Long‐chain fatty acids promote opening of the reconstituted mitochondrial permeability transition pore

Abstract

Adenine nucleotide translocase^porin^hexokinase complex isolated from rat brain, when reconstituted into phospholipid-cholesterol vesicles, exhibits all properties of the mitochondrial permeability transition pore [Beutner, G., Ru «ck, A., Riede, B., Welte, W. and Brdiczka, D. (1996) FEBS Lett. 396, 189^195]. In the present work, the effect of long-chain fatty acids on such reconstituted pore was examined. Opening of the pore was measured by leakage of either malate or fluorescein sulphonate entrapped inside the vesicles. It was found that myristate and oleate in the presence of 50 or 100 W WM Ca 2+ produced a partial release of the probes in a dose-dependent way. A dicarboxylic fatty acid analogue, that appeared inactive as protonophore in intact mitochondria, exerted no effect on pore opening in the reconstituted system. 100 W WM Ca 2+ alone was without effect. Pore opening by fatty acids in the reconstituted system was partly prevented by cyclosporin A. The pore opening also occurred when the vesicles were incubated in the presence of pancreatic phospholipase A 2 . In this case, the opening was decreased by cyclosporin A or serum albumin. These results indicate that long-chain fatty acids elicit opening of the permeability transition pore reconstituted in phospholipid vesicles in a similar way as in intact mitochondria [Wie°ckowski, M.R.

Betulinic acid induces cytochrome c release and apoptosis in a Bax/Bak-independent, permeability transition pore dependent fashion

“…Importantly, some tumor cells have been shown to contain higher cholesterol levels in their mitochondrial membrane and lowering this sensitizes them to apoptosis [32]. More importantly, reconstituted PT pores in artificial membranes localize to cholesterol areas and can be opened using long chain fatty acids [45], indicating that lipids have a strong modulating effect on the pore.…”

Transglutaminase Type II Is Involved in the Pathogenesis of Endotoxic Shock

“…Heart tissues from WT and TG2 Ϫ/Ϫ animals were removed immediately after sacrifice and homogenized in 250 mM sucrose, 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, and protease inhibitor (Roche), and mitochondria were isolated (33). Approximately 20 g of proteins was separated on a 5-12% precast SDS-polyacrylamide gel (Invitrogen Life Technologies) and transferred to nitrocellulose membrane.…”

The permeability transition and BCL-2 family proteins in apoptosis: co-conspirators or independent agents?

“…The inner and outer mitochondrial membranes appear to interact in specialized regions called the 'contact sites', which are enriched in adenine nucleotide translocase (ANT) of the IMM and the voltage-dependent anion channel (VDAC) of the OMM bound to cytosolic hexokinase I. Reconstitution of PTP activity after partial purification of these hexokinase-bound complexes led to the idea that the PTP may be formed by ANT and VDAC molecules interacting at these sites, [10][11][12][13] possibly along with ancillary regulatory proteins such as cyclophilin D (CyP-D), hexokinase and the peripheral benzodiazepine receptor (PBR) (a review of the data supporting this view of the molecular composition of the PTP can be found in Crompton 14 ). However, conclusive evidence that the ANT is not essential for PTP formation was obtained in a detailed analysis of mitochondria lacking all ANT isoforms, which revealed that a Ca 2 þ -dependent PT took place.…”