; BAM15; Electron transport chain; FCCP; Mitochondria; PH sensor; Proton transport; Uncoupling protein.

Figure 1:. BAM15 activates H⁺ current mainly through AAC.

A. Graph representing ratio of AAC1-dependent H⁺ current (grey) and the non-specific protonophoric activity (light red) from previous characterization induced by long chain fatty acid arachidonic acid (AA), FCCP, BAM15 and DNP (Bertholet et al., 2019; Bertholet et al., 2022). B. H⁺ current densities at −160 mV in heart mitoplasts with different BAM15 concentrations. Data are mean ± s.e.m. n = 7–8 mitoplasts for all conditions. C. H⁺ current densities at −160 mV in heart mitoplasts with different FCCP concentrations. Data are mean ± s.e.m. n = 4–7 mitoplasts for all conditions. D to G. Representative H⁺ current induced by 250 nM BAM15 (D, red trace), 1 μM BAM15 (D, blue trace), and 5 μM BAM15 (F, red trace) in heart mitoplasts. Representative H⁺ current induced by 250 nM FCCP (E, red), 1 μM FCCP (E, blue), and 2 μM FCCP (G, red) in heart mitoplasts. The voltage ramp protocol is shown above the traces. H. Scheme representing the mode of action of BAM15, which is mainly to induce H⁺ transport through AAC, and the mode of action of FCCP, which partly induces H⁺ transport through AAC and has high protonophoric activity.

Figure 2:. Mitochondrial pHluorin expression to evaluate the mitochondrial matrix H⁺ pool.

A1. Generation of a Super-Ecliptic pHluorin sensor (SE-pHluorin) targeted to the mitochondrial matrix. The Cox8 SE-pHluorin coding region and WPRE non-coding region used to enhance RNA expression and stability, were removed by BamHI -XhoI digestion of the Addgene plasmid 58500 (Venkatachalam V, Cohen AE, 2014) and inserted into a homemade plasmid (Calmettes et al., JGP 2013; John et al., PlosOne 2023), which contained the CMV promoter. A2. The same vector was used to generate the AAC1 and AAC1 A123D mutant constructs. B. Representative images of the changes in fluorescence induced by addition of 2 μM FCCP in WT C2C12 cells. In most experiments, we only recorded the ratio values shown in panel C and did not acquire images. Each experiment included measurements taken simultaneously from 2–5 individual cells in the same field of view. A region of interest (ROI) was drawn around each cell, and fluorescence ratios were recorded in real time from these ROIs. Upon addition of FCCP, between image #1 and image #3, a decrease in the fluorescence ratio from 1.19 to 1.15 was observed. Following removal of FCCP, addition of 20 mM NH₄Cl induced recovery of the fluorescence ratio (image #4), corresponding to re-alkalinization of the matrix (increase in light intensity). C. Representative traces of the fluorescence ratio integrated over the ROI in each cell (shown in panel B), with varying expression levels of SE-pHluorin (a, b), and changes upon addition of FCCP and NH₄Cl. The 535/480 nm emission ratio is plotted as a function of time. A downward trend of the ratio corresponds to a decrease in matrix pH, and an upward trend indicates an increase in pH. These 535/480 ratio values were recorded and used for further analysis. The level of pH sensor expression may affect the absolute value of the emission ratio (compare traces a and b), but the time course of the changes in emission ratio remains the same. This suggests that the pH sensor does not have a strong effect in buffering the matrix pH. The numbers shown along the traces (1 to 4) correspond to the image number in panel B. D. Changes in the emission ratio as a function of pH and fit of the data using a Hill equation. The C2C12 cells used to generate this calibration curve were first permeabilized with 100 μM β-escin and then exposed to solutions with varying pH values. The fit yielded a pH_0.5 of 7.49, consistent with that obtained by others in permeabilized cells (Boffi et al., 2018). These ratio values can be used to estimate changes in pH in the matrix under different experimental conditions. More details on this dose-response curve are presented in Supplementary Figure 2.

Figure 3:. Mitochondrial matrix acidification due to BAM15 induces alkalinization.

A. Representative time course of the changes in fluorescence evoked by 500 nM BAM15 in WT C2C12 cells expressing SE-pHluorin targeted to the mitochondrial matrix. Addition of BAM15 evoked an initial phase of acidification that was followed by a phase of re-alkalinization. Number 1 corresponds to the acidification phase, and number 2 corresponds to the re-alkalinization phase. 20 mM NH₄Cl was added at the end of each experiment to validate the measurements and for data normalization. n=9. 20 mM NH₄Cl was added at the end of each experiment to assess the responsiveness of the sensor and for data normalization. B. Fit of the experimental data in panel A (dotted box), using the sum of an exponential and a sigmoidal function. The blue dots are the experimental data, and the red line corresponds to the fit. C. Bar graph showing the correlation between the amplitude and time course of the exponential and sigmoidal functions. Mean ± s.e.m. n=31 (BAM15 100 nM: n=4, BAM15 500 nM: n=9, BAM15 5 μM: n=18). D. Deconvolution of the fit in B into its two components. The orange trace corresponds to the acidification phase, and the green trace corresponds to the re-alkalinization phase. Note the same time scale is used in panels B and D. This analysis shows that the fluorescence changes induced by BAM15 comprise an exponential acidification that triggers a sigmoidal re-alkalinization phase. E. Correlation between the acidification time constant and the halfway point of the re-alkalinization phase. n=31 (BAM15 100 nM: n=4, BAM15 500 nM: n=9, BAM15 5 μM: n=18). F. Correlation between the amplitude of the exponential phase and the difference between the maximum and minimum of the sigmoid. n=31 (BAM15 100 nM: n=4, BAM15 500 nM: n=9, BAM15 5 μM: n=18).

Figure 4:. BAM15 induces H⁺ fluxes across the IMM that rely on AAC and the ATP synthase.

A. Representative time course of the changes in fluorescence evoked by 500 nM BAM15 in WT C2C12 cells expressing SE-pHluorin targeted to the mitochondrial matrix. This caused acidification of the matrix (phase 1) followed by a phase of alkalinization (phase 2). n=9. B. Representative time course of fluorescence changes induced by 500 nM BAM15 in DKO C2C12 cells expressing SE-pHluorin. BAM15 induced a slow acidification phase but no re-alkalinization phase. n = 18. C. Superimposition of traces shown in panels A and B to illustrate the evolution over time of fluorescence changes caused by 500 nM BAM15 in WT (A) and DKO (B) cells. This superimposition shows that the acidification phase caused by BAM15 is faster in WT cells than in DKO cells when acidification is adjusted to a single exponential. However, with the use of a two-time-constant exponential adjustment for acidification in WT cells, the values are comparable to those obtained with DKO cells, 31.85-+/280.57=ז (n=10) for WT cells and 49-+/235.33=ז (n=18) for DKO cells. The Y-axis represents arbitrary units. D. Representative time course of the changes in SE-pHluorin fluorescence in WT C2C12 cells elicited by 500 nM BAM15 after pretreatment with 10 μM oligomycin. n=11. E. Time course of the changes in SE-pHluorin fluorescence in DKO C2C12 cells elicited by 500 nM BAM15 after pretreatment with 10 μM oligomycin. n=9. 20 mM NH₄Cl was added at the end of each experiment to assess the responsiveness of the sensor and for normalization of the data.

Figure 5:. Reintroduction of functional AAC1 in DKO C2C12 cells re-establishes the re-alkalinization phase.

A. Diagram of the lentivirus vector used to reintroduce mouse AAC1 in DKO C2C12 cells. A bicistronic construct was used to express AAC1 and GFP together and avoid tagging AAC1. B. Left panel: Representative bright field image overlaid with a GFP fluorescent image showing that about 95% of C2C12 DKO cells were GFP positive. Right panel: quantification of GFP-positive cells among DKO cells transduced with cytosolic GFP as a control (grey bar) and DKO cells transduced with the bicistronic construct AAC1 (green bar). Data are mean ± s.e.m. n=4 coverslips per condition. C. Western blot analysis of total cell extract protein marker, Na⁺/K⁺ ATPase, AAC1, and mitochondrial biomass marker TOM20 expression. AAC1 was only detected after it had been reintroduced via lentivirus transduction. n=3 per group. D. DKO C2C12 cells (red line) developed a very small OCR, for which oligomycin application had no effect. BAM15 induced OCR due to its protonophoric activity. OCR was reduced in the absence of AAC, but with AAC1 reintroduction (blue line), basal respiration was recovered and oligomycin reduced OCR. These data demonstrate that the ADP/ATP exchange is functional with the re-introduced AAC1. The maximal respiration also increased with the application of BAM15 injection. AAC1 was thus capable of both its functions, ADP/ATP exchange and H⁺ transport. Data are mean ± s.e.m. n=13 wells for DKO and DKO+AAC1. See Supplementary Figure 5 for OCR data interpretation. E. Representative time course of the changes in SE-pHluorin fluorescence evoked by BAM15 in DKO C2C12 cells in which AAC1 had been reintroduced. For these experiments, the cells were transfected with the two plasmids for WT AAC1 and SE-pHluorin. Both phases, acidification and re-alkalinization, were observed, demonstrating that AAC1 is required for the re-alkalinization phase. n=12. F. Representative time course of the changes in SE-pHluorin fluorescence evoked by BAM15 in DKO C2C12 cells expressing the non-functional A123D AAC1 mutant. For these experiments, cells were transfected with the two plasmids for A123D AAC1 and SE-pHluorin. Under these conditions, an acidification phase was elicited by BAM15, but no re-alkalinization phase was observed. n=11. 20 mM NH₄Cl was added at the end of each experiment to assess the responsiveness of the sensor and for data normalization.

Figure 6:. Differences in mitochondrial pH profile with BAM15 and FCCP treatment.

A. Representative time course of the changes in fluorescence evoked by 500 nM BAM15 in WT C2C12 cells expressing SE-pHluorin targeted to the mitochondrial matrix. This caused acidification of the matrix (phase 1), followed by a phase of alkalinization (phase 2). n=9. B. Representative time course of the changes in fluorescence evoked by 5 μM BAM15 in WT C2C12 cells expressing SE-pHluorin. At this concentration, BAM15 still elicited acidification of the matrix, followed by re-alkalinization. n=18. C. Representative time course of the changes in fluorescence evoked by 200 nM FCCP in WT C2C12 cells expressing SE-pHluorin. At this concentration, FCCP was able to elicit acidification of the matrix, which was followed by a re-alkalinization. n=12. D. Representative time course of the changes in SE-pHluorin fluorescence in WT C2C12 cells after addition of 2 μM FCCP. At this concentration, FCCP could still elicit acidification of the matrix, but the phase of re-alkalinization was no longer present. n=18. E. Representative time course of the changes in SE-pHluorin fluorescence in WT C2C12 cells after addition of 75 nM FCCP. n=4. 20 mM NH₄Cl was added at the end of each experiment to assess the responsiveness of the sensor and for data normalization.

Figure 7:. H⁺ accumulation in the matrix due to ETC inhibition leads to the ATP synthase reversal activity.

A. Representative time course of the changes in SE-pHluorin fluorescence evoked by 6 μM antimycin A in WT C2C12 cells. Antimycin A induced acidification of the matrix, followed by a phase of re-alkalinization, as observed with BAM15 treatment. n=53. B. Representative time course of the changes in SE-pHluorin fluorescence evoked by 6 μM antimycin A in DKO C2C12 cells. In these cells, antimycin A induced a phase of acidification but no phase of re-alkalinization. n=25. C. Representative time course of the changes in SE-pHluorin fluorescence evoked by 6 μM antimycin A in WT C2C12 cells after pretreatment with10 μM oligomycin. Under these conditions, an acidification phase occurred, but inhibition of the ATP synthase activity blocked the phase of re-alkalinization. n=21. D. Representative time course of the changes in SE-pHluorin fluorescence evoked by 7.5 μM rotenone in WT C2C12 cells. Rotenone induced acidification of the matrix, followed by a re-alkalinization phase, similar to that observed with antimycin A. n=11. E. Diagram representing each mitochondrial compound target. 20 mM NH₄Cl was added at the end of each experiment to assess the responsiveness of the sensor and for data normalization.