Mitochondrial DNA: The Heart of the Matter Wednesday, February 11, 2015 607a 3061-Pos Board B491 Cardiolipin Reorganization and Phase Transition Induced by Dynamin- Related Protein 1 Facilitates Mitochondrial Membrane Fission Natalia Stepanyants, Patrick Macdonald, Rajesh Ramachandran. Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA. Cardiolipin (CL) is a unique, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells. Dynamin-related protein 1 (Drp1), a member of the dynamin superfamily of large GTPases, maintains the balance of mitochon- drial division and fusion by rapidly catalyzing mitochondrial fission. Although recent studies have indicated a role for CL in stimulating Drp1 self-assembly as well as GTPase activity on the mitochondrial surface, the exact mechanism by which CL functions in membrane fission remains unclear. Here we use a variety of fluorescence spectroscopic and imaging approaches, together with model membranes, to demonstrate that Drp1 and CL function cooperatively in effect- ing membrane fission in three distinct steps: (i) Drp1’s preferential association with unconstrained, fluid-phase CL molecules located at a high spatial density in the membrane bilayer, (ii) CL’s reorganization in concert with Drp1 self- assembly, and (iii) CL’s rapid phase transition from a lamellar, bilayer structure to an inverted hexagonal, non-bilayer configuration in the presence of Drp1 and GTP, resulting in the creation of localized membrane constrictions that are primed for fission. We propose that Drp1 thus catalyzes mitochondrial division. 3062-Pos Board B492 VDAC3 Forms Typical Voltage-Gated, Anion-Selective, and Tubulin- Sensitive Channels Oscar Teijido Hermida1, Adam J. Kuszak2, Susan K. Buchanan3, Sergey M. Bezrukov1, Tatiana K. Rostovtseva1. 1Program on Physical Biology, NICHD, NICHD, National Institutes of Health (NIH), Bethesda, MD, USA, 2NIDDK, National Institutes of Health (NIH), Bethesda, MD, USA, 3 Program on Physical Biology, NICHD, NIDDK, National Institutes of Health (NIH), Bethesda, MD, USA. The Voltage Dependent Anion Channel (VDAC) forms the major pathway for metabolites across the mitochondrial outer membrane (MOM). Mammalian mitochondria have three isoforms of this protein: VDAC1, VDAC2, and VDAC3 which may play different roles in the regulation of mitochondrial func- tions. VDAC1 is the most studied isoform, since it is the major protein of the MOM in most cells, followed by VDAC2. Both, VDAC1 and VDAC2 recon- stituted into planar membranes form voltage-dependent anion-selective chan- nels whose conductance is modulated by dimeric tubulin. As counterpart, the poor channel-forming ability of VDAC3 together with its low proficiency in restoring normal growth in pore-less yeast led to hypothesize that this isoform does not form channels and could be just a modulator of the other two isoforms. We developed an improved protocol of purification and refolding of mouse re- combinant VDAC3 that allowed us to fully characterize channels formed by VDAC3 reconstituted in planar lipid membranes and to compare its channel properties with those of VDAC1 and VDAC2. We observed the typical VDAC channel behavior of VDAC3, such as high conductive open state (3.5-4.1 nS in 1M KCl) and multiple low conductive states at potentials R 540mV, selectivity for anions with PCl -/PK þ¼ 1.7, and typical voltage- gating. This suggests a structural homology of VDAC3 with the other two iso- forms. Interestingly, the most distinct characteristic of VDAC3 is its signifi- cantly lower sensitivity to the blockage by tubulin, which would lead to an increased permeability of this channel to ATP and other metabolites. This might explain why the down-regulation of VDAC3 expression among the three VDAC isoforms in some cancer cells causes the most drastic decrease in their mitochondrial membrane potential, or why VDAC3 is predominantly ex- pressed in the short-term high energy demanding cells, such as spermatozoa. 3063-Pos Board B493 Channeling of Mitochondrial Energy in Cardiac and Cancer Cells by the Metabolically-Dependent Outer Membrane Potential Victor V. Lemeshko. Escuela de Fisica, Universidad Nacional de Colombia, Medellin, Colombia. Mitochondria are the main source of energy in eukaryotic non-proliferating aer- obic cells. In cancer cells, significant part of the energy metabolism is sup- ported by the aerobic glycolysis. Oxidative phosphorylation metabolites cross the mitochondrial outer membrane through the voltage dependent anion channel (VDAC). The possibility of the voltage-gating functioning of VDAC under physiological conditions and proposed mechanisms of generation of the outer membrane potential (OMP) require further study. In this work, we present a computational thermodynamic analysis of a possible generation of OMP by the creatine kinase(CK)-VDAC and VDAC-hexokinase(HK) com- plexes, as well as by their intermembrane complexes with the adenine nucleo- tide translocator (ANT). The CK-VDAC and VDAC-HK complexes function as direct steady-state voltage generators, using the free energy of kinase reac- tions, while the ANT-CK-VDAC and ANT-VDAC-HK complexes allow the application of a part of the sum of the corresponding ‘‘kinase voltage’’ and the inner membrane potential to the outer membrane. The developed computa- tional models demonstrate a high probability of generation of the metabolically-dependent OMP with magnitudes high enough to close free VDACs. We suggest that in case of cardiac cells, the CK-VDAC-mediated channeling of mitochondrial energy, earlier postulated by Saks and Wallimann, leads to the metabolically-dependent generation of a high OMP during systole, thus causing electrical closure of free VDACs and essentially avoiding the mitochondrial ATP usage by non-contraction system consumers. In case of cancer cells, that have a very high percentage of hexokinase attached to mito- chondria, the electrical closure of free VDACs by OMP allows the preferential VDAC-HK-mediated channeling of the mitochondrial ATP to initiate the aer- obic glycolysis by an anti-turbo mode. (Research grants #111852128625 and #520154531565, Colciencias). 3064-Pos Board B494 Alpha-Synuclein Blocks VDAC Suggesting Mechanism of Mitochondrial Regulation and Toxicity in Parkinson Disease Philip A. Gurnev1,2, Tatiana K. Rostovtseva2, David P. Hoogerheide2,3, Olga Protchenko4, Thai Leong Yap5, Jennifer C. Lee5, Sergey M. Bezrukov2. 1Physics Department, UMass-Amherst, Amherst, MA, USA, 2Section on Molecular Transport, PPB/NICHD/NIH, Bethesda, MD, USA, 3Center for Neutron Research, NIST, Gaithersburg, MD, USA, 4Liver Diseases Branch, NIDDK/NIH, Bethesda, MD, USA, 5 Laboratory of Protein Conformation and Dynamics, NHLBI/NIH, Bethesda, MD, USA. Alpha-synuclein (a-syn), an intrinsically disordered neuronal protein, is impli- cated in the etiology of Parkinson disease (PD) and a number of other neurode- generative dementias. Though recent research demonstrates the involvement of a-syn in a variety of mitochondrial dysfunction in neurodegeneration, the mo- lecular mechanism of a-syn toxicity and its effect on neuronal mitochondria remain vague. We demonstrate a functional interaction between a-syn and the voltage-dependent anion channel (VDAC), the major conduit for ATP and other bioenergetics metabolites in the mitochondrial outer membrane. We found that at nanomolar concentrations, the full-length a-syn, its 45 amino acid C-terminal truncated mutant, as well as b- and a-synuclein isoforms induce reversible and highly voltage-dependent blockage of VDAC reconsti- tuted into planar lipid bilayers. Binding parameters varied with each isoform, revealing the key role of the negatively charged C-terminal of synucleins in blocking a positively charged pore of VDAC. Synuclein-blocked states of VDAC differ in ionic selectivity, implying the existence of several conforma- tion states of synuclein molecules in the channel pore. We propose a model of a-syn interaction with VDAC, in which the negatively charged C-terminus of a-syn enters the net-positive channel pore, providing a steric block for ATP flux. Experiments with a yeast strain deficient in VDAC1 demonstrate that a-syn toxicity in yeast depends on VDAC, revealing a-syn interaction with the channel in living cells. Thus, our findings show the long-sought physiolog- ical and pathophysiological roles for monomeric a-syn, which reconcile previ- ous observations of various synuclein effects on mitochondrial bioenergetics. 3065-Pos Board B495 Mitochondrial DNA: The Heart of the Matter Meagan McManus1, Martin Picard1, Alessia Angelin1, Prasanth Potluri1, Jagat Narula2, Douglas Wallace1. 1CHOP/UPENN, Philadelphia,, PA, USA, 2Mt Sinai, New York, NY, USA. Cardiac conduction defects and cardiomyopathy are commonly reported in pa- tients with inborn errors of metabolism harboring pathogenic mutations in either mitochondrial or nuclear DNA (mtDNA and nDNA, respectively). We hypothesized that the pathophysiology of inborn errors of metabolism varies due to synergistic heterozygosity between the two genomes. We examined a 13-generation Mennonite pedigree with autosomal recessive cardiomyopathy due a mutation in the adenine nucleotide translocator-1 (ANT1). Substantial variability in the progression of heart disease segregated with maternal lineage, and the severity of cardiomyopathy correlated with the mtDNA haplogroups (Strauss, et al 2013). To determine the causative nature of this correlation, we examined the influence of inherited mtDNA mutations on ANT1- cardiomyopathy in the mouse. We introduced homoplasmic mtDNA ND6 or COI missense mutations into the mouse female germ line, generating mice with complex I or IV deficiency, respectively, and analyzed Ant1-dependent cardiomyopathy on the different mtDNA backgrounds. On wt mtDNA back- ground, the Ant1-/- mice developed a distinctive concentric dilated cardiomy- opathy, characterized by substantial myocardial hypertrophy and ventricular dilation. Both COI and ND6 mtDNA mutations accelerated Ant1-/- age- dependent cardiomyopathy, as evidenced by ultrastructrural abnormalities, bio- energetic defects, mtROS production, sensitized mitochondrial permeability transition, increased mtDNA damage, and heart failure, which ultimately 608a Wednesday, February 11, 2015 attenuated the lifespan of Ant1-deficient mice. Our results prove mtDNA dic- tates the penetrance of age-related cardiomyopathy and mammalian lifespan. Therefore, therapeutics that most effectively preserve mitochondrial DNA and bioenergetics will provide the most promise for healthy aging. 3066-Pos Board B496 Activation of the Mitochondrial Permeability Transition Pore Leads to the Increase in Amount of C-Subunit of ATP Synthase Associated with Channel- Forming Complex of Polyhydroxybutyrate and Inorganic Polyphosphate Pia A. Elustondo1, Alexander Negoda1, Alejandro M. Cohen1, Evgeny Pavlov2. 1 Physiology and Biophysics, Dalhousie University, Halifax, NS, Canada, 2 Department of Basic Sciences, College of Dentistry, New York Universty, New York, NY, USA. Low permeability of the mitochondrial inner membrane is critical for maintain- ing the mitochondrial electrochemical potential - the driving force for ATP pro- duction. Acute stress conditions, lead to the increase in the mitochondrial inner membrane permeability due to the opening of the permeability transition pore (PTP). PTP allows free movement of ions and small molecules leading to mito- chondrial depolarization, ATP depletion and cell death. Recent studies suggest that C-subunit of the mitochondrial ATP synthase plays a central role in PTP. Previous work in our laboratory showed that mitochondria contain non-protein complex composed of polyhydroxybutyrate, inorganic polyphosphate and cal- cium that forms an ion channel with properties resembling PTP. Here we explore the possibility of interactions between these non-protein components and C-subunit during the induction of PTP. To induce PTP,isolated energized mitochondria were treated with calcium. Control mitochondria were treated with calcium either in the presence of ruthe- nium red, inhibitor of calcium uptake or Cyclosporin A, inhibitor of PTP. This was followed by a water-free chloroform extraction of channel forming fraction of PTP. Components of the extract were analyzed using immunoblot analysis. We found significantly increased amount of C-subunit associated with channel forming fraction extracted from mitochondria with activated PTP. In contrast, C-subunit was not detectable in the extract when Ruthenium Red was present and significantly decreased in the presence of Cyclosporin A or in the absence of calcium. These results show that C-subunit is likely an interacting partner of the pore- forming complex of polyphosphate, calcium and polyhydroxybutyrate. We hy- pothesize that fully functional PTP requires calcium-induced formation of the pore made of both the complexed polymers and the c-subunit of ATP synthase 3067-Pos Board B497 Small-Molecule PKD Inhibitor Prevents Mitochondrial Fragmentation and Dysfunction during Gq-Protein Coupled Receptor Stimulation in Car- diac Cells Bong Sook Jhun, Xiaole Xu, Jyotsna Mishra, Stephen Hurst, Jin O-Uchi, Shey-Shing Sheu. Thomas Jefferson University, Philadelphia, PA, USA. Regulation of mitochondrial morphology and dynamics is crucial for the main- tenance of various cellular functions in cardiomyocytes. Abnormal mito- chondrial morphologies concomitant with mitochondrial dysfunction are frequently observed both in human heart failure (HF) and in animal HF models. However, it is still unclear which cardiac signaling pathways regulate mitochon- drial morphology and function under pathophysiological conditions. Recent reports suggest that Gq-protein coupled receptor (GqPCR) signaling pathways are critical for the development and progression of HF. Therefore, we hypoth- esize that GqPCR stimulation induces mitochondrial fragmentation and dysfunc- tion, which initiates cardiomyocyte death. We found that protein kinase D (PKD) activated by GqPCR signaling was translocated to outer mitochondrial membrane (OMM) observed by Western blot analysis of cytosolic and mitochondria-enriched fractionated proteins and by live cell imaging of fluores- cence resonance energy transfer (FRET). We also found that GqPCR-mediated PKD activation induced mitochondrial fragmentation, leading to increased reactive oxygen species (ROS) generation as well as increased mitochondrial permeability transition pore (mPTP) opening, which initiates apoptotic signaling activation and cardiomyocyte death. These morphological and func- tional changes in cardiac mitochondria were mediated via PKD-dependent phos- phorylation of mitochondrial fission protein, Dynamin-Like Protein 1 (DLP1) at S637. Moreover, pretreatment with a novel potent PKD inhibitor CRT0066101 effectively inhibited GqPCR-mediated PKD translocation to OMM, DLP1 phosphorylation at S637, mitochondrial fragmentation, ROS generation and mPTP activation. In conclusion, we demonstrate that GqPCR stimulation induces mitochondrial fragmentation and dysfunction through PKD-dependent phosphorylation of DLP1 at S637, which likely contributes to cardiomyocyte injury. Thus, small-molecule PKD inhibitor may become a novel and potent therapeutic for preventing cardiac cell injury and death during HF. 3068-Pos Board B498 EPR Data Support the Existence of a Symmetric BH3-in-Groove Homo- dimer in Oligomeric BAK Tirtha Mandal, Kyoung Joon Oh. Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA. The BAX or BAK oligomeric pore formation in the mitochondrialouter membrane isacriticalstepinapoptosis,yettheirstructuresarenotclearlyunderstood.Czabotar et al. (Cell 2013, 152, 519) reported a crystal structure of a water-soluble tetramer of the BAX fragment (helices a2-a5) fused to the green-fluorescent protein (GFP), inwhichthea2-a3extendedhelicesandthea5helix,respectively,werejuxtaposed to each other, in an anti-parallel orientation, forming a symmetric ‘‘BH3-in-groove homodimer (BGH).’’ We have constructed a GFP-BAK fusion protein using the a2-a5 helices of mouse BAK, designated as GFP-BAKa2-a5, which also forms a soluble tetramer. To determine whether the BGH exists in the BAK oligomers in the membrane or not, we spin labeled the C-terminal hexahistidine-tagged solu- ble form of mouse BAK (helices a1-a8) at residues 84, 122, 128 and 135 and the corresponding residues in GFP-BAKa2-a5. We then compared the continuous wave (CW) EPR spectra of the spin-labeled residues from the tetrameric GFP- BAKa2-a5 with those from the oligomeric BAK in membrane. Spin labeled resi- due 122R1, located in the loop interconnecting helices a4 and a5 in the homology model of the GFP-BAKa2-a5 tetramer, displayed a mobile lineshape. The corre- sponding residue in the oligomeric BAK also had a remarkably similar lineshape, indicating that the two residues are in similar structural environments. Residues 84R1, 128R1 and 135R1, located at the anti-parallel helical interfaces in the BGH also had remarkably similar immobile lineshapes both in the GFP-BAKa2- a5 tetramer and in the oligomeric BAK in membrane, further strengthening the above conclusion. The intra-dimer distances between 84R1 spin label pairs in the GFP-BAKa2-a5 and the oligomeric BAK, determined by the double electron electron resonance (DEER) method, also support this interpretation. 3069-Pos Board B499 Modulation of Membrane Interactions of Anti-Apoptotic Regulator Bcl-xL by Lipids Mauricio Vargas-Uribe, Mykola V. Rodnin, Alexey S. Ladokhin. Biochemistry and Molecular Biology, KUMC, Kansas City, KS, USA. The Bcl-2 family of proteins (e.g., pro-apoptotic Bax and anti-apoptotic Bcl-xL) regulates the mitochondrial outer membrane permeabilization during the early stages of apoptosis. The prevalent Embedded Together Model of Bcl-2 action suggests that the membrane environment is critical for their proper functional interactions, consistent with the increasing evidence of lipids being involved in the regulation of apoptotic response. In this study, we apply a collection of fluorescence-based methods to investigate the effect of various lipids on the pH-triggeredmembraneinteractionsofBcl-xL.Theinitialmembraneassociation was studied using a FRET assay with donor-labeled Bcl-xL and acceptor-labeled vesicles, while the insertion/refolding of Bcl-xL into the membrane was moni- toredusingtheenvironment-sensitive probeNBDselectivelyattachedinthemid- dle of hydrophobic helix a6. Our results demonstrate that the lipid composition affects the pH-dependence of both initial membrane association and subsequent insertion/refolding of Bcl-xL. We found that a linear correlation exists between the membrane surface potential created by anionic lipids and the pKa of mem- brane binding, suggesting that the initial step is controlled by an electrostatic mechanism. The effect of lipids on the membrane insertion/refolding step is more complex and appears to be influenced by the size of the lipid headgroup. Thekinetics ofboth themembrane associationandmembrane insertion/refolding is affected by the presence of non-bilayer forming lipids commonly found in mitochondria. While the presence of phosphatidylethanolamine accelerated the process, addition of lysophosphatidylcholine had the opposite effect, suggesting that mechanical properties of the bilayer also play a role. Taken together our results indicate that lipids can modulate the membrane interactions of Bcl-xL in multiple ways, providing an additional regulatory mechanism that ensures proper control of a complex cascade of apoptotic reactions leading to cell death or survival. NIHGM-069783, Fulbright-CONICYT, BRTP. 3070-Pos Board B500 Binding of Pro-Apoptotic Protein Bax to Cytoprotective UDCA and TUDCA Tânia Sousa1, Ana Coutinho2, Soojay Banerjee3, Rui Castro4, Richard Youle3, Cecı́lia Rodrigues4, Manuel Prieto1, Fábio Fernandes1. 1Centro de Quı́mica-Fı́sica Molecular and Institute of Nanoscience and Nanotechnology, Instituto Superior Tecnico, Universidade de Lisboa, Lisbon, Portugal, 2 Dep. Quı́mica e Bioquı́mica, Faculdade de Ciências da Universidade de Lisboa, Ins, Lisbon, Portugal, 3 NINDS, National Institutes of Health, Bethesda, MD, USA, 4Faculdade de Farmácia da Universidade de Lisboa, Lisbon, Portugal. Hydrophobic bile acids, such as deoxycholic acid (DCA), strongly induce apoptosis in both hepatic and non-hepatic cells while hydrophilic bile acids, Cardiolipin Reorganization and Phase Transition Induced by Dynamin-Related Protein 1 Facilitates Mitochondrial Membrane Fission VDAC3 Forms Typical Voltage-Gated, Anion-Selective, and Tubulin-Sensitive Channels Channeling of Mitochondrial Energy in Cardiac and Cancer Cells by the Metabolically-Dependent Outer Membrane Potential Alpha-Synuclein Blocks VDAC Suggesting Mechanism of Mitochondrial Regulation and Toxicity in Parkinson Disease Mitochondrial DNA: The Heart of the Matter Activation of the Mitochondrial Permeability Transition Pore Leads to the Increase in Amount of C-Subunit of ATP Synthase A ... Small-Molecule PKD Inhibitor Prevents Mitochondrial Fragmentation and Dysfunction during Gq-Protein Coupled Receptor Stimul ... EPR Data Support the Existence of a Symmetric BH3-in-Groove Homodimer in Oligomeric BAK Modulation of Membrane Interactions of Anti-Apoptotic Regulator Bcl-xL by Lipids Binding of Pro-Apoptotic Protein Bax to Cytoprotective UDCA and TUDCA