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798-Pos Board B584Single Molecule Cross-Talk Transferred from a Molecular Event to anOrgan FunctionNelson P. Barrera.Pontificia Universidad Catolica de Chile, Santiago, Chile.At a single molecule level, the mechanism underlying the simultaneous acti-vation of several interacting transduction pathways is poorly understood;largely due to the lack of suitable experimental models. In addition structuraland functional studies based on physicochemical modelling should be pursuedin order to predict the magnitude and timing of crosstalk in cellular responses.It is shown here that the cross-talk in ciliary beat frequency (CBF) of oviduc-tal ciliated cells induced by ATP and adenosine is triggered by the activationof plasma membrane IP3 receptors. Later on this effect is transferred intoa synergistic egg transport across the oviduct. The rate of synergism is max-imal at the IP3 receptor activation and Ca2þ concentration increase which de-cays exponentially until the physiological end-point. These results representthe first dynamic studies for tracking transfer of synergism from molecular re-sponses to cells and tissue responses. Funded by Millennium ScienceInitiative.

Mitochondria in Cell Life & Death

799-Pos Board B585A Putative Role of Voltage-Dependent Anion Channel in IschemiaOscar Teijido Hermida, Sergey M. Bezrukov, Tatiana K. Rostovtseva.National Institutes of Heath (NIH), Bethesda, MD, USA.Ischemia is characterized by the inhibition of ATP production via oxidativephosphorylation due to oxygen deprivation, which leads to mitochondria depo-larization. Impaired mitochondrial respiration involves the shift to an anaerobicmetabolism and ionic imbalance that leads to a decrease of cytosolic pH andgeneration of reactive oxygen species (ROS). The voltage-dependent anionchannel (VDAC) is the most abundant protein in the mitochondrial outer mem-brane, which regulates the passage of ATP, ADP, and other mitochondrial re-spiratory substrates across this membrane. Motivated by these facts, we areinterested in establishing a possible functional link between the physiologicalfeatures of ischemia and VDAC. It has been reported that cytosolic acidifica-tion protects cells from death during early ischemic stress. However, the mech-anism of this protection is still unclear. Ischemia is followed by a reperfusionstage in which pH is restored to neutral leading to cell death. In experimentswith VDAC reconstituted into planar lipid membranes we show that VDACvoltage-induced closure increases at low pH. Interestingly, the channel fullyre-opens after the pH is restored to neutral. The cytoskeleton protein, dimerictubulin regulates mitochondrial membrane potential by blocking VDAC per-meability for ATP (Sheldon et al., 2011; Gurnev et. al, 2011). We foundthat, synergistically with the effect on gating, acidification significantly pro-motes VDAC blockage by tubulin, and speculate here that acidic pH-inducedVDAC inhibition could potentially be involved in the protective effect of aci-dosis and also in the mitochondria depolarization usually detected duringischemia.A possible effect of ROS on VDAC permeability was assessed by probingVDAC functioning in planar membranes containing oxidized lipids. We havefound that oxidized lipids affect VDAC interaction with tubulin. The relevanceof these observations for the mechanisms of ischemia and apoptosis isdiscussed.

800-Pos Board B586VDAC Regulation by Tubulin and its Physiological ImplicationsTatiana K. Rostovtseva, Kely L. Sheldon, Philip A. Gurnev,Sergey M. Bezrukov.NICHD, NIH, Bethesda, MD, USA.A significant portion of the mitochondria outer membrane (MOM) controlfunctions is realized through the voltage-dependent anion channel (VDAC)that constitutes a major pathway for ATP/ADP across MOM. We have re-cently found that dimeric tubulin induces reversible blockage of VDAC re-constituted into planar lipid membrane and dramatically reducesmitochondrial respiration. The voltage sensitivity of the blockage equilib-rium is truly remarkable. It is described by an effective ‘‘gating charge’’of more than ten elementary charges, thus making the blockage reactionas responsive to the applied voltage as the most voltage-sensitive channelsof electrophysiology. Analysis of the tubulin-blocked state demonstratedthat although this state is still able to conduct small ions, it is impermeableto ATP and other multi-charged anions because of the reduced aperture and

inversed selectivity. The findings, obtained in the channel reconstitution as-say, were supported by experiments with isolated mitochondria and humanhepatoma cells. Thus, regulation of VDAC by tubulin that was first discov-ered in vitro, was then related to the action of this protein in vivo. The mech-anism of VDAC-tubulin interaction appears to be more complex than wasinitially thought; in particular, it is very sensitive to the membrane lipidcomposition. Tubulin adsorption on the membrane affects the on-rate oftubulin-VDAC binding and depends on the lipid packing stress as well ason the membrane surface charge. Immediate physiological implications in-clude new insights into the function of serine/threonine kinase signalingpathways, microtubule activity in the case of the highly dynamic microtu-bule network in cancerogenesis and cell proliferation, and the role of lipidsin mitochondrial dynamics and MOM permeabilization characteristic ofapoptosis.

801-Pos Board B587Tubulin-Blocked State of VDAC Probed by Polymer Partitioning andBilayer Surface ChargePhilip A. Gurnev1, Marıa Queralt Martın2, Vicente M. Aguilella2,Tatiana K. Rostovtseva1, Sergey M. Bezrukov1.1National Institute of Child Health and Human Development, Bethesda,MD, USA, 2Laboratory of Molecular Biophysics, Department of Physics,Universitat Jaume I, 12071, Castellon de la Plana (Castellon), Spain.Recently reported functional interaction between voltage-dependent anionchannel of the outer mitochondrial membrane, VDAC, and dimeric tubulinis observed as reversible channel blockage. The tubulin-blocked state isonly partially closed for small ions and, in 1 M KCl solutions, its conductancestays at about 40 % of that for the open channel. Reconstituting VDAC intoplanar lipid membranes we have studied the nature of the tubulin-blockedstate of the channel using partitioning of poly-(ethylene glycol)s, reversal po-tential measurements, and manipulation of the membrane surface charge. Wefound that judged by polymer partitioning an estimated reduction of the open-state channel radius upon tubulin blockage is about 26 %, but the channel se-lectivity reverses from anionic to cationic. In the limit of low salt concentra-tions (down to 0.05 M KCl) the current of the blocked state is decreased bypositive and increased by negative charge of membrane lipids; open state ofthe VDAC is virtually insensitive to the surface charge. This behavior canbe understood taking into account the cationic selectivity of the blocked stateand the increased proximity of ion pathway(s) to the nearest lipid headgroupsas compared with the open state of the channel. Interestingly, the sensitivity ofthe tubulin-VDAC blockage to the applied transmembrane voltage character-ized by the effective gating charge practically does not depend on salt concen-tration in the bulk or on lipid charge, staying within the range of about 10–14elementary charges.

802-Pos Board B588A Novel Mitochondrial Potassium Channel in Embryonic HippocampalMitochondraAnna Kajma1, Piotr Bednarczyk2, Adam Szewczyk1.1Nencki Institute of Experimental Biology, Warsaw, Poland,2Warsaw University of Life Science, Warsaw, Poland.Since 1991, when in Nature Inoue and co-workers described the first potassiumchannel in the inner mitochondrial membrane, ATP-regulated (mitoKATP),four other channels were found. ATP-regulated (mitoKATP), large-conductance calcium activated (mitoBKCa), voltage-dependent (mitoKv1.3),intermediate-conductance calcium-activated potassium channel (mitoIKCa),were identified by the electrophysiological techniques. The last, TWIK-related acid-sensitive potassium channel (TASK-3) was identified with theuse of immunofluorescence methods.Patch-clamp single channel studies on mitochondria isolated from embryonicrat hippocampus revealed the presence of the potassium channel which has out-wardly rectifying activity at the symmetrical conditions (150mM/150mMKCl). The channel displayed a conductance of 61 pS, at positive voltages,and also strong voltage dependence. Patch-clamp studies at the mitoplast-attached mode showed that this channel was not sensitive to the classical acti-vators and inhibitors of the mitochondrial potassium channels, but regulated bythe pH and arachidonic acid.In summary, by the single channel recordings, we characterized for the firsttime an ion channel which was cation selective, permeable to potassium ionsand displayed voltage sensitivity. The channel not correspond to the potassiumion channels described earlier in the inner mitochondrial membrane.Grant sponsor: 793/N-DAAD/2010/0 and Nencki Institute of ExperimentalBiology