Alpha-Synuclein Blocks VDAC Suggesting Mechanism of Mitochondrial Regulation and Toxicity in Parkinson Disease 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 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