1 F-ATPase of D. melanogaster Forms 53 pS Channels Responsible for Mitochondrial Ca 2+ -induced Ca 2+ Release Sophia von Stockum ‡ , Valentina Giorgio ‡ , Elena Trevisan ‡ , Giovanna Lippe § , Gary D. Glick ¶ , Michael A. Forte ǀǀ , Caterina Da-Rè ** , Vanessa Checchetto ** , Gabriella Mazzotta ** , Rodolfo Costa ** , Ildikò Szabò ** and Paolo Bernardi ‡1 Running title: Drosophila F-ATPase forms 53 pS channels ‡ Department of Biomedical Sciences, University of Padova and Consiglio Nazionale delle Ricerche Neuroscience Institute, Padova, Italy, § Department of Food Science, University of Udine, I-33100 Udine, Italy, ¶ Department of Chemistry, Graduate Program in Immunology, University of Michigan, Ann Arbor, Michigan 48109, the ǀǀ Vollum Institute, Oregon Health and Sciences University, Portland, Oregon 97239 and the ** Department of Biology, University of Padova, Italy Address correspondence to Paolo Bernardi, E-mail: bernardi@bio.unipd.it, Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, I-35121 Padova, Italy, Fax: 39-049-827-6049 Background: The Ca 2+ -induced Ca 2+ release channel (mCrC) of Drosophila mitochondria is similar to the permeability transition pore (PTP). Results: mCrC is modulated by PTP effectors and Drosophila F-ATPase forms 53 pS channels. Conclusion: F-ATPase mediates Ca 2+ -induced Ca 2+ release in Drosophila mitochondria. Significance: Channel formation by F-ATPases has been conserved in evolution, but species-specific features exist that may underscore different roles in different organisms. Mitochondria of D. melanogaster undergo Ca 2+ - induced Ca 2+ release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 pS in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca 2+ and H + . We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423 and Mg 2+ /ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S 2 R + cells sensitizes the mCrC to Ca 2+ but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53 pS channels activated by Ca 2+ and thiol oxidants, and inhibited by Mg 2+ /-imino ATP. These findings indicate that the mCrC is the “PTP” of D. melanogaster, and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species. Mitochondria of D. melanogaster possess an array of Ca 2+ transport pathways, i.e. the Ca 2+ uniporter MCU, the Na + /Ca 2+ exchanger NCLX and a Na + - insensitive Ca 2+ efflux system (1) that display the same features as those observed in mammalian mitochondria (2-5). An important difference, however, exists. In mammalian mitochondria Ca 2+ and Pi induce opening of the permeability transition pore (PTP 2 ), a 500 pS channel that forms from the F- ATPase under conditions of oxidative stress (6). The PTP is permeable to sucrose, as is the pore of yeast mitochondria (7,8) where Pi has an inhibitory effect and F-ATPase forms 300 pS channels (9). Also in D. melanogaster Ca 2+ opens a mitochondrial permeability pathway resulting in depolarization and Ca 2+ release (1); yet this Ca 2+ - induced Ca 2+ release channel (mCrC, which like in yeast is inhibited rather than activated by Pi) is impermeable to sucrose, suggesting that its size may be considerably smaller than that of the PTP (1). The recent demonstration that the PTP forms from the F-ATPase (6,9) provides a new framework to analyze the nature of the mCrC of D. melanogaster. Here we have assessed whether F-ATPase dimers purified from Drosophila mitochondria possess channel activity. Our findings provide novel information on the channel function of F-ATPases, establish that the mCrC is the “PTP” of D. melanogaster, and shed new light http://www.jbc.org/cgi/doi/10.1074/jbc.C114.629766 The latest version is at JBC Papers in Press. Published on December 30, 2014 as Manuscript C114.629766 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on February 2, 2017 http://www.jbc.org/ Downloaded from