Mitochondria play a pivotal role in cell survival and tissue development by virtue of their role in energy metabolism, regulation of cellular Ca 2+ homeostasis and apoptosis. Given this multifactorial role, Ca 2+ homeostasis, metabolism, and bioenergetics function as an integrated system since energy conservation is used to drive each process. Mitochondrial energy conservation (ATP production) requires the respiration-driven formation of a proton electrochemical potential difference (delta mu H) across the inner mitochondrial membrane (IMM), which is created by proton pumping by the respiratory complexes. Maintenance of the gradient demands a low permeability of the IMM to protons, charged species and solutes. Yet, mitochondria in vitro can easily undergo an IMM permeability increase to solutes with molecular masses of about 1,500 Da or lower. This permeability change, called the permeability transition (PT), is regulated by the opening of a membrane pore, the mitochondrial permeability transition pore (PTP). PTP opening in vitro has dramatic consequences on mitochondrial function (e.g., collapse of the delta mu H and depletion of pyridine nucleotides) and structure (release of cytochrome c) that lead to respiratory inhibition. This process has long been studied as a potential target for mitochondrial dysfunction in vivo and as a mediator of programmed cell death (PCD) through the release of cytochrome c and other intermembrane proteins active on the apoptotic machinery. However, despite detailed functional characterization over the last 30 years, the molecular components forming the PTP have been not been definitively established nor has the precise role of the PTP in vivo been defined. This proposal is based in the synergy possible through the combination of novel approaches available in our two laboratories. Our specific plans include the following aims:
Aim 1 : In screens for chemical inhibitors of the PTP, we have identified Ro 68-3400 in functional assays as a high affinity (nM) blocker of the PTP through covalent modification of isoform 1 of mammalian VDAC (VDAC1). Similar experiments have also demonstrated that yeast VDAC1 is specifically targeted by this compound. We plan to use our experience with both mammalian and yeast VDAC to pin-point the structural requirements for high affinity association of VDAC with this compound, examine other mammalian VDAC isoforms for their ability to be modified by Ro 68-3400 and test the sensitivity of mitochondria treated with this novel PTP blocker to proteins in the BCL-2 family.
Aim 2 : Traditionally, the PTP has been considered to be a dynamic multiprotein complex formed at inner/outer membrane contact sites through the interaction of the adenine nucleotide translocator (ANT) of the IMM, VDAC in the OMM and a matrix regulatory protein, mitochondrial cyclophilin D (CyP-D). However, evidence implicating the ANT in the PTP complex has not been supported by recent data. Therefore, in this aim we plan to take advantage of Ro 68-3400 as a specific tool to further define the core components forming the PTP, with a specific focus on the identification of the IMM partner for VDAC in the pore complex.
Aim 3 : Inhibition by cyclosporin A (CsA) and non-immunosuppressive analogs has become the standard diagnostic tool for the characterization of the PTP in isolated mitochondria, in living cells, and in vivo. The target of CsA in these studies, CyP-D, is the only component of the PTP whose role has been definitively established. The goal of this aim is to unambiguously resolve basic questions related to the influence of CyP-D on the PTP, the participation of the PTP in specific aspects of the apoptotic program, and its role in specific pathological processes of significance to human, disease through the use of mice in which the expression of CyP-D and MVDAC1 have been eliminated by """"""""knock-out"""""""" strategies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Zatz, Marion M
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Oregon Health and Science University
Schools of Medicine
United States
Zip Code
Roy, Sudeshna; Šileikyt?, Justina; Neuenswander, Benjamin et al. (2016) N-Phenylbenzamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore. ChemMedChem 11:283-8
Carraro, Michela; Bernardi, Paolo (2016) Calcium and reactive oxygen species in regulation of the mitochondrial permeability transition and of programmed cell death in yeast. Cell Calcium 60:102-7
Šileikyt?, Justina; Forte, Michael (2016) Shutting down the pore: The search for small molecule inhibitors of the mitochondrial permeability transition. Biochim Biophys Acta 1857:1197-1202
Bernardi, Paolo; Di Lisa, Fabio; Fogolari, Federico et al. (2015) From ATP to PTP and Back: A Dual Function for the Mitochondrial ATP Synthase. Circ Res 116:1850-62
Roy, Sudeshna; Šileikyt?, Justina; Schiavone, Marco et al. (2015) Discovery, Synthesis, and Optimization of Diarylisoxazole-3-carboxamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore. ChemMedChem 10:1655-71
von Stockum, Sophia; Giorgio, Valentina; Trevisan, Elena et al. (2015) F-ATPase of Drosophila melanogaster forms 53-picosiemen (53-pS) channels responsible for mitochondrial Ca2+-induced Ca2+ release. J Biol Chem 290:4537-44
Bernardi, Paolo; Forte, Michael (2015) Commentary: SPG7 is an essential and conserved component of the mitochondrial permeability transition pore. Front Physiol 6:320
Bernardi, Paolo; Di Lisa, Fabio (2015) The mitochondrial permeability transition pore: molecular nature and role as a target in cardioprotection. J Mol Cell Cardiol 78:100-6
Bernardi, Paolo; Rasola, Andrea; Forte, Michael et al. (2015) The Mitochondrial Permeability Transition Pore: Channel Formation by F-ATP Synthase, Integration in Signal Transduction, and Role in Pathophysiology. Physiol Rev 95:1111-55
Šileikyt?, Justina; Blachly-Dyson, Elizabeth; Sewell, Randall et al. (2014) Regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral benzodiazepine receptor (Translocator Protein of 18 kDa (TSPO)). J Biol Chem 289:13769-81

Showing the most recent 10 out of 31 publications