Regulation of the Inner Membrane Mitochondrial Permeability Transition by the Outer Membrane Translocator Protein (Peripheral Benzodiazepine Receptor)

Justina Šileikytė,Valeria Petronilli,Alessandra Zulian,Federica Dabbeni-Sala,Giuseppe Tognon,Peter Nikolov,Paolo Bernardi,Fernanda Ricchelli

doi:10.1074/jbc.m110.172486

Abstract

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

Highlights

Nel called the permeability transition pore (PTP)
The long-standing idea that the PTP may form at innerouter membrane contact sites and that it may be constituted by the adenine nucleotide translocator (ANT) in the inner mitochondrial membrane (IMM) and the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane (OMM) has not been confirmed by genetic ablation of these proteins [2,3,4], yet the PT can be regulated by proteins that interact with the OMM such as hexokinase [5, 6] and by ligands of the OMM translocator protein of 18 kDa, TSPO [7,8,9,10,11,12,13,14,15,16,17]
The PT of mitoplasts maintains most of the basic features of the PT observed in mitochondria (Ca2ϩ dependence, desensitization by cyclosporin A (CsA), activation by oxidants, inactivation by hematoporphyrin IX (HP) at low irradiation times), indicating that the PT is fundamentally an IMM event that can occur in the absence of an intact OMM

Summary

EXPERIMENTAL PROCEDURES

Reagents—HP, PP, deuteroporphyrin IX (DP), and coproporphyrin III (CP) were obtained from Frontier Scientific (Logan, UT), and stock solutions were prepared in dimethyl sulfoxide. Preparation of Mitoplasts—The mitochondrial suspension was added to solutions of varying digitonin concentrations in isolation buffer at a final protein concentration of 20 mg/ml. Porphyrin Uptake and Photosensitization of Mitochondria and Mitoplasts—Mitochondria and mitoplasts were suspended in standard medium, and porphyrins were added under gentle stirring at room temperature. The rates of permeabilization were studied after irradiation times ranging from 10 to 240 s at 40 W/m2 (total light doses between 0.04 and 0.96 J/cm2) Under these conditions, (i) porphyrins were bound to mitochondria and mitoplasts as monomers, the only species that is appreciably photoactive [39]; (ii) no measurable effects were induced in the dark (data not shown); and (iii) the molar absorption at the irradiation wavelength used (365 nm) was very similar for all porphyrins (difference within 10%), which reflects an equivalent number of absorbed photons (data not shown). Observations were made with an FEI Tecnai F12 transmission electron microscope

RESULTS

20 Ϯ 2 25 Ϯ 4 30 Ϯ 5 10 Ϯ 3

DISCUSSION