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H+ transport is an integral function of the mitochondrial ADP/ATP carrier

Nature volume 571, pages 515–520 (2019)Cite this article

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Abstract

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H+ transport. The AAC-mediated H+ current requires free fatty acids and resembles the H+ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H+ leak, but does not completely inhibit it. This suggests that the H+ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H+ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.

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Fig. 1: Pharmacological and biophysical properties of IH.
Fig. 2: AAC is required for IH.
Fig. 3: Adenine nucleotide transport by AAC.
Fig. 4: Nucleotide exchange negatively regulates IH.
Fig. 5: Mitochondrial uncoupling requires AAC.

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Data availability

All data used to support the conclusions of this study are included in this article. Full all-point electrophysiological traces are available from the corresponding author upon request.

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Acknowledgements

We thank S. Bal Craquin and the members of the Y.K. laboratory for helpful discussions. This work was supported by NIH grants R01GM107710 and R01GM118939 to Y.K. and grants NS021328, MH108592, OD010944 (NIH), and W81XWH-16-1-0401 (DOD) to D.C.W. as well as a Canadian Institutes of Health Research postdoctoral fellowship to L.K. B.M.S. received funding from the JPB Foundation.

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Authors and Affiliations

  1. Department of Physiology, University of California San Francisco, San Francisco, CA, USA

    Ambre M. Bertholet, Andriy Fedorenko & Yuriy Kirichok

  2. Dana-Farber Cancer Institute & Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Edward T. Chouchani, Lawrence Kazak, Jonathan Z. Long, Sara Vidoni, Ryan Garrity & Bruce M. Spiegelman

  3. Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Alessia Angelin & Douglas C. Wallace

  4. Department of Pathology, University of Florida College of Medicine, Gainesville, FL, USA

    Joonseok Cho & Naohiro Terada

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Contributions

A.M.B., A.F. and Y.K. conceived the project and designed experiments. A.M.B. performed all electrophysiological experiments, except A.F. performed pilot experiments and provided data for Extended Data Figs. 1–3. D.C.W. provided AAC1 knockout mice, and J.C. and N.T. provided AAC2 hypomorphic mice and consulted on their use. A.A., A.M.B. and D.C.W. performed respirometry on cardiac mitochondria. E.T.C., L.K., A.M.B., R.G., J.Z.L. and B.M.S. conducted respirometry in C2C12 cells and mitochondria. S.V. performed mitochondrial biomass analysis in C2C12 cells. A.M.B. and Y.K. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Yuriy Kirichok.

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B.M.S. serves as a Consultant to Calico Life Sciences. The other authors declare no competing interests.

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Peer review information Nature thanks Paolo Bernardi, Kevin Foskett, Clay Semenkovich and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 FA-dependent IH in the IMM and plasma membrane.

a, Left, a diagram of patch-clamp recording from a vesicle of the whole IMM (mitoplast). After forming a gigaohm seal between the patch pipette and the mitoplast, the IMM patch under the pipette is broken by applying short pulses of high voltage (200–500 mV, 5–30 ms) combined with light suction to gain access into the mitoplast through the pipette. In this configuration, called the ‘whole-mitoplast’ configuration, the interior of the mitoplast (mitochondrial matrix) is perfused with the pipette solution. The bath is also perfused to control the experimental solution on the cytosolic side of the IMM. The voltage across the IMM is set using the patch-clamp amplifier. Directions of currents flowing across the IMM: inward currents (flowing into the mitoplast) are negative, while outward currents are positive. Right, an example of an IH current trace recorded in the whole-mitoplast mode. The voltage protocol used to induce the currents is shown above the traces. All indicated voltages are within the mitochondrial matrix relative to the bath (cytosol). The voltage of the bath solution is defined to be zero. Baseline (zero current level) as well as negative (inward) and positive (outward) currents are indicated. b, IH induced in a skeletal muscle mitoplast by 1.5 µM (IMM, upper panel, n = 11) or 15 µM (IMM, lower panel, n = 3) AA. Voltage protocol is shown above the traces. Bath (cytosolic side of the IMM) and pipette (matrix side) pH are indicated in the pipette–mitoplast diagram. c, As in b but using the plasma membrane (PM, n = 4 at 1.5 µM, n = 4 at 15 µM) of HEK293 cells. d, IH current densities in the IMM of skeletal muscle and PM of HEK293 cells at 1.5 (n = 11 for IMM, n = 4 for PM) and 15 µM AA (n = 3 for IMM, n = 4 for PM). IH measured at −160 mV. Data represent mean ± s.e.m.

Source data

Extended Data Fig. 2 UCP1-independent IH in various mouse tissues.

a, IH induced in mitoplasts from heart (n = 6), liver (n = 5), and brown fat (UCP1−/− mice, n = 6) by application of 1.5 µM AA on the cytosolic side of the IMM. The voltage protocol used is shown above the traces, and the bath and pipette pH values are indicated in the pipette–mitoplast diagram. b, IH induced in mitoplasts from skeletal muscle of wild-type (n = 7), UCP2−/− (n = 8), and UCP3−/− (n = 11) mice by application of 1.5 µM AA on the cytosolic side of the IMM. c, IH current densities in skeletal muscle mitoplasts from wild-type (n = 7), UCP2−/−(n = 8), and UCP3−/− (n = 11) mice, measured at −160 mV as in b. Data represent mean ± s.e.m. d, Representative skeletal muscle mitochondrial IH induced by 1.5 µM AA before (red) and after application of 1 mM GDP (blue) (n = 4). e, Mitochondrial IH recorded in the absence of added FA (control, black) was deactivated by addition of 10 mM MβCD to the bath (n = 10).

Source data

Extended Data Fig. 3 H+ selectivity of mitochondrial IH.

a, Left, representative mitochondrial IH recorded at ΔpH = 1 in response to the voltage step protocol indicated above (skeletal muscle mitoplast, n = 6); ΔV = 40 mV. A holding potential of −60 mV (close to the EH) was selected to minimize H+ current and depletion of the proton buffer between applications of voltage steps. Red dotted line indicates zero current. Right, I/V curve corresponding to the current traces in the left panel (skeletal muscle mitoplasts, n = 6). Note the reversal potential. The pH values in the pipette and bath solutions are indicated on the diagram. b, Left, mitochondrial IH recorded at ΔpH = 1.5 in response to the voltage step protocol indicated above (skeletal muscle mitoplast, n = 3); ΔV = 60 mV. Holding potential was −90 mV (close to the Nernst H+ equilibrium potential EH). Right, I/V curve corresponding to the current traces in the left panel (skeletal muscle mitoplasts, n = 6). c, Left, mitochondrial IH recorded at ΔpH = −0.5 in response to the voltage step protocol indicated above (skeletal muscle mitoplasts, n = 4); ΔV = 40 mV. Holding potential was 0 mV. Right, I/V curve corresponding to the current traces in the left panel. All currents were induced by 1.5 µM AA (skeletal muscle mitoplasts, n = 6). d, IH reversal potentials (Vrev) compared to EH. Linear fitting of Vrev (red) and EH at 24 °C (black) versus transmembrane ΔpH; pH 6/7, n = 6; pH 6/7.5, n = 3; pH 6.5/6, n = 4 skeletal muscle mitoplasts. Data represent mean ± s.e.m.

Source data

Extended Data Fig. 4 AAC-dependent and -independent currents induced by FA.

a, Current induced by 4 μM PA (red) was inhibited by 1 μM CATR (blue). Control current is shown in black. Representative experiments performed in heart mitoplasts, n = 4. b, The same experiment performed with 100 μM lauric acid (LA), n = 5. c, Top, currents induced by 2 μM of AA (green), PA (blue), or LA (red) in the same mitoplast. Control current is shown in black. Heart mitoplasts, n = 4. Bottom, mean IH current densities at −160 mV induced in heart mitoplasts by 2 μM of AA (n = 6), PA (n = 7), or LA (n = 4) as in experiment shown above. Data represent mean ± s.e.m. d, Left, IH induced by 2 μM AA (red) was inhibited by 4 μM BKA (blue). Control currents are shown in black. Representative experiment performed in a heart mitoplast (n = 4). Right, inhibition of IH induced by 2 µM AA in heart mitoplasts by 4 µM BKA. Remaining IH measured at −160 mV is shown as a percentage of control, n = 4. Paired t-test, two-tailed. Data represent mean ± s.e.m. e, Current induced by 2 μM AA sulfonate before (red) and after (blue) addition of 1 μM CATR. Representative experiment performed in a heart mitoplast, n = 6. f, Current induced by 2 μM AA sulfonate before (red) and after (blue) addition of 50 μM mersalyl. Representative experiment performed in a heart mitoplast, n = 4. g, Currents induced by 2 μM AA before (red) and after (blue) addition of 50 μM mersalyl. Note that only the outward current was inhibited. Representative experiment performed in a heart mitoplast, n = 6. h, The outward current activated by 2 µM AA (red) is inhibited by 50 μM mersalyl (blue) and is restored by 1 mM DTT (green). Control current is shown in black. Heart mitoplasts, n = 4. i, Whole-mitoplast current before (control, black) and after application of 2 μM AA (red), and upon washout of AA (blue). Heart mitoplasts, n = 6. j, IH induced by 2 μM AA (red) was inhibited by 1 μM CATR (blue). Control current is shown in black. Symmetrical pH 6.0. Heart mitoplasts, n = 4. k, Inhibition of the inward IH induced by 2 µM AA in skeletal muscle, heart, liver, and kidney by 1 µM CATR. Skeletal muscle (n = 22), heart (n = 18), liver (n = 4), and kidney (n = 7) for both control and CATR treatment. Remaining inward current measured at −160 mV is shown as a percentage of control. Paired t-test, two-tailed. Data represent mean ± s.e.m. l, Inhibition of the outward current induced by 2 µM AA in skeletal muscle, heart, liver, and kidney by 1 µM CATR. Remaining outward current measured at +100 mV is shown as a percentage of control. Skeletal muscle (n = 21), heart (n = 17), liver (n = 4), and kidney (n = 7) for both control and CATR treatment. Paired t-test, two-tailed. Data represent mean ± s.e.m.

Source data

Extended Data Fig. 5 FA-dependent IH via AAC is potentiated by oxidation.

a, c, e, IH activated by 2 µM AA (red) was then potentiated by oxidizers 250 µM tBHP, 100 µM 4-HNE, or 20 µM TBT (blue). IH potentiated by oxidizers was inhibited by CATR (green). Control current is shown in black. Bar graphs show ratio of IH amplitudes at −160 mV before and after addition of oxidizer. Heart mitoplasts. Note that TBT and 4-HNE, but not tBHP, inhibited the AAC-independent outward current observed at positive membrane potentials. a, n = 4; c, n = 3; e, n = 5 for all experimental conditions. Paired t-test, two-tailed. Data represent mean ± s.e.m. b, d, f, Currents before (control, black) and after (red) application of 250 µM tBHP (n = 3), 20 µM TBT (n = 3), or 100 µM 4-HNE (n = 3).

Source data

Extended Data Fig. 6 FA-dependent currents in AAC1 knockout and AAC2 hypomorphic mice.

a, b, Representative currents induced by 2 µM AA in wild-type (left) and AAC2 hypomorphic (middle) mitoplasts from heart (n = 9 for wild-type, n = 9 for hypomorphic; a) and kidney (n = 4 for wild-type, n = 5 for hypomorphic; b). Right, IH current densities at −160 mV for wild-type (n = 10 for heart, n = 5 for kidney) and AAC2 hypomorphic mitoplasts (n = 10 for heart, n = 6 for kidney). Data are mean ± s.e.m. c–e, Densities of the outward current measured at +100 mV for wild-type and AAC1−/− mitoplasts from heart (c; n = 14 for wild-type, n = 10 for AAC1−/−), skeletal muscle (d; n = 21 for wild-type, n = 12 for AAC1−/−), and kidney (e; n = 5 for wild-type, n = 6 for AAC1−/−). Mann–Whitney U-test, two-tailed. Data are mean ± s.e.m. f, g, Densities of the outward current measured at +100 mV for wild-type (n = 9 for heart, n = 5 for kidney) and AAC2 hypomorphic (n = 10 for heart, n = 5 for kidney) mitoplasts from heart (f) and kidney (g). Mann–Whitney U-test, two-tailed. Data are mean ± s.e.m. h, Inhibition of the outward current induced by 2 µM AA in AAC1−/− heart mitoplasts by 1 µM CATR (n = 5, control and CATR). Remaining outward current measured at +100 mV is shown as a percentage of control. Paired t-test, two-tailed. Data are mean ± s.e.m. i, Left, inhibition of inward IH induced by 2 µM AA in AAC1−/− skeletal muscle mitoplasts by 1 µM CATR (n = 10, control and CATR). Remaining IH measured at −160 mV is shown as a percentage of control. Right, inhibition of the outward current induced by 2 µM AA in in AAC1−/− skeletal muscle mitoplasts by 1 µM CATR (n = 9, control and CATR). Remaining current measured at +100 mV is shown as a percentage of control. Paired t-test, two-tailed. Data are mean ± s.e.m. j, Two representative experiments in which the IH induced by 2 µM AA in AAC1−/− mitoplasts from skeletal muscle was the smallest (left, n = 4) and the largest (right, n = 3). IH induced by 2 μM AA (red) was inhibited by 1 μM CATR (blue). Control current shown in black.

Source data

Extended Data Fig. 7 Interaction of FA anions with AAC.

a, IH induced by 2 µM AA (red) was inhibited by 66 ± 2% (n = 4, skeletal muscle mitoplasts) by 5 µM AA-sulf (blue). Data are mean ± s.e.m. b, Current induced by 5 µM AA-sulf (left, red) or 1 µM AA (right, red) was inhibited by 1 µM CATR (blue). Control currents are shown in black. A lower concentration of AA was used to induce comparable currents with AA-sulf. Heart mitoplasts, n = 4. c, d, Currents recorded before (control, black) and after addition of 5 µM AA-sulf (c) or 10 mM C6-sulf (d) to bath (red). Brown fat mitoplast (UCP1, left), heart mitoplast (AAC, right), n = 4 for each. Currents were measured at pH 6.0 to inhibit the production of FA by phospholipase A2 (PLA2) associated with the brown fat IMM and to ensure that UCP1 currents were activated by exogenously applied FA anions only. e, Current before (control, black) and after (red) application of 50 mM C6-sulf to the bath. Pipette solution contained 50 mM C6-sulf. Symmetrical pH 6.0. Heart mitoplasts, n = 3. f, Current before (control, black) and after (red) application of 5 µM AA-sulf to the bath. Pipette solution contained 10 µM AA-sulf. Bath AA-sulf was kept at 5 µM because higher concentrations disrupted the IMM. Symmetrical pH 6.0. Heart mitoplasts, n = 3. g, Proposed model of FA-dependent IH via AAC. Without FA, AAC is impermeable for H+ (1). When FA binds in the AAC translocation pathway, its protonatable headgroup enables H+ binding and transport (2). FA can activate IH with AAC in either the c- or m-state (2, 3). Because the SBS is positively charged and retains its structure with c–m conformational change, the negatively charged head of FA is likely to interact with the SBS, while the hydrophobic carbon tail may protrude into the membrane and/or be stabilized by hydrophobic interactions within AAC (2, 3).

Extended Data Fig. 8 Adenine nucleotide exchange by AAC.

a, The alternating access mechanism of adenine nucleotide transport by AAC. AAC is shown in green, and its SBS (overall positively charged) located in the middle of the membrane is shown in blue. Cytosolic ADP binds to AAC in the c-state (1). AAC transitions to the m-state, and ADP is released into the matrix (2, 3). Matrix ATP binds to AAC in the m-state (4). AAC transitions to the c-state, and ATP is released into the cytosol (5). b, AAC current activated by 1 mM ADP (red) is inhibited by 1 μM CATR (blue). Pipette solution contained 1 mM ATP. Heart mitoplast, n = 3. Control trace is in black. c, Inhibition of the inward ADP/ATP exchange current via AAC by 1 µM CATR. Heart mitoplast, n = 6 (control and CATR treatment). Paired t-test, two-tailed. Data are mean ± s.e.m. Remaining inward current measured at −160 mV is shown as a percentage of control. See also Fig. 3a. d, Inhibition of the outward ATP/ADP exchange current via AAC by 1 µM CATR. Heart mitoplast, n = 5 (control and CATR treatment). Paired t-test, two-tailed. Data are mean ± s.e.m. Remaining outward current measured at −100 mV is shown as a percentage of control. See also Fig. 3b. e, Inhibition of the inward IH induced by 2 µM AA by 1 µM CATR after ADP pre-treatment. Heart mitoplast, n = 7 (control and CATR treatment). Paired t-test, two-tailed. Data are mean ± s.e.m. Remaining IH measured at −160 mV is shown as a percentage of control. See also Fig. 3e. f, Current before (control, black) and after (red) addition of 2 µM AA to bath. Subsequent addition of 1 µM CATR (blue) inhibited IH. Pipette solution contained 4 µM AA. Heart mitoplast, n = 4. g, Control current (black) and current after addition of 1 mM ADP to the bath solution (red). AA (2 µM) was added to the bath solution at the end of the experiment (blue). Pipette solution contained 4 µM AA. Heart mitoplasts, n = 4.

Source data

Extended Data Fig. 9 Regulation of FA-dependent IH by nucleotides.

a, Explanation of transient inhibition of IH by cytosolic adenine nucleotides. AAC in the c-state, with an FA anion in the translocation pathway, mediates IH (1). Cytosolic ADP3− binds in the c-state and expels the FA anion or blocks the translocation pathway, leading to inhibition of IH (2). Upon AAC conformation change, ADP dissociates into the matrix (pipette) solution (3). The FA anion re-associates with AAC in the m-state, restoring IH (4 and 5). Cytosolic ADP cannot inhibit IH while AAC is in the m-state (5). See also Fig. 4a. b, Proposed mechanism of inhibition of IH by adenine nucleotide exchange. AAC in the c-state, with an FA anion in the translocation pathway, mediates IH (1). Cytosolic ADP3− binds in the c-state and expels the FA anion or blocks the translocation pathway, leading to inhibition of IH (2). The resultant continuous exchange of cytosolic and matrix adenine nucleotides inhibits FA anion binding and IH (3–5). ATP (and not ADP) is shown as a matrix adenine nucleotide to reflect physiological conditions. See also Fig. 4b. c, Remaining IH after inhibition by different concentrations of ADP applied to both sides of the IMM to induce continuous adenine nucleotide exchange via AAC as in e. ADP/ADP exchange was used to avoid contaminating IH with ADP/ATP exchange current. Heart and skeletal muscle mitoplasts, n = 5 (control and 10 μM ADP), n = 8 (control and 100 μM ADP, n = 9 (control and 1 mM ADP). Data are mean ± s.e.m. d, IH via UCP1 is inhibited by 100 µM Mg2+-free ADP (top, n = 5) and 1 mM Mg2+-free ADP (bottom, n = 3). IH activated by 2 µM AA is shown before (red) and after inhibition by ADP (blue). In the beginning of the experiment, before the application of AA, the endogenous membrane FA were removed by a 30–40-s pre-treatment with 10 mM MβCD (black, control). All recording solutions contained 1 µM CATR to reduce the contribution of AAC to the IH measured. Pipette solution contained either 100 µM ADP (top) or 1 mM ADP (bottom) to match the recording conditions for AAC (e). Brown fat mitoplasts. e, IH via AAC is inhibited by 100 µM Mg2+-free ADP (top, n = 8) and 1 mM Mg2+-free ADP (bottom, n = 8). IH activated by 2 µM AA is shown before (red) and after inhibition by ADP (blue). Pipette solution contained either 100 µM ADP (top) or 1 mM ADP (bottom) to achieve symmetrical [ADP] on both sides of IMM. Heart mitoplasts. f, Mean densities of IH via UCP1 (dark grey) and AAC (light grey) in control samples (brown fat, n = 11 and heart, n = 9) and in the presence of 100 µM ADP (brown fat, n = 5 and heart, n = 8) or 1 mM ADP (brown fat, n = 3 and heart, n = 8) on both sides of the IMM. IH amplitudes were measured at −160 mV. The same data as in Fig. 4e. Data represent mean ± s.e.m.

Source data

Extended Data Fig. 10 Phenotypes associated with AAC deficiency.

a, Representative OCRs of isolated heart mitochondria from wild-type (left, n = 3 wells) and AAC1−/− mice (right, n = 4 wells). As indicated by the arrows, first oligomycin and then either PA (50 μM, light orange and 100 μM, dark orange) or buffer (black) were added, followed by FCCP and rotenone. Higher PA concentrations were used than for electrophysiological experiments, because in suspensions of isolated mitochondria and in the presence of albumin, the effective concentration of PA is markedly lower. FCCP-induced uncoupled respiration in wild-type and AAC1−/− mitochondria validated their respiration capacity. Data represent mean ± s.e.m. This experiment was repeated with independent mitochondrial isolations from wild-type (n = 4) and AAC1−/− (n = 3) mice with the same results. b, Basal OCR of isolated heart mitochondria from wild-type (n = 24 wells) and AAC1−/− (n = 18 wells) mice. Mann–Whitney U-test, two-tailed. Data represent mean ± s.e.m. c, Representative immunoblots of wild-type (n = 5) and DKO (n = 7) C2C12 cells for: NDUFB8 (complex I, CI), SDHA (complex II, CII), core 2 subunit (complex III, CIII), CIV-I subunit (complex IV, CIV), and ATP5A (complex V, CV), TOM20, and the loading control (plasma membrane Na+/K+ ATPase). For gel source data see Supplementary Fig. 1. d, Basal and ADP-stimulated OCR of mitochondria from wild-type (basal, ADP 100 μM, and ADP 200 μM, n = 20) and DKO (n = 16 for basal, n = 16 for ADP 100 μM, and n = 17 for ADP 200 μM) C2C12 cells. Mann–Whitney U-test, two-tailed. Data represent mean ± s.e.m. e, Representative confocal micrographs of wild-type (top, n = 45 cells) and DKO (bottom, n = 45 cells) C2C12 cells immunolabelled with TOM20 (green) and tubulin (red) antibodies. Insets show magnified areas from the same images. f, Mitochondrial biomass per cell in wild-type and DKO C2C12 cells, calculated as a ratio between TOM20 signal and the total area of the cell; n = 45 per group. Data represent mean ± s.e.m. g, Comparison of ratio between mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) in wild-type (n = 6) and DKO (n = 6) C2C12 cells. Data represent mean ± s.e.m. h, Kinetic study of extracellular acidification rate (ECAR) in wild-type (n = 22) and DKO (n = 22) C2C12 cells under basal conditions and upon addition of oligomycin to the respiration medium. Note that inhibition of mitochondrial ATP production in DKO cells with oligomycin did not affect ECAR, whereas in wild-type cells, oligomycin potently stimulated ECAR. Data represent mean ± s.e.m.

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Supplementary Figure 1: Gel source data.

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Bertholet, A.M., Chouchani, E.T., Kazak, L. et al. H+ transport is an integral function of the mitochondrial ADP/ATP carrier. Nature 571, 515–520 (2019). https://doi.org/10.1038/s41586-019-1400-3

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