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A Novel Ion Channel in ATP Synthase C-Subunit Ring: Gatekeeper of Life and Death
310a
Monday, February 29, 2016
Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7CypD binding prevented Ca2þ- and ROS-induced DJm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site. 1521-Pos Board B498 A Novel Ion Channel in ATP Synthase C-Subunit Ring: Gatekeeper of Life and Death Nelli Mnatsakanyan, Han-A Park, Jing Wu, Paige Miranda, Elizabeth A. Jonas. Internal Medicine, Yale University, New Haven, CT, USA. Mitochondria play a central role in ageing, cardiac disease, cell death and cancer. Different pro-death signals lead to the permeabilization of the outer and inner mitochondrial membranes. Permeabilization of the inner mitochondrial membrane is facilitated by the mitochondrial permeability transition pore (mPTP), which is a key player in activation of cell death pathways. mPTP is a voltage and Ca2þ-dependent high conductance channel. The brief opening of mPTP may serve physiological functions, but its prolonged opening leads to swelling of the mitochondrial matrix, rupture of outer and inner membranes and eventually to cell death. In spite of critical roles played by mPTP, its molecular nature remains mysterious and was a subject of scientific debate for many years. We recently demonstrated that the membrane-embedded c-subunit ring of F1FO-ATP synthase can form an uncoupling channel with biophysical characteristics of mPTP, nevertheless the conformational changes in ATP synthase leading to the opening of the c-subunit channel are still poorly understood. We have used site-directed mutagenesis accompanied by patch clamp recording as well as protein-protein interaction studies to gain a new understanding of the gating mechanism of the c-subunit leak channel. We have also expressed wild-type and mutant c-subunit channels in primary hippocampal neurons to study the role of the c-subunit channel in neuronal cell death, neuronal function and survival. We present a novel structural-functional model of mPTP gating by highlighting the molecular mechanisms and conformational changes in the F1FO-ATP synthase necessary to open the c-subunit channel. 1522-Pos Board B499 Is the C-Subunit Ring of the F1Fo ATP Synthase the Elusive Mitochondrial Permeability Transition Pore? Wenchang Zhou, Corrine Nief, Jose´ D. Faraldo-Go´mez. National Heart, Lung and Blood Institute, Bethesda, MD, USA. Certain metabolic conditions induce an increase in the permeability of the mitochondrial inner membrane, which, if prolonged, ultimately leads to cell death. It is believed that at the onset of this so-called permeability transition, the opening of an ion channel causes the inner membrane to become depolarized, which halts ATP production and has other deleterious effects such as the release of mitochondrial Ca2þ. The molecular identity of this channel, refered to as Mitochodrial Permeability Transition Pore (MPTP) remains to be conclusively established and has been the subject of much controversy. A recent electrophysiological study purports to have demonstrated that the elusive MPTP is, surprisingly, the lumen of the c-subunit ring of the F1Fo ATP synthase, under conditions in which the catalytic domain, F1, becomes detached from the ring. Here, we examine this claim through theoretical calculations of the ion conductance of two representative c-rings, which differ in their c-subunit stoichiometry and thus in the width of their lumen. These calculations, which are based on fully-atomistic molecular dynamics simulations, clearly demonstrate that the lumen of the c-ring cannot possibly sustain the conductance levels associated with the MPTP, even under the assumption that the lumen is fully hydrated, rather than occupied by lipid molecules. 1523-Pos Board B500 Mitochondrial Permeability Transition Pore (mPTP) Formation Requires the Participation of c-Subunit of ATP-Synthase, Polyhydroxybutyrate (PHB) and Inorganic Polyphosphate (polyP) Pia A. Elustondo1, Nelli Mnatsakanyan2, Zakharian Eleonora3, Elizabeth A. Jonas2, Evgeny Pavlov4. 1 Physiology and Biophysics, Dalhousie University, Halifax, NS, Canada, 2 Internal Medicine, Yale University, New Haven, CT, USA, 3Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL, Canada, 4Department of Basic Sciences, New York University, New York, NY, USA. Mitochondrial Permeability Transition Pore (mPTP) is a channel in the mitochondrial inner membrane. Opening of mPTP during acute stress conditions following ischemia-reperfusion is the principal molecular event leading to cell death and tissue damage. We demonstrated previously that a highly puri-
fied mitochondrial fraction containing c-subunit of ATP synthase (c-subunit), polyP and PHB possesses channel activity with properties resembling mPTP as seen in native mitochondrial membranes. Importantly, we have been able to purify this fraction mainly from mitochondria with calcium-activated mPTP. When mitochondria were exposed to the mPTP blocker Cyclosporine A, the components of the channel forming fraction were reduced to control levels. Here we investigate the molecular details of the interactions between components of the channel-forming complex. Using immunoblot and mass spectrometry approaches we demonstrate that c-subunit purified from intact mitochondria is closely associated with PHB. Furthermore, we are able to reconstitute mPTP-like channel activity in artificial lipid bilayers by combining purified mammalian c-subunit with synthetic polyP in the presence of calcium. We propose that the c-subunit-PHB-polyP complex is sufficient and essential for formation of the mPTP channel. According to our hypothesis, during cellular stress conditions, calcium induces formation of the complex between the c-subunit, PHB and polyP. This is a critical event required for mPTP activation. 1524-Pos Board B501 Contribution of Inorganic Polyphosphate Towards Regulation of Mitochondrial Free Calcium M. de la Encarnacion Solesio Torregrosa1, Lusine Demirkhanyan2, Eleonora Zakharian2, Evgeny Pavlov1. 1 Basic Sciences, NYU-College of Dentistry, NYC, NY, USA, 2Department of Cancer Biology and Pharmacology, University of Illinois, Peoria, IL, USA. Calcium signaling plays a key role in the regulation of multiple processes in mammalian mitochondria, ranging from the physiological regulation of cellular bioenergetics to the induction of stress-induced cell death. While the total concentration of calcium inside the mitochondria can increase by several orders of magnitude, the concentration of bioavailable, free calcium in mitochondria is maintained within the micromolar range by the mitochondrial calcium buffering system. It is established that this calcium buffering system involves participation of orthophosphate. However, the mechanisms of its function are not yet understood. Specifically, it is not clear how orthophosphate is capable to regulate free calcium concentration in the micromolar range. We hypothesized that inorganic polyphosphate, a biological polymer, made of up to 100 orthophosphates, plays a significant role in this regulation on mitochondria. We used confocal fluorescent microscopy to measure the relative levels of free calcium in this organelle, in cultured hepatoma (HepG2) cells with variable levels of inorganic polyphosphate. We found that the depletion of inorganic polyphosphate leads to the significantly lower levels of mitochondrial free calcium concentration, under conditions of pathological calcium overload. We conclude that inorganic polyphosphate is a previously unrecognized integral component of the mitochondrial calcium buffering system. 1525-Pos Board B502 Formation of Polyphosphate-Poly-Beta-Hydroxybutyrate Granule-Like Complexes in Heart Failure Myocytes Lusine Demirkhanyan1, Ian P. Palmer2, Walter Boyd3, Claus S. Sondergaard3, Kristin Grimsrud4, Leigh G. Griffiths4, Julie Bossuyt2, Donald M. Bers2, Eleonora Zakharian1, Elena N. Dedkova2. 1 Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL, USA, 2Department of Pharmacology, School of Medicine, University of California, Davis, CA, USA, 3Department of Surgery, School of Medicine, University of California, Davis, CA, USA, 4 School of Veterinary Medicine, University of California, Davis, CA, USA. Inorganic polyphosphate (polyP) is a naturally occurring polyanion made of ten to several hundred orthophosphates linked together by phosphoanhydride bonds. Specific physiological roles of polyP vary dramatically depending on its size, concentration, tissue and subcellular localization. Recently we reported that mitochondria of rabbit ventricular myocytes contain significant amounts (280 5 60 pmol/mg of protein) of polyP with an average length of 25 orthophosphates, and that polyP is involved in Ca2þ-dependent activation of the mitochondrial permeability transition pore (mPTP). Using DAPI as a probe for polyP, we showed that polyP levels depend on the activity of the mitochondrial respiratory chain. Here, we visualized intracellular polyP distribution in cardiomyocytes using the affinity of the Xpress epitope-tagged recombinant polyphosphate-binding domain (PPBD) of E. coli exopolyphosphatase and immunocytochemical approach. Primarily mitochondrial location of polyP was demonstrated in both control and heart failure (HF) myocytes from two different HF models: (1), non-ischemic rabbit HF model induced by combined aortic insufficiency and stenosis and (2), targeted microbead embolization of the first diagonal branch of left anterior descending coronary artery in Yucatan mini-pigs. However, enhanced formation of polyP dense spots was observed in
Monday, February 29, 2016
Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7CypD binding prevented Ca2þ- and ROS-induced DJm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site. 1521-Pos Board B498 A Novel Ion Channel in ATP Synthase C-Subunit Ring: Gatekeeper of Life and Death Nelli Mnatsakanyan, Han-A Park, Jing Wu, Paige Miranda, Elizabeth A. Jonas. Internal Medicine, Yale University, New Haven, CT, USA. Mitochondria play a central role in ageing, cardiac disease, cell death and cancer. Different pro-death signals lead to the permeabilization of the outer and inner mitochondrial membranes. Permeabilization of the inner mitochondrial membrane is facilitated by the mitochondrial permeability transition pore (mPTP), which is a key player in activation of cell death pathways. mPTP is a voltage and Ca2þ-dependent high conductance channel. The brief opening of mPTP may serve physiological functions, but its prolonged opening leads to swelling of the mitochondrial matrix, rupture of outer and inner membranes and eventually to cell death. In spite of critical roles played by mPTP, its molecular nature remains mysterious and was a subject of scientific debate for many years. We recently demonstrated that the membrane-embedded c-subunit ring of F1FO-ATP synthase can form an uncoupling channel with biophysical characteristics of mPTP, nevertheless the conformational changes in ATP synthase leading to the opening of the c-subunit channel are still poorly understood. We have used site-directed mutagenesis accompanied by patch clamp recording as well as protein-protein interaction studies to gain a new understanding of the gating mechanism of the c-subunit leak channel. We have also expressed wild-type and mutant c-subunit channels in primary hippocampal neurons to study the role of the c-subunit channel in neuronal cell death, neuronal function and survival. We present a novel structural-functional model of mPTP gating by highlighting the molecular mechanisms and conformational changes in the F1FO-ATP synthase necessary to open the c-subunit channel. 1522-Pos Board B499 Is the C-Subunit Ring of the F1Fo ATP Synthase the Elusive Mitochondrial Permeability Transition Pore? Wenchang Zhou, Corrine Nief, Jose´ D. Faraldo-Go´mez. National Heart, Lung and Blood Institute, Bethesda, MD, USA. Certain metabolic conditions induce an increase in the permeability of the mitochondrial inner membrane, which, if prolonged, ultimately leads to cell death. It is believed that at the onset of this so-called permeability transition, the opening of an ion channel causes the inner membrane to become depolarized, which halts ATP production and has other deleterious effects such as the release of mitochondrial Ca2þ. The molecular identity of this channel, refered to as Mitochodrial Permeability Transition Pore (MPTP) remains to be conclusively established and has been the subject of much controversy. A recent electrophysiological study purports to have demonstrated that the elusive MPTP is, surprisingly, the lumen of the c-subunit ring of the F1Fo ATP synthase, under conditions in which the catalytic domain, F1, becomes detached from the ring. Here, we examine this claim through theoretical calculations of the ion conductance of two representative c-rings, which differ in their c-subunit stoichiometry and thus in the width of their lumen. These calculations, which are based on fully-atomistic molecular dynamics simulations, clearly demonstrate that the lumen of the c-ring cannot possibly sustain the conductance levels associated with the MPTP, even under the assumption that the lumen is fully hydrated, rather than occupied by lipid molecules. 1523-Pos Board B500 Mitochondrial Permeability Transition Pore (mPTP) Formation Requires the Participation of c-Subunit of ATP-Synthase, Polyhydroxybutyrate (PHB) and Inorganic Polyphosphate (polyP) Pia A. Elustondo1, Nelli Mnatsakanyan2, Zakharian Eleonora3, Elizabeth A. Jonas2, Evgeny Pavlov4. 1 Physiology and Biophysics, Dalhousie University, Halifax, NS, Canada, 2 Internal Medicine, Yale University, New Haven, CT, USA, 3Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL, Canada, 4Department of Basic Sciences, New York University, New York, NY, USA. Mitochondrial Permeability Transition Pore (mPTP) is a channel in the mitochondrial inner membrane. Opening of mPTP during acute stress conditions following ischemia-reperfusion is the principal molecular event leading to cell death and tissue damage. We demonstrated previously that a highly puri-
fied mitochondrial fraction containing c-subunit of ATP synthase (c-subunit), polyP and PHB possesses channel activity with properties resembling mPTP as seen in native mitochondrial membranes. Importantly, we have been able to purify this fraction mainly from mitochondria with calcium-activated mPTP. When mitochondria were exposed to the mPTP blocker Cyclosporine A, the components of the channel forming fraction were reduced to control levels. Here we investigate the molecular details of the interactions between components of the channel-forming complex. Using immunoblot and mass spectrometry approaches we demonstrate that c-subunit purified from intact mitochondria is closely associated with PHB. Furthermore, we are able to reconstitute mPTP-like channel activity in artificial lipid bilayers by combining purified mammalian c-subunit with synthetic polyP in the presence of calcium. We propose that the c-subunit-PHB-polyP complex is sufficient and essential for formation of the mPTP channel. According to our hypothesis, during cellular stress conditions, calcium induces formation of the complex between the c-subunit, PHB and polyP. This is a critical event required for mPTP activation. 1524-Pos Board B501 Contribution of Inorganic Polyphosphate Towards Regulation of Mitochondrial Free Calcium M. de la Encarnacion Solesio Torregrosa1, Lusine Demirkhanyan2, Eleonora Zakharian2, Evgeny Pavlov1. 1 Basic Sciences, NYU-College of Dentistry, NYC, NY, USA, 2Department of Cancer Biology and Pharmacology, University of Illinois, Peoria, IL, USA. Calcium signaling plays a key role in the regulation of multiple processes in mammalian mitochondria, ranging from the physiological regulation of cellular bioenergetics to the induction of stress-induced cell death. While the total concentration of calcium inside the mitochondria can increase by several orders of magnitude, the concentration of bioavailable, free calcium in mitochondria is maintained within the micromolar range by the mitochondrial calcium buffering system. It is established that this calcium buffering system involves participation of orthophosphate. However, the mechanisms of its function are not yet understood. Specifically, it is not clear how orthophosphate is capable to regulate free calcium concentration in the micromolar range. We hypothesized that inorganic polyphosphate, a biological polymer, made of up to 100 orthophosphates, plays a significant role in this regulation on mitochondria. We used confocal fluorescent microscopy to measure the relative levels of free calcium in this organelle, in cultured hepatoma (HepG2) cells with variable levels of inorganic polyphosphate. We found that the depletion of inorganic polyphosphate leads to the significantly lower levels of mitochondrial free calcium concentration, under conditions of pathological calcium overload. We conclude that inorganic polyphosphate is a previously unrecognized integral component of the mitochondrial calcium buffering system. 1525-Pos Board B502 Formation of Polyphosphate-Poly-Beta-Hydroxybutyrate Granule-Like Complexes in Heart Failure Myocytes Lusine Demirkhanyan1, Ian P. Palmer2, Walter Boyd3, Claus S. Sondergaard3, Kristin Grimsrud4, Leigh G. Griffiths4, Julie Bossuyt2, Donald M. Bers2, Eleonora Zakharian1, Elena N. Dedkova2. 1 Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL, USA, 2Department of Pharmacology, School of Medicine, University of California, Davis, CA, USA, 3Department of Surgery, School of Medicine, University of California, Davis, CA, USA, 4 School of Veterinary Medicine, University of California, Davis, CA, USA. Inorganic polyphosphate (polyP) is a naturally occurring polyanion made of ten to several hundred orthophosphates linked together by phosphoanhydride bonds. Specific physiological roles of polyP vary dramatically depending on its size, concentration, tissue and subcellular localization. Recently we reported that mitochondria of rabbit ventricular myocytes contain significant amounts (280 5 60 pmol/mg of protein) of polyP with an average length of 25 orthophosphates, and that polyP is involved in Ca2þ-dependent activation of the mitochondrial permeability transition pore (mPTP). Using DAPI as a probe for polyP, we showed that polyP levels depend on the activity of the mitochondrial respiratory chain. Here, we visualized intracellular polyP distribution in cardiomyocytes using the affinity of the Xpress epitope-tagged recombinant polyphosphate-binding domain (PPBD) of E. coli exopolyphosphatase and immunocytochemical approach. Primarily mitochondrial location of polyP was demonstrated in both control and heart failure (HF) myocytes from two different HF models: (1), non-ischemic rabbit HF model induced by combined aortic insufficiency and stenosis and (2), targeted microbead embolization of the first diagonal branch of left anterior descending coronary artery in Yucatan mini-pigs. However, enhanced formation of polyP dense spots was observed in