1 Scientific RepoRts | 6:26700 | DOI: 10.1038/srep26700 www.nature.com/scientificreports Alterations in voltage-sensing of the mitochondrial permeability transition pore in ANT1-defcient cells Judit Doczi 1,2 , Beata torocsik 1 , Andoni echaniz-Laguna 3 , Bénédicte Mousson de Camaret 4 , Anatoly starkov 5 , Natalia starkova 6 , Aniko Gál 7 , Mária J Molnár 7 , Hibiki Kawamata 5 , Giovanni Manfredi 5 , Vera Adam-Vizi 1 & Christos Chinopoulos 1,2 the probability of mitochondrial permeability transition (mpt) pore opening is inversely related to the magnitude of the proton electrochemical gradient. the module conferring sensitivity of the pore to this gradient has not been identifed. We investigated mPT’s voltage-sensing properties elicited by calcimycin or H 2 o 2 in human fbroblasts exhibiting partial or complete lack of ANT1 and in C2C12 myotubes with knocked-down ANT1 expression. mPT onset was assessed by measuring in situ mitochondrial volume using the ‘thinness ratio’ and the ‘cobalt-calcein’ technique. De-energization hastened calcimycin-induced swelling in control and partially-expressing ANT1 fbroblasts, but not in cells lacking ANT1, despite greater losses of mitochondrial membrane potential. Matrix Ca 2+ levels measured by X-rhod-1 or mitochondrially-targeted ratiometric biosensor 4mtD3cpv, or ADP-ATP exchange rates did not difer among cell types. ANT1-null fbroblasts were also resistant to H 2 o 2 - induced mitochondrial swelling. Permeabilized C2C12 myotubes with knocked-down ANT1 exhibited higher calcium uptake capacity and voltage-thresholds of mpt opening inferred from cytochrome c release, but intact cells showed no diferences in calcimycin-induced onset of mPT, irrespective of energization and ANT1 expression, albeit the number of cells undergoing mPT increased less signifcantly upon chemically-induced hypoxia than control cells. We conclude that ANT1 confers sensitivity of the pore to the electrochemical gradient. Mitochondria that are subject to calcium overload exhibit a permeability transition mediated by a pore forming in the inner mitochondrial membrane 1,2 . Te identity of the structural components of this pore has been until recently unknown; in the past three years though, the c-rings of the ATP synthase 3,4 , and the interface within ATP synthase dimers 5 are being strongly favoured for flling this gap of knowledge 6,7,2 , but still with a number of questions unanswered, reviewed in 8–10 . Nonetheless, over the past three decades extensive amount of eforts have focused on the regulation and functional characteristics of this phenomenon 11 , and an inverse correlation of the probability of pore opening to the magnitude of the electrochemical gradient has been thoroughly char- acterized 12–15 . Te molecular entity responsible for the voltage-dependence of the pore has not been identifed, though the tuning of this sensor by the oxidation-reduction state of vicinal thiols as well as the contribution of critical arginines has been reported 16–19 . Relevant to this, glutathione depletion in cultured neurons by mon- ochlorobimane was shown to initiate bioenergetic defciency that was mediated by inhibition of ANT 20 . ANT exhibits a number of thiols that are amenable to oxidation by several agents 21,22 , some of which are unmasked in 1 Department of Medical Biochemistry, Semmelweis University MtA-Se Laboratory for neurobiochemistry, Budapest, 1094, Hungary. 2 MTA-SE Lendület Neurobiochemistry Research Group, Budapest, Hungary. 3 Département de Neurologie, Hôpitaux Universitaires, Hôpital de Hautepierre, 67098 Strasbourg cedex, France. 4 Service des Maladies Héréditaires du Métabolisme, Centre de Biologie et de Pathologie Est, CHU Lyon, 69677 Bron cedex, France. 5 Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY 10065, USA. 6 Icahn School of Medicine at Mount Sinai, Department of Hematology and Medical Oncology, New York, NY 10029, USA. 7 Institute of Genomic Medicine and Rare Disorders, Semmelweis University, Budapest, 1083, Hungary. Correspondence and requests for materials should be addressed to C.C. (email: chinopoulos.christos@eok.sote.hu) received: 10 May 2015 Accepted: 09 May 2016 Published: 25 May 2016 opeN