Mitochondria generate heat due to H+ leak (IH) across their inner membrane1. IH results from the action of long-chain fatty acids on uncoupling protein 1 (UCP1) in brown fat2,3,4,5,6 and ADP/ATP carrier (AAC) in other tissues1,7,8,9, but the underlying mechanism is poorly understood. As evidence of pharmacological activators of IH through UCP1 and AAC is lacking, IH is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP)10,11. Although protonophores show potential in combating obesity, diabetes and fatty liver in animal models12,13,14, their clinical potential for treating human disease is limited due to indiscriminately increasing H+ conductance across all biological membranes10,11 and adverse side effects15. Here we report the direct measurement of IH induced by DNP, FCCP and other common protonophores and find that it is dependent on AAC and UCP1. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain fatty acids, and find that they overlap with the putative ADP/ATP-binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent IH through AAC. Thus, common protonophoric uncouplers are synthetic activators of IH through AAC and UCP1, paving the way for the development of new and more specific activators of these two central mediators of mitochondrial bioenergetics.
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The MATLAB code for the mathematical model, force field and docking parameters for DNP, FCCP, BAM15, SF6847, CATR and the homology model of m-state bovine AAC1 are available at Zenodo (https://doi.org/10.5281/zenodo.5058463).
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We thank D. C. Wallace for providing AAC1-knockout mice; A. Angelin for performing pilot mitochondrial respiration studies; and B. M. Spiegelman for the help with Seahorse respiration studies. This work was supported by NIH grants R01GM107710, R01GM118939 and R35GM136415 to Y.K., R01GM089740 and R01GM137109 to M.G., R01NS098772 to N.B., as well as a grant from the UCSF Program for Breakthrough Biomedical Research (PBBR) to M.G. and Y.K. A.M.B. was supported by an American Heart Association Career Development Award 19CDA34630062 and NIH grant R35GM143097. P.B. was supported by an American Heart Association Post-Doctoral Fellowship 18POST33960587. Simulations were carried out, in part, at the UCSF Wynton Cluster on hardware made possible through NIH grants 1S10OD021596 and R01GM089740 to M.G.
Y.K., M.G., P.B. and J.S. are shareholders of Equator Therapeutics. Y.K. is an advisor to Equator Therapeutics. Y.K. is shareholder of YourChoice Therapeutics. E.T.C. is a founder, board member and equity holder in Matchpoint Therapeutics. The other authors declare no competing interests.
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Extended data figures and tables
a, IH densities induced by different DNP concentrations in heart IMM at −160 mV (n = 15 for 50 µM, n = 9 for 100 µM, n = 8 for 200 µM, n = 14 for 500 µM). Data are mean ± SEM. b, IH induced by 50 μM DNP in heart IMM before and after application 4 μM BKA (left). Remaining fraction of IH induced by 50 μM DNP at −160 mV after inhibition by 4 μM BKA, n = 3 (right). Data are mean ± SEM. c, IH induced by 500 μM DNP on the background of 10 mM MβCD in heart IMM before and after application of 1 µM CATR (left). IH densities induced by 50 µM DNP alone (n = 15) and 500 µM DNP on the background of 10 mM MβCD (n = 6) (right). Data are mean ± SEM. d, IH induced by 250 nM FCCP in heart IMM was inhibited by 1 μM CATR. e, Remaining fraction of FCCP-induced IH at -160 mV after inhibition by 1 μM CATR. Control (100% IH) is shown in gray. Data are mean ± SEM. Paired t-test, two-tailed, control vs. 1 µM CATR, n = 6.
a, IH induced by 25 nM SF6847 across WT heart IMM before and after application 1 μM CATR. b, IH induced by 25 nM SF6847 in AAC1−/− heart IMM. c, Remaining fraction of IH induced by 25 nM SF6847 at −160 mV after inhibition by 1 μM CATR. Data are mean ± SEM. Paired t-test, two-tailed, control vs CATR, n = 6. d, IH densities induced by 25 nM SF6847 at −160 mV in WT (n = 10) and AAC1−/− (n = 5) heart IMM. IH was measured at −160 mV. Data are mean ± SEM. Mann–Whitney U-test, two-tailed, WT vs AAC1−/−. e, IH induced by 250 nM BAM15 across WT heart IMM before and after application 1 μM CATR. f, IH induced by 250 nM BAM15 in AAC1−/− heart IMM. g, Remaining fraction of IH induced by 250 nM BAM15 at −160 mV after inhibition by 1 μM CATR. Data are mean ± SEM. Paired t-test, two-tailed, control vs CATR, n = 4. h, IH densities induced by 250 nM BAM15 at −160 mV in WT (n = 5) and AAC1−/− (n = 5) heart IMM. IH was measured at −160 mV. Data are mean ± SEM. Mann–Whitney U-test, two-tailed, WT vs AAC1−/−. i, j, Representative K+ currents induced by 100 nM valinomycin across heart IMM in WT (i) and AAC1−/− (j). k, K+ current densities induced by 100 nM valinomycin at −160 mV in WT and AAC1−/− heart IMM (n = 5). Data are mean ± SEM.
a, Oxygen consumption rate (OCR) of mitochondria isolated from WT and DKO C2C12 cells in the presence of oligomycin as measured with the Seahorse Analyzer. Each point corresponds to an individual respiration well, n = –60 (WT), and n = 56 (DKO). Mann–Whitney U-test, two-tailed. Data represent mean ± SEM. b, c OCR increase observed in mitochondria isolated from WT and DKO C2C12 cells after addition of DNP, n = 20, WT and n = 17, DKO (b) or BAM15, n = 10, WT and n = 10, DKO (c) in the presence of oligomycin. Each point corresponds to OCR change in an individual well of the Seahorse Analyzer, n = 17 – 20 (DNP) and n = 10 (BAM15). Data are mean ± SEM. Mann–Whitney U-test, two-tailed. d, OCR time course of isolated mitochondria from WT (n = 20 wells, blue) and DKO C2C12 cells (n = 17 wells, red). Basal respiration (1), addition of oligomycin (2), addition of DNP (3) and addition of rotenone/antimycin (4). Data represent mean ± SEM. e, Basal OCR of mitochondria isolated from WT or DKO C2C12 cells measured with a Clark electrode (n = 22, WT and n = 14, DKO). Mann–Whitney U-test, two-tailed. Data represent mean ± SEM. f, OCR increase above basal, observed using a Clark electrode after addition of DNP to mitochondria isolated from WT and DKO C2C12 cells; n = 3 (WT) and n = 5 (DKO) for 5 µM; n = 5 (WT) and n = 4 (DKO) for 12.5 µM. Data are mean ± SEM. Mann–Whitney U-test, two-tailed. g, Left panel: Immunoblots of WT (n = 5) and DKO (n = 5) C2C12 cells showing expression of the electron-transport chain complexes complex I (CI) – complex V (CV), TOM20, and the loading control (plasma membrane Na+/K+ ATPase). Right panel: The same immunoblots at longer exposure for the bands corresponding to complexes I-V. h, TOM20 protein expression level relative to Na+/K+ ATPase for the immunoblot shown in (g). Data shown as mean ± SEM, n = 5. Two-tailed unpaired t-test, WT vs DKO. i–m, Protein expression levels for complexes I-V relative to TOM20 for the immunoblots shown in (g). Data shown as mean ± SEM, n = 5. Two-tailed unpaired t-test, WT vs DKO. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 4 Respiration induced by mitochondrial uncouplers in intact cells depends on AAC.
a, Oxygen consumption rate (OCR) of WT (n = 20) and DKO C2C12 cells (n = 16) before and after addition of oligomycin (oligo). Two-tailed unpaired t-test, WT vs DKO. b–d, OCR increase observed in WT and DKO C2C12 cells upon application of various concentrations of FCCP (n = 4), DNP (n = 4), and BAM15 (n = 4). All OCRs were measured in the presence of oligomycin. Two-tailed unpaired t-test, WT vs DKO. e, AAC-dependent fraction of uncoupled respiration at various concentrations of FCCP, DNP, and BAM15. All data shown as mean ± SEM, n = 4. f, OCR increase observed in WT and DKO C2C12 cells upon application of various concentrations of valinomycin. All OCRs were measured in the presence of oligomycin. Nigericin (1 µM) was added to reduce matrix K+ accumulation and mitochondrial swelling caused by valinomycin27. Two-tailed unpaired t-test, WT vs DKO, n = 4.
a, DNP-induced IH at various ATP concentrations on the cytosolic face of the IMM of brown fat. IH was activated with either 500 μM DNP (left panel) or 5 mM DNP (middle panel) applied in the presence of 10 mM MβCD. Control currents were recorded in 10 mM MβCD. Right panel is the dose-dependence of IH inhibition by ATP at 500 μM DNP (red, n = 5) and 5 mM DNP (blue, n = 6). Current amplitudes were measured at −160 mV. Data are mean ± SEM. b, Left panel, IH induced by 50 µM DNP in control (1), upon transient inhibition by bath ADP (2), and upon subsequent recovery in the presence ADP (3). Heart mitoplast. Middle panel, DNP-dependent IH time course of left panel. IH was measured at −160 mV. Right panel, ADP inhibition of IH at points (2) and (3) in left and middle panels. Data are mean ± SEM. Paired t-test, two-tailed, ADP (2) vs. control, n = 4. c, IH activated by 50 µM DNP was inhibited by bath ADP. Pipette solution contained 1 mM ADP. Heart mitoplast. Middle panel, IH time course of the left panel. Right panel, ADP inhibition of IH in point (2) as in left and middle panels. Data are mean ± SEM. Paired t-test, two-tailed, n = 4.
Extended Data Fig. 6 Binding mode comparisons of CATR, ADP, small molecule uncouplers, and fatty acid.
a–d, Comparison of binding poses in the AAC1 c-state (2C3E) cavity of (a) docked ADP and docked DNP; (b) crystallographic CATR and docked DNP; (c) FA (arachidonic acid) pose from MD simulation and docked ADP; and (d) FA pose from MD simulation and crystallographic CATR. TM helices 1 and 6 of AAC1 are hidden. ADP and CATR are shown as thinner sticks with mauve carbon atoms. DNP and FA are shown as thicker sticks with yellow carbon atoms. e, Full final docked DNP, FCCP, BAM15, and SF6847 poses in their deprotonated and protonated forms (left) as in Fig. 4c and titratable heavy atoms (pink spheres) from these poses (right). TM helix 1 of AAC1 is hidden. f, ADP binding mode to the c-state as predicted from docking. g, Simulation snapshot showing arachidonic acid (solid spheres) bound to the TM5/6 fenestration of AAC1 in the c-state superimposed with the docked pose of DNP (transparent spheres). h, Simulation snapshot showing two arachidonic acid molecules bound simultaneously to the AAC1 c-state cavity, one in the TM5/6 fenestration and the other in the small molecule uncoupler site.
Extended Data Fig. 7 Docking reveals a common binding site for small molecules and nucleotide in both c- and m-states of AAC1.
a–j, Final docking poses to the AAC1 c-state (a–e) and m-state (f–j) structures of deprotonated forms of DNP, FCCP, BAM15, and SF6847, as well as ADP, respectively. In e and j ADP phosphate groups are hidden for clarity but were included in the docking. The same protein sidechains are shown as sticks in all panels.
a, Illustration of the simulation setup used to assess binding of negatively charged DNP to the c-state of AAC1. DNP is shown in sphere representation in the aqueous region of the simulation box. AAC1 is shown as a cyan ribbon, with TM1 and TM6 hidden, and lipid molecules are shown as sticks. The Cζ atom of AAC1 residue R234 at the base of the c-state cavity is shown as a black sphere. Binding was tracked in b–d by monitoring the distance from the DNP centre of mass to AAC1 R234 Cζ atom indicated by the dotted line. b, Trajectories of negatively charged DNP initially placed in solution far from the binding site. c, Trajectories of neutral DNP, initially placed in the binding site. d, Trajectories of negatively charged DNP initially in the binding site, with an applied −160 mV membrane potential. In each plot, the two different colored traces are measurements made from two independent simulation trajectories. Simulations in c were initiated from docking poses, while those under an applied −160 mV potential in d were initiated from the final snapshots of the DNP binding simulations in b.
a, Arachidonic acid transiently bound to the AAC1 region identified as the DNP/small molecule binding site in contact with protein residue Y186; snapshots are from two independent simulation trajectories. View is from the membrane with TM5 & TM6 of AAC1 hidden to show the cavity. b, Top view of structures in panel a viewed from the cytoplasm. c, Final states of four arachidonic and three palmitic acids bound to the fenestration between AAC1 helices TM5 & TM6; snapshots are from 7 independent simulation trajectories. d, Top view of structures in panel c. In panels a–d, AAC1 is shown as a cyan ribbon and FAs are shown as sticks with yellow carbon and red oxygen atoms. e, Side view and f, top view of a single structure from panels c and d with FA atoms shown as spheres and bilayer lipids shown as sticks. Carbon atoms 1–6, 7–12, and 13–20 of the arachidonic acid are colored yellow, blue, and pink, to highlight the parts that are inside the AAC1 cavity, in the TM5/TM6 fenestration, and interacting with bilayer lipids, respectively.
Extended Data Fig. 10 Calculation of membrane potential profiles and mathematical model results for AAC1.
a, Membrane potential profile (V(z)) across c-state (left) and energy values along the central pore for a monovalent cation (|e|·V(z)) (right). The profiles were computed under a −160 mV applied voltage using Poisson-Boltzmann theory (see Materials and Methods) on a configuration extracted from the equilibrated MD simulations. Colour gradients represent isocontours of linearly spaced scalar values ranging from 0 mV (upper region of graphic) to −160 mV (lower region). The water filled cavity facing upward supports a minor fraction of the field with the majority of the field focused across the closed matrix gate of the c-state. These values are quantified in the right panel showing the energy of interaction for a monovalent cation moving through the membrane field. b–e, Steady state single-transporter currents as a function of voltage (b, c), pKa (d), and rate k23 of proton transfer from the uncoupler to matrix pathway (e) for the 3-state model shown in Fig. 4g using values in Supplementary Table 2 unless otherwise indicated in the panel. The electrostatic potential in the cavity (Eelec) was varied: 10 kBT (blue), 8 kBT (gold), 6 kBT (green) (b and d). The fraction of the membrane voltage traversed by a H+ entering from the cytosol was varied: f = 0.1 (blue), 0.25 (gold), and 0.5 (green) (c). The energy of the proton in the matrix pathway (E1) was varied: 0 kBT (blue), 5 kBT (gold), 10 kBT (green) (e).
Supplementary Fig. 1: gel source data; Supplementary Note 1: mathematical model of uncoupler-aided H+ permeation through AAC; Supplementary Note 2: Supplementary Discussion; Supplementary Table 1: parameters for electrostatic calculations; Supplementary Table 2: base mathematical model parameters; and Supplementary Table 3: molecular dynamics simulations.
DNP binding to AAC1 in two independent molecular dynamics simulations. Two independent 500 ns simulation trajectories of AAC1 (cyan ribbon) with charged DNP (yellow/blue/red sticks), shown side by side. Distances from DNP to the bottom of the AAC1 cavity are plotted in Extended Data Fig. 8b. The transparent, fixed DNP molecule represents the DNP pose identified from docking. White spheres are selected lipid atoms indicating the extent of the hydrophobic interior of the lipid bilayer.
Arachidonic acid binding to AAC1 in two independent molecular dynamics simulations. Two independent 5 µs simulation trajectories of AAC1 (cyan ribbon) with arachidonic acid (yellow and red spheres), shown side by side. Each shows the arachidonic acid binding first to the DNP/uncoupler site, and then to the fenestration between TM5 and TM6. White spheres are selected lipid atoms indicating the extent of the hydrophobic interior of the lipid bilayer.
The AAC1 c-state can accommodate two FA molecules simultaneously. A 1 µs simulation of two arachidonic acid molecules (yellow and red spheres) simultaneously occupying the DNP/uncoupler site and the FA site between TM5 and TM6. AAC1 is shown as a cyan ribbon and is viewed from the cytoplasmic side of the membrane. Sidechains of protein residues Lys22, Arg79, Tyr186 and Arg279 are shown as sticks.
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Bertholet, A.M., Natale, A.M., Bisignano, P. et al. Mitochondrial uncouplers induce proton leak by activating AAC and UCP1. Nature 606, 180–187 (2022). https://doi.org/10.1038/s41586-022-04747-5