MICU1-dependent Threshold and Cooperativity of Mitochondrial Ca2+ Uptake in the Liver Wednesday, February 6, 2013 655a Mitochondria in Cell Life and Death 3363-Pos Board B518 A Step Forwardin Understanding the Mechanismof VDAC Voltage-Gating Oscar Teijido Hermida1, Shay Rappaport1, Rachna Ujwal2, Jeff Abramson2, Vicente M. Aguilella3, Sergey M. Bezrukov1, Tatiana K. Rostovtseva1. 1NICHD, National Institutes of Health (NIH), Bethesda, MD, USA, 2David Geffen School of Medicine, UCLA, Los Angeles, CA, USA, 3 Universitat Jaume I, Castello de la Plana, Spain. The voltage-dependent anion channel (VDAC) governs the exchange of ions and metabolites between the mitochondria and the rest of the cell. In its open state VDAC exhibits high conductance and selectivity for anions that facilitates the passage of ADP, ATP, and other metabolites. At increased voltages (>30mV) VDAC switches to lower conducting states, termed as ‘‘closed’’ states. Closed states are cation-selective and impermeable for ATP. The voltage-induced transition from the open to closed states is referred to as voltage-gating. Although it is well established that VDAC voltage-gating in- volves large structural rearrangements, the precise molecular mechanism of this process is still under debate. We investigated VDAC voltage-gating by sys- tematically titrating VDAC charge residues and by using thermodynamic and kinetic approaches to study opening and closing of the channel. All the models proposed so far agree that N-terminal region plays a key role in VDAC voltage- gating. According to the original idea, the N-terminal region is a part of a mo- bile voltage sensor domain, which slides in and out of the channel lumen in re- sponse to the applied voltage. The alternative models consider independent movement of the N-terminal region upon gating. In order to test the role of VDAC N-terminal region in voltage-gating, we engineered a double Cys mu- tant of murine VDAC1 that cross-links the a-helix to the b-strand 11 of the pore wall. The cross-linked VDAC1 reconstituted into planar lipid membranes exhibited typical voltage gating, which suggests that the N-terminal a-helix is located inside the pore of VDAC in the open state and remains associated with the pore wall during voltage gating. Our findings support a model where b-bar- rel is not rigid but undergoes a conformational change that leads to a partial constriction upon transition to the closed states. 3364-Pos Board B519 Novel Mechanism of Mitochondrial Respiration Control through Competition between Hexokinase-2 and Tubulin for VDAC Binding Kely L. Sheldon1, Coert J. Zuurbier2, Sergey M. Bezrukov1, Tatiana K. Rostovtseva1. 1The Natl. Institutes of Health, Bethesda, MD, USA, 2University of Amsterdam, Amsterdam, Netherlands. The voltage dependent anion channel (VDAC) is involved in regulation of me- tabolite flux across the mitochondrial outer membrane (MOM). Hexokinse II (HK2) is known to bind the MOM where it phosphorylates glucose into glucose-6-phosphate (G6P). High expression of HXK2 is a common phenotype of many cancers, where its concentration can be 200 times of that in noncan- cerous cells, and is implicated in the Warburg effect. It is believed that VDAC serves as a HXK2 binding site in the MOM. The 15 amino acid N-ter- minal sequence of HXK2 is responsible for mitochondrial binding and, when conjugated to TAT (TAT-HK2), binds to mitochondria with higher affinity than native HXK2, causing HXK2 detachment. We have previously found that dimeric tubulin reversibly binds and partially blocks VDAC inhibiting me- tabolite flux across the MOM. Now we show that this binding can be attenuated by TAT-HXK2 peptide as well as by full length HXK2. We have found that TAT-HXK2 and recombinant full length HXK2 inhibit tubulin blockage of VDAC reconstituted into planar lipid bilayers without altering characteristic channel properties such as single channel conductance and selectivity. Binding of HXK2 to VDAC is verified by the generation of high-frequency excess cur- rent noise without channel closure. HXK2 bound to VDAC prevents subse- quent tubulin binding, but only when added before tubulin, and inhibits tubulin-induced VDAC blockage in a dose dependent manner. Moreover, G6P, which is known to cause HXK2 detachment from the MOM, fully re- verses the inhibition of tubulin-VDAC binding. This suggests that HXK2 de- tachment from VDAC (and hence the MOM) is caused by a HXK2 conformational change upon G6P binding. Thus we propose a novel mecha- nism of mitochondrial respiration control in cancer cells through the competi- tion between HXK2 and tubulin for VDAC binding. 3365-Pos Board B520 Reprogramming of Mitochondrial Ca 2D Handling in MICU1-Deficient HeLa Cells Tünde Golenár1, György Csordás1, Erin L. Seifert1, Cynthia Moffat1, Fabiana Perocchi2, Yasemin Sancak2, David Weaver1, Vamsi K. Mootha2, György Hajnóczky1. 1 Thomas Jefferson University, Philadelphia, PA, USA, 2 Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA. Recent studies have revealed MCU as the pore forming domain and MICU1 as a critical Ca2þ-sensitive regulator of the mitochondrial Ca2þ uniporter. How- ever, the exact role of MICU1 in Ca2þ transport remains to be addressed. Our previous studies showed that prolonged down-regulation of MICU1 in HeLa cells (shMICU1) promotes mitochondrial Ca 2þ uptake at low [Ca 2þ ], which unexpectedly, fails to effectively increase matrix [Ca 2þ ]. To determine the source of discrepancy between the mitochondrial Ca 2þ uptake and the ma- trix [Ca2þ] phenotypes, first we simultaneously monitored ruthenium red- sensitive clearance of added Ca2þ from the cytoplasm and the corresponding matrix [Ca2þ] response in permeabilized shMICU1 cells. Under conditions of similar cytoplasmic Ca 2þ clearance, shMICU1 cells showed a smaller matrix [Ca 2þ ] increase than the control, indicating enhanced buffering of Ca 2þ in the matrix. Enhanced Ca 2þ binding in the matrix likely reflects alkalinization and enhanced phosphate transport. To test if upregulation of Ca2þ buffering is di- rectly linked to MICU1 depletion, we also assessed mitochondrial Ca2þ han- dling after 72hr silencing of MICU1 (siMICU1). In siMICU1cells both mitochondrial Ca 2þ uptake and the matrix [Ca 2þ ] rise were effectively stimu- lated at low Ca 2þ levels. Thus, upregulation of matrix Ca 2þ buffering seems to be a component of an adaptive response to sensitization of mitochondrial Ca 2þ uptake in shMICU1. The adaptive response is likely to be important to attenuate some MICU1-depletion induced cellular impairments that we found to manifest as attenuated mitochondrial ATP production and cell proliferation. 3366-Pos Board B521 MICU1-dependent Threshold and Cooperativity of Mitochondrial Ca 2D Uptake in the Liver György Csordás1, Erin L. Seifert1, Tünde Golenár1, Cynthia Moffat1, Sergio de la Fuente Perez1, David Weaver1, Roman Bogorad2, Victor Koteliansky2, Vamsi K. Mootha3, György Hajnóczky1. 1Thomas Jefferson University, Philadelphia, PA, USA, 2Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA, 3 Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA. Recent studies have revealed MCU as the pore forming domain and MICU1 as a critical Ca2þ-sensitive regulator of the mitochondrial Ca2þ uniporter. How- ever, the mechanism of the complex Ca2þ dependence of the uniporter activity remains elusive. Our previous studies showed that prolonged down-regulation of MICU1 in HeLa cells causes lower threshold and decreased cooperativity of mitochondrial Ca 2þ uptake. To study the functional significance of the effects of MICU1 we used hepatocytes harvested from the liver of mice exposed to in vivo silencing (4 weeks). Silencing of MICU1 or MCU resulted in >80% decrease in their respective mRNA levels. Silencing of MICU1 caused a left- ward-shifted dose response and decreased cooperativity of mitochondrial Ca2þ uptake in both permeabilized and intact hepatocytes. By contrast, silenc- ing of MCU resulted in slower Ca 2þ uptake in the entire range of Ca 2þ concen- trations without change in threshold. Mitochondrial respiration and cellular ATP content were unaffected in media containing both glycolytic and mito- chondrial fuels in either MICU1 or MCU-deficient hepatocytes. However, si- lencing of MICU1 caused an augmented loss of ATP when the cells were confined to oxidative metabolism and an enhanced sensitivity to mitochondrial Ca 2þ overload and permeabilization. During stimulation with vasopressin, a Ca 2þ mobilizing hormone, both MICU1 and MCU-deficient cells displayed an attenuated mitochondrial matrix [Ca 2þ ] increase and stimulation of respira- tion. Collectively, these results show that keeping the gate of MCU closed by MICU1 at low [Ca2þ] is required to maintain healthy mitochondria, and MICU1-mediated control of MCU (cooperativity?) is required to support the propagation of short-lasting calcium spikes and oscillations to the mitochondria and the ensuing physiological stimulation of oxidative metabolism. 3367-Pos Board B522 Targeting Mcl-1 and Bak as a Therapeutic Tool to Selectively Induce Apoptosis in Hepaptocellualr Carcinoma Nima Niknejad, Soumya Sinha Roy, Eric Knudsen, György Hajnóczky. Thomas Jefferson University, Philadelphia, PA, USA. In this study we seek to identify novel drug targets to induce apoptosis in he- patocellular carcinoma (HCC) cells thus providing opportunities to develop novel treatments to improve the prognosis of liver cancer patients. Several ap- optotic pathways are mediated through cleavage of Bid (a BH3 domain-only, pro-apoptotic protein) to produce truncated Bid (tBid). tBid induces apoptosis through induction of outer mitochondrial membrane (OMM) permeabilization by activation of pro-apoptotic Bak that resides in the OMM or cytoplasmic Bax. Due to its localization, Bak can mediate the early phase of the response to tBid. We have recently demonstrated that OMM targeting of Bak and the sensitivity to tBid-induced OMM permeabilization is dependent on the expression of A Step Forward in Understanding the Mechanism of VDAC Voltage-Gating Novel Mechanism of Mitochondrial Respiration Control through Competition between Hexokinase-2 and Tubulin for VDAC Binding Reprogramming of Mitochondrial Ca2+ Handling in MICU1-Deficient HeLa Cells MICU1-dependent Threshold and Cooperativity of Mitochondrial Ca2+ Uptake in the Liver Targeting Mcl-1 and Bak as a Therapeutic Tool to Selectively Induce Apoptosis in Hepaptocellualr Carcinoma