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, 289 (20), 13769-81

Regulation of the Mitochondrial Permeability Transition Pore by the Outer Membrane Does Not Involve the Peripheral Benzodiazepine Receptor (Translocator Protein of 18 kDa (TSPO))

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Regulation of the Mitochondrial Permeability Transition Pore by the Outer Membrane Does Not Involve the Peripheral Benzodiazepine Receptor (Translocator Protein of 18 kDa (TSPO))

Justina Šileikytė et al. J Biol Chem.

Abstract

Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances. Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a role in the mitochondrial permeability transition pore (PTP), which has been associated with cell death in many human pathological conditions. Here, we critically assess the role of TSPO in the function of the PTP through the generation of mice in which the Tspo gene has been conditionally eliminated. Our results show that 1) TSPO plays no role in the regulation or structure of the PTP, 2) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, 3) outer mitochondrial membrane regulation of PTP activity occurs though a mechanism that does not require TSPO, and 4) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from control mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP.

Keywords: Drug Action; Gene Knockout; Mitochondria; Mitochondrial Permeability Transition; Photodynamic Effect; Porphyrin.

Figures

FIGURE 1.
FIGURE 1.
Generation and characterization of liver-specific TSPO-null mitochondria. A, outline of the creation of TspoloxP mice and outline of final genetic structure, with the location of primers used for genotyping and analysis indicated. See “Experimental Procedures” for details. B, Western blot analysis of mitochondria isolated from Tspo+/+, TspoloxP, and AlbCre;TspoloxP mouse livers. Ndufs1 antibody was used as a loading control. C, [3H]PK11195 binding to TspoloxP (black symbols) and AlbCre;TspoloxP liver mitochondria (red symbols) was assessed as described under “Experimental Procedures.” D, Scatchard plots of experiments described in C. Error bars, mean ± S.D. of three independent experiments performed in duplicate. B refers to bound, and F refers to free.
FIGURE 2.
FIGURE 2.
Morphological and functional features of TSPO-null liver mitochondria. A, transmission electron microscopy of TspoloxP (left) and AlbCre;TspoloxP mouse livers (right); bar, 500 nm. B, oxygen consumption rate (OCR) of mitochondria of the indicated genotypes incubated with succinate plus rotenone (squares) or glutamate plus malate (circles). Where indicated, 4 mm ADP, 2.5 μg/ml oligomycin, 4 μm FCCP, and 2 μm antimycin plus 2 μm rotenone (circles only) were added. Error bars, mean ± S.D. C, typical recording traces of Rh123 fluorescence changes in TspoloxP (black trace) and AlbCre;TspoloxP (red trace) liver mitochondria. Where indicated, 0.5 mg/ml mitochondria, 5 mm succinate, and 0.5 μm FCCP were added. a.u., absorbance units.
FIGURE 3.
FIGURE 3.
Mitochondrial Ca2+ retention capacity of TspoloxP and AlbCre;TspoloxP mitochondria; effect of TSPO ligands. TspoloxP (A and C) and AlbCre;TspoloxP (B and D) mouse liver mitochondria (0.5 mg/ml) were suspended in AB supplemented with 5 mm succinate plus 1 μm rotenone and 0.5 μm Calcium Green-5N and loaded with a train of Ca2+ pulses. A and B, in traces b, the medium was supplemented with 1 μm CsA. C and D, in traces b, c, and d, the mitochondrial suspensions were supplemented with 100 μm PK11195, 100 μm Ro5-4864, and 8 μm PP IX, respectively, immediately prior to Ca2+ additions. Representative traces of at least three independent experiments are shown. a.u., absorbance units.
FIGURE 4.
FIGURE 4.
Mitochondrial Ca2+ retention capacity of TspoloxP and AlbCre;TspoloxP mitochondria; effect of Pi, arachidonic acid, PhAsO, and Cu(OP)2. A–D, liver mitochondria from TspoloxP (black symbols) or AlbCre;TspoloxP mice (red symbols) were energized with 5 mm glutamate plus 2.5 mm malate in the presence of the indicated concentrations of Pi, arachidonic acid, PhAsO, or Cu(OP)2. In B–D, the Pi concentration was 1 mm. Values are the mean ± S.D. (error bars) of at least three independent experiments performed in duplicate.
FIGURE 5.
FIGURE 5.
Effect of NEM, Cu(OP)2, and PhAsO on mitochondria and mitoplasts. 0.5 mg/ml Tspo+/+ liver mitochondria (trace a) or mitoplasts (trace b) (A) and TspoloxP (trace a) or AlbCre;TspoloxP (trace b) liver mitochondria (B) were suspended in assay buffer supplemented with 5 mm succinate plus 1 μm rotenone; where indicated, 150 μm Ca2+, 0.5 mm NEM, and 5 μm alamethicin were added. C–F, 0.5 mg/ml TspoloxP (C and E) or AlbCre;TspoloxP mouse liver mitochondria (D and F) in the same buffer as in A and B were supplemented with 15 μm Ca2+ and 5 μm Cu(OP)2 (C and D) or 15 μm PhAsO (E and F) (traces a). In traces b (C and D) 0.2 mm MBM+ was added prior to Cu(OP)2. E and F, medium was supplemented with 1 μm CsA. Representative traces of three independent experiments are shown.
FIGURE 6.
FIGURE 6.
Effect of irradiation on the rate of permeabilization of TspoloxP or AlbCre;TspoloxP mitochondria. TspoloxP (black symbols) or AlbCre;TspoloxP (red symbols) liver mitochondria (0.5 mg/ml) were treated with 0.5 μm PP IX and kept in the dark or irradiated for the indicated time prior to the addition of 220 μm Ca2+, and mitochondrial volume changes were followed. Permeabilization rates were normalized to those of non-irradiated mitochondria. Error bars, S.D. of three independent experiments. a.u., absorbance units.
FIGURE 7.
FIGURE 7.
Effect of ischemia/reperfusion on LDH release in hearts from TspoloxP and Myh6Cre/Esr1;TspoloxP mice. A, tamoxifen was administered to mice as indicated, followed by sacrifice and heart isolation 3 days after the last tamoxifen injection. B, top, Western blot analysis of heart homogenates prepared from Myh6Cre/Esr1;TspoloxP mice treated with vehicle (lanes 1 and 2) or tamoxifen (lanes 3 and 4); each lane refers to one mouse. Bottom, LDH activity released in the coronary effluent during postischemic reperfusion (percentage of total LDH). Values are mean ± S.D. (error bars) of six independent experiments.

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