1. Mechanism of Fatty-Acid-Dependent UCP1 Uncoupling in Brown Fat Mitochondria 
Andriy Fedorenko, Polina V. Lishko, Yuriy Kirichok 
Cell 
Volume 151, Issue 2, Pages (October 2012)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Electrophysiological Properties of UCP1 Current
(A) Transmitted, fluorescent, and superimposed images (left to right) of BAT mitoplasts isolated from mice expressing CFP in the mitochondrial matrix (false green color). White arrows, IMM; red arrows, remnants of outer membrane.
(B) Whole-mitoplast putative UCP1 current before (control, red) and after (black) the addition of 1 mM GDP to the bath solution. The voltage protocol is indicated at the top. The pipette-mitoplast diagram indicates the recording conditions. The mitoplast membrane capacitance was 1.1 pF.
(C) Putative UCP1 current (control, red) is potentiated by 3 μM arachidonic acid (AA, blue) and inhibited by 0.25% BSA (black). The mitoplast membrane capacitance was 1.0 pF.
(D) Representative whole-mitoplast currents recorded from wild-type (black) and UCP1−/− (red) mitoplasts.
(E) Current-voltage dependence of IUCP1. Amplitudes were measured at the beginning of the voltage steps shown in Figure S1D; n = 5.
(F) IUCP1 reversal potentials (Vrev) compared to H+ equilibrium potentials (EH) predicted by the Nernst equation. The red line indicates the linear fitting of IUCP1 reversal potentials versus ΔpH; n = 3–10. The black line indicates EH calculated by the Nernst equation at 24°C.
(G) Whole-mitoplast IUCP1 before (control, red), after the addition of 4 μM oleoyl-CoA to the bath solution (blue), and after the subsequent application of 1 mM GDP (black).
(H) Left panel: IUCP1 at different symmetrical pH values. Representative traces recorded from different mitoplasts are shown. Right panel: Mean IUCP1 densities in different symmetrical pH values; n = 4–12. Amplitudes were measured upon stepping from 0 to −160 mV as in the left panel.
Error bars represent standard error of the mean (SEM). See also Figure S1.
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4. Figure 2 UCP1 Is Activated by Endogenous Membrane LCFAs
(A) Left panel: Representative time course of IUCP1 amplitude in control (1), upon application of 0.5% BSA (2), and with 0.5% BSA and 1 mM GDP (3). AA (5 μM) was applied at the end to verify that IUCP1 can still be activated (4). Pipette solution contained 0.5% BSA. Amplitudes were measured upon stepping from 0 to −160 mV (see right panel). Right panel: IUCP1 traces recorded at times 1, 2, 3, and 4 as indicated in the left panel.
(B) The same experiment as in (A) but performed with 15 mM αCD in the bath and pipette solutions.
(C) Left panel: Representative time course of IUCP1 amplitude in control (1) and upon the application (2) and subsequent washout (3) of 10 mM αCD at pH 8.0. Right panel: IUCP1 traces recorded at times 1, 2, and 3 as indicated in the left panel.
(D) The same experiment as in (C) but performed at symmetrical pH 6.0.
See also Figure S2.
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5. Figure 3 Regulation of IUCP1 by Lysophospolipids
(A) Left panel: Representative time course of the IUCP1 amplitude in control (1), upon the application of 4 μM oleoyl-lysoPC (2), and the subsequent application of 1 mM GDP (3). IUCP1 amplitudes were measured upon stepping from 0 to −160 mV (see right panel). Right panel: IUCP1 traces recorded at times 1, 2, and 3 as indicated in the left panel.
(B) Left panel: Representative time course of the IUCP1 amplitude after the extraction of endogenous LCFAs with 10 mM αCD, reactivation of IUCP1 with 1 μM OA (1), the subsequent addition of 4 μM oleoyl-lysoPC (2), and the application of 1 mM GDP (3). The pipette solution contained 15 mM αCD to extract endogenous membrane LCFAs. Right panel: IUCP1 traces recorded at times 1, 2, and 3 as indicated in the left panel.
(C) The same experiment as in (A) but performed with 4 μM oleoyl-lysoPA instead of oleoyl-lysoPC.
(D) The same experiment as in (B) but performed with 4 μM oleoyl-lysoPA instead of oleoyl-lysoPC.
See also Figure S3.
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6. Figure 4 Alkylsulfonates Are UCP1 Transport Substrates
Left panels: Representative IUCP1 recorded after the extraction of endogenous membrane LCFAs with αCD (control, red), after subsequent application of the indicated concentration of Cn-sulfonate (blue), and upon adding 1 mM GDP (black) at symmetrical pH 6.0. The structure of the activating Cn-sulfonate is shown near the currents induced. Right panels: Same as the left panels, except that the pipette solution contained the same concentration of Cn-sulfonate as applied to the bath (10 μM C18 in A, 100 μM C11 in B, 1 mM C8 in C, 10 mM C6 in D, and 50 mM C3 in E). The calibration bar relates to all traces. A zero current level is indicated by the green dotted line in (C) and (D). See also Figure S4.
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7. Figure 5 H+ Transport by UCP1 Is Coupled to Transport of LCFA Anions
(A) Comparison of reversal potentials (Vrev) of the IUCP1 induced by C6-sulfonate, with C6-sulfonate equilibrium potentials (EC6) predicted by the Nernst equation. The red line indicates the linear fitting of IUCP1 reversal potentials; Vrev versus −log [C6]o/[C6]i, n = 3–6. The black line indicates EC6 calculated by the Nernst equation at 24°C.
(B) Upper panel: The IUCP1 activated by endogenous membrane LCFAs before (control, red) and after the application of 50 μM C11-sulfonate either alone (blue) or in combination with 1 mM GDP (black). Lower panel: The same experiment as in the upper panel but with 5 μM C18-sulfonate.
(C) IUCP1 in 10 mM αCD (control, red) and in 10 mM αCD plus 3.5 mM DBLA (blue) at symmetrical pH 6.0 (upper panel), pH 7.0 (middle panel), and pH 8.0 (lower panel).
Error bars represent SEM. See also Figure S5.
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8. Figure 6 Asymmetry of LCFA Binding to UCP1
(A) IUCP1 recorded with 2 mM C11-sulfonate in the pipette. Representative IUCP1 recorded upon extraction of endogenous membrane LCFAs with αCD (control, red), in 100 μM C11-sulfonate (blue), and after the addition of 1 mM GDP (black).
(B) Upper panel: IUCP1 recorded with 10 mM αCD in the pipette. Representative IUCP1 recorded upon extraction of endogenous membrane LCFAs with αCD (control, red), in 100 μM C11-sulfonate (blue), and after the addition of 1 mM GDP (black). Lower panel: IUCP1 recorded with 2 mM C11-sulfonate in the pipette. Representative IUCP1 recorded upon extraction of endogenous membrane LCFAs with αCD (control, red), in 10 mM αCD (blue), and after the addition of 1 mM GDP (black).
(C) Amplitudes of the inward (negative) and outward (positive) UCP1 currents induced by various alkylsulfonates added to the bath (blue) or pipette (red) solutions. IUCP1 was recorded with the same alkylsulfonate concentrations using the same voltage protocol as in Figures 4A–4D. IUCP1 was measured in the beginning of the second (−50 mV, inward IUCP1) and third (+50 mV, outward IUCP1) voltage steps. The leak current remaining after application of 1 mM GDP was subtracted.
(D) IUCP1 recorded with 50 mM C6-sulfonate (upper panel) and 10 mM αCD (lower panel) in the pipette solution. Representative IUCP1 in 10 mM αCD (red), in 50 mM C6-sulfonate (blue), and after the addition of 1 mM GDP (black). The zero current level is indicated by the dotted line.
(E) I/V curves of IUCP1 induced by1 μM OA at pH 7.0 (black) and pH 8.0 (red). The pipette solution contained 15 mM αCD, pH 7.0. The IUCP1 amplitudes were measured as indicated in Figure S6B.
(F) The dose dependence of IUCP1 inhibition by ATP at two different concentrations of activating OA. IUCP1 was activated either with 0.2 mM OA mixed with 10 mM MβCD (red curve) or with 2 mM OA mixed with 10 mM MβCD (black curve). Amplitudes were measured upon stepping from 0 to −160 mV as in Figures S6C and S6D.
Error bars represent SEM. See also Figure S6.
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9. Figure 7 LCFA-Shuttling Model of UCP1 Operation
(A) The simplest mechanism of steady H+ IUCP1 induced by LCFAs. UCP1 operates as a symporter that transports one LCFA and one H+ per the transport cycle. First, the LCFA anion binds to UCP1 on the cytosolic side at the bottom of a hypothetical cavity (1). H+ binding to UCP1 occurs only after the LCFA anion binds to UCP1 (1). The H+ and the LCFA are translocated by UCP1 upon conformational change, and H+ is released on the opposite side of the IMM, whereas the LCFA anion stays associated with UCP1 due to the hydrophobic interactions established by its carbon tail (2). The LCFA anion then returns to initiate another H+ translocation cycle (3). Charge is translocated only in step 3 when the LCFA anion returns without the H+.
(B) The mechanism of transient IUCP1 induced by low-pKa LCFA analogs. A low-pKa LCFA analog can be translocated by UCP1 similar to an LCFA anion. However, the low pKa of the LCFA analog prevents the binding of H+ to UCP1. The negatively charged low-pKa LCFA analog shuttles within the UCP1 translocation pathway in response to the transmembrane voltage, producing transient currents.
(C) The mechanism of steady IUCP1 induced by short-chain low-pKa fatty-acid analogs. The hydrophobic tail is too short to anchor the fatty analog to UCP1, and the analog is translocated through UCP1, producing a steady current. In contrast to LCFAs, the short-chain low-pKa fatty-acid analogs can bind to UCP1 on both sides of the IMM.
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10. Figure S1
Electrophysiological Properties of UCP1 Current, Related to Figure 1
(A) Proposed models of UCP1 operation (from left to right): (1) H+ channel activated by allosteric binding of LCFAs; (2) OH− channel activated by allosteric binding of LCFAs; (3) the H+ buffering model; (4) the fatty-acid cycling model.
(B) Mean normalized amplitudes of the whole-mitoplast putative UCP1 current measured before (control) and after addition of 3 μM arachidonic acid (AA), oleic acid (OA), docosahexaenoic acid (DHA), palmitoleic acid (PA), or 0.25% BSA to the bath solution; n = 4–18. Current amplitudes were measured upon stepping from 0 to −160 mV (see voltage protocol in Figure 1C) and normalized to the control value. The leak current was not subtracted.
(C) The whole-mitoplast putative UCP1 current (control, red) is inhibited by 10 mM αCD (blue). The voltage protocol is indicated at the top. The pipette-mitoplast diagram indicates the recording conditions.
(D) The whole-mitoplast IUCP1 in response to voltage steps from a holding potential of 0 mV; ΔV = 20 mV.
(E) Inside-out IUCP1 in response to voltage steps from a holding potential of 0 mV, ΔV = 40 mV. The BSA-insensitive leak current was subtracted.
(F) Left panel: Whole-mitoplast IUCP1 recorded at ΔpH = 0.5 in response to the voltage step protocol indicated at the top of the panel; ΔV = 10 mV. A holding potential of −30 mV (close to the EH) was selected to minimize IUCP1 between applications of voltage steps and to eliminate depletion of the proton buffer inside the mitoplast (see Experimental Procedures). A zero current level is indicated by the green dotted line. Right panel: The I/V curve corresponding to the current traces in the left panel. Note the reversal potential. The pH values in the pipette and bath solutions are as indicated on the diagram.
(G) Left panel: Whole-mitoplast IUCP1 recorded at ΔpH = 1 in response to the voltage step protocol indicated at the top of the panel; ΔV = 20 mV. A holding potential of −60 mV (close to EH) was selected to minimize depletion of proton buffer inside the mitoplast between voltage steps. A zero current level is indicated by the red dotted line. Right panel: The I/V curve corresponding to the current traces in the left panel. Note the reversal potential. The pH values in the pipette and bath solutions are as indicated on the diagram.
(H) Representative time-course activation of the whole-mitoplast current by 3 μM AA in wild-type (WT, black) and UCP1−/− (red) mice. Current amplitudes were measured upon stepping from 0 to −160 mV as in Figure 1C.
Error bars represent SEM.
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11. Figure S2 UCP1 Is Activated by LCFAs, Related to Figure 2
(A) Representative IUCP1 in control (red), upon extraction of endogenous LCFAs with 0.25% BSA (blue), and in 1 mM GDP (black).
(B) Representative IUCP1 upon extraction of endogenous LCFAs with 10 mM MβCD (red), after application of 2 mM OA mixed with 10 mM MβCD (blue), and upon application of 2 mM OA/10 mM MβCD with 1 mM GDP (black).
(C) Left panel: Representative time course of IUCP1 amplitude in control (1) and after the application (2) and subsequent washout (3) of 0.5% BSA at pH 7.0. IUCP1 amplitudes were measured upon stepping from 0 to −160 mV (see right panel). Right panel: IUCP1 traces recorded at times 1, 2, and 3 as indicated in the left panel.
(D) Graph showing the fraction of IUCP1 recovered after deactivation with 10 mM αCD at different symmetrical pH values.
Error bars represent SEM.
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12. Figure S3 Regulation of IUCP1 by PLA2 Inhibitors, Related to Figure 3
(A) Left panel: Representative time course of the IUCP1 amplitude in control (1), after the extraction of endogenous LCFAs with 10 mM αCD (2), after the reactivation of IUCP1 by endogenous membrane LCFAs upon washout of αCD (3), and upon subsequent application of 4 μM oleoyl-lysoPC (4). IUCP1 amplitudes were measured upon stepping from 0 to −160 mV (see right panel). Right panel: IUCP1 traces recorded at times 1, 2, 3, and 4 as indicated in the left panel.
(B) Left panel: Representative time course of IUCP1 amplitude in control (1), upon the application of 50 μM pyrrophenone (Pyr, 2), and with the subsequent application 1 mM GDP (3). IUCP1 amplitudes were measured upon stepping from 0 to −160 mV (see right panel). Right panel: IUCP1 traces recorded at times 1, 2, and 3 as indicated in the left panel.
(C) Left panel: Representative time course of the IUCP1 amplitude after the extraction of endogenous LCFAs with 10 mM αCD, the reactivation of IUCP1 with 1 μM of OA (1), the subsequent addition of 50 μM pyrrophenone (2), and the application of 1 mM GDP (3). The pipette solution contained 15 mM αCD to extract endogenous LCFAs. IUCP1 amplitudes were measured upon stepping from 0 to −160 mV (see right panel). Right panel: IUCP1 traces recorded at times 1, 2, and 3 as indicated in the left panel.
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13. Figure S4
Currents Caused by Low-pKa LCFA Analogs in Wild-Type and UCP1−/− Mitoplasts, Related to Figure 4
(A) Left panel: Representative whole-mitoplast IUCP1 recorded upon extraction of endogenous membrane LCFAs with αCD (control, red) and after subsequent activation with 100 μM C11-sulfonate (blue). The experiment was performed at symmetrical pH 6.0. These traces are also shown in Figure 4B, left panel. Voltage protocol and structure of C11-sulfonate are shown at the top of the panel. The pipette-mitoplast diagram indicates the recording conditions. Right panel: IUCP1 activated by 40 μM lauric acid (C11-carboxylate) under the same recording conditions as in the left panel.
(B–D) Currents induced by 100 μM C11-sulfonate, 1 mM C8-sulfonate, and 10 mM C6-sulfonate, respectively, in UCP1−/− mitoplasts. See (A) for recording conditions and calibration bars.
(E and F) IUCP1 induced by 5 μM perfluorotridecanoic acid and 20 μM dodecyl sulfate, respectively, in wild-type mitoplasts. See (A) for recording conditions and calibration bars.
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14. Figure S5
IUCP1 at Different Transmembrane Gradients of C6-Sulfonate, Related to Figure 5
IUCP1 carried by C6-sulfonate at pH 6.0 in the presence of 5 mM C6-sulfonate in the pipette and varying concentrations of C6-sulfonate (50 mM, 15 mM, and 5 mM) in the bath solution. The voltage protocol is shown at the top, and the pipette-mitoplast diagram indicates the recording conditions. The zero current level is indicated by the dotted line.
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15. Figure S6 Asymmetry of Substrate Binding to UCP1, Related to Figure 6
(A) The mean amplitude of IUCP1 carried by C6-sulfonate, recorded with either 50 mM C6-sulfonate in the pipette, 50 mM C6-sulfonate in the bath, or symmetrical 50 mM C6-sulfonate, n = 4. IUCP1 was measured in the beginning of the second (−50 mV, inward IUCP1) and third (+50 mV, outward IUCP1) voltage steps; see Figure 6D.
(B) Left panel: Representative IUCP1 recorded with 15 mM αCD in the pipette solution and 1 μM OA in the bath solution. Both bath and pipette solutions had pH values of 7.0. The voltage step protocol is indicated at the top of the panel; ΔV = 40 mV. Right panel: IUCP1 recorded from the same mitoplast after the pH in the bath solution was changed to 8.0. The holding potential was changed to −60 mV to eliminate depletion of the proton buffer inside the mitoplast between voltage steps. The zero current level is indicated by the dotted lines. The pipette-mitoplast diagram indicates the recording conditions. The IUCP1 amplitude measured at the beginning of the voltage steps was used to plot the UCP1 I/V curves in Figure 6E.
(C) Representative IUCP1 induced by 0.2 mM OA mixed with 10 mM MβCD in control (red), and in the presence of 100 μM (blue) and 1 mM (black) ATP. Note that the blue and the black traces coincide.
(D) Representative IUCP1 induced by 2 mM OA mixed with 10 mM MβCD in control (red), and in the presence of 100 μM (blue) and 1 mM (black) ATP.
Error bars represent SEM.
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16. Figure S7
Cl− Conductance of the IMM of BAT Is Primarily UCP1 Independent, Related to Figure 1
(A) Representative whole-mitoplast currents recorded from a wild-type mitoplast in symmetrical 150 mM Cl− in control (red) and after the addition of 1 mM GDP (black).
(B) The same experiment as in (A), but each trace represents an average of 30–40 original current traces to smooth out fluctuations in the outward Cl− current mediated by the large-conductance 108 pS anion channel (IMAC).
(C) Representative whole-mitoplast currents recorded from a UCP1−/− mitoplast in symmetrical 150 mM Cl− in control (red) and after addition of 1 mM GDP (black).
(D) The same experiment as in (C), but each trace represents an average of 30–40 original current traces to smooth out fluctuations in the outward Cl− current mediated by the large-conductance 108 pS anion channel (IMAC).
(E) Representative whole-mitoplast IUCP1 in a bath solution containing 2 mM (black) or 75 mM (red) Cl−. Bath and pipette solutions contain 1 mM Mg2+ to inhibit the IMAC current. Note the absence of large-conductance IMAC openings at positive potentials even in the presence of 75 mM Cl− in the bath (red trace).
(F) Current-voltage dependence of IUCP1 in a bath solution containing 2 mM (black) or 75 mM (red) Cl− (n = 5). The IUCP1 was recorded at symmetrical pH 7.0. IUCP1 was measured with a voltage step protocol from the holding potential of 0 mV (not shown). Current amplitudes were measured at the beginning of voltage steps.
Error bars represent SEM.
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