Dimers of mitochondrial ATP synthase form the permeability transition pore

Abstract

Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase.

Fig. 1.

CyPD interacts with OSCP and is displaced by Bz-423. (A) Extracts and immunoprecipitates of BHM with anti-OSCP or b or d subunit antibodies were immunoblotted as indicated. Lane 1, mitochondria; lane 2, immunoprecipitates with OSCP (Left), b (Center), and d (Right) antibodies; lanes 3, IgG antibody. (B) Total cell extracts (Left) and complex V immunoprecipitates (Right) of mitochondria from cells either untreated (lane 1) or treated with scrambled siRNA (lane 2) or OSCP siRNA (lane 3) probed for F₁ α,β-subunits, OSCP, and CyPD. (C) Heart mitochondria were treated with the indicated concentrations of Bz-423 (μM), immunoprecipitated with anticomplex V antibodies, and immunoblotted with antibodies against β-subunit or CyPD. Ratio between CyPD and β-band intensities is reported (n = 3 ± SE). *P ≤ 0.02; **P = 0.0015, Student t test.

Fig. 2.

Bz-423 decreases the mitochondrial Ca²⁺ retention capacity. Isolated WT (A and B) or Ppif^−/− mouse liver mitochondria (C) were incubated in the presence of 1 (open symbols) or 5 mM (closed symbols) Pi⋅Tris and Bz-423 as indicated. In B only, 1.6 μM CsA was added. Extramitochondrial Ca²⁺ was monitored, and CRC was determined by stepwise addition of 10 µM Ca²⁺ pulses. The measured CRC (i.e., the amount of Ca²⁺ accumulated before onset of Ca²⁺-induced Ca²⁺ release) was normalized to that obtained in absence of Bz-423 (CRC₀), and data are average of triplicate experiments ± SE. Absolute CRC values (nmol Ca²⁺/mg protein) at 1 mM Pi were 120 ± 0, 160 ± 20, and 166.7 ± 30.6 for A, B, and C, respectively; absolute CRC values (nmol Ca²⁺/mg protein) at 5 mM Pi were 86.7 ± 11.5, 126.7 ± 30.6, and 160 ± 20 for A, B, and C, respectively (n = 3 ± SD).

Fig. 3.

ATP synthase catalysis and OSCP knockdown affect the Ca²⁺ sensitivity of the PT. (A) Membrane potential was measured in mitochondria incubated with respiratory substrate and ADP plus an ATP-consuming system (trace a) or no substrate, ATP, and an ATP-regenerating system (trace b) as detailed in Materials and Methods. Where indicated, 2 mg mouse liver mitochondria (MLM), 1 µg/mL oligomycin (oligo), and 1 µM carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP). (B) Conditions for traces a and b were exactly as in A, but Ca²⁺ was measured. (C) The incubation medium contained respiratory substrate and 1 µg/mL oligomycin, and it was supplemented with 0.4 mM ADP (trace a) or 0.4 mM ATP (trace b); one experiment representative of three is shown. (D) Scrambled siRNA- or OSCP siRNA-treated cells (closed and open bars, respectively) were permeabilized with digitonin, and their CRC was measured in the presence of an ATP-regenerating system. Data are average ± SD of nine independent determinations per condition. *P = 0.0025 (Student t test). The blots display the levels of OSCP and F₁ β-subunits in the two batches of cells used (lanes 1, scrambled siRNA; lanes 2, OSCP siRNA).

Fig. 4.

Purification of F_OF₁ ATP synthase. (A) BHM were subjected to BNE to separate oligomers (O), dimers (D), and monomers (M) of ATP synthase, which were identified by Coomassie blue (lane 1) and in-gel activity staining (lane 2). Dimers and monomers were excised, eluted, subjected to SDS/PAGE, and stained with colloidal Coomassie (B), or they were transferred to nitrocellulose and tested for respiratory complexes and ATP synthase, subunit-β of F₁, ANT, VDAC, and CyPD (C). T, D, and M refer to total extract, dimer, and monomer, respectively. One experiment representative of three is shown.

Fig. 5.

Dimers of F_OF₁ ATP synthase generate currents matching MMC-PTP. (A) A bilayer experiment in 50 mM KCl, 1 mM Pi, and 0.3 mM Ca²⁺ (Ca²⁺ only in trans; V_cis = −60 mV). After addition of dimeric ATP synthase, no activity could be observed (Upper) until immediately after the addition of 0.1 mM Bz-423 to the trans side (Lower; arrow). When monomers were used, the recording was identical to Upper, which was not modified by the addition of Bz-423. (B) A similar experiment in the presence of 0.1 mM PhAsO in trans. Activity (Upper) was elicited by the addition of Bz-423 as in A and inhibited by 0.1 mM AMP-PNP in trans (Lower; arrow). Corresponding current amplitude histograms from gap-free 60-s traces are shown in Right. (C) Current traces (150 mM KCl; V_cis as indicated) with dimeric ATP synthase and 0.3 mM Ca²⁺, 0.1 mM Bz-423, and 50 μM PhAsO added to the trans side. Note numerous substates. (D, Left) Activity (V_cis = −80 mV) recorded as in C (first trace) and after sequential additions of Mg²⁺ (0.6 mM) and ADP (0.6 mM) to the trans side; (D, Right) corresponding amplitude histograms from gap-free 100-s traces. Representative experiments are shown of a total of 28 experiments performed under various conditions using six different ATP synthase dimer preparations.