functional coupling. The pH spikes were abrogated by protonophores and their frequency strongly decreased by respiratory chain inhibitors (rotenone: -57%, antimycin: -96%) or by inhibition of the ATP- synthase (oligomycin: -52%). Conversely, inhibition of the adenine nucleotide exchanger (ANT) by actractyloside increased spike frequency by 510%. Normal pH spikes were observed in cells pre- treated with the SERCA-ATPase inhibitor thapsigargin, indicating that the pH elevations did not require calcium release from intracellular stores. Simultaneous ψ mt and pH mito measurements revealed con- comitant depolarization and basification transients. Superoxide flashes with similar properties were previously reported in individual mitochondria with a circularly permutated YFP (Wang et al., 2008, Cell 134). Since this probe is known to be pH-sensitive, the signals reported as superoxide flashes might have been due to pH spikes. Alternatively, superoxide flashes could generate pH spikes via Fenton and dismutation reactions, although we did not detect ROS elevations with the mitochondrial ROS sensor roGFP. In summary, we show that individual mitochondria in intact HeLa cells undergo spontaneous basification transients. The pH spikes are not due to calcium release from stores, but require functional OXPHOS machinery. Supported by grant 3100A0-118393 from the Swiss National Science Foundation. doi:10.1016/j.bbabio.2010.04.357 15L.4 Mitochondrial cholesterol and cell death José C. Fernández-Checa Liver Unit, Hospital Clinic, CIBEREHD, IDIBAPS, and IIBB-CSIC, Barcelona, Spain; and Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA E-mail: checa229@yahoo.com Cholesterol is a critical component of biological membranes, which determines their structural and biophsycial properties. Its distribution within membranes is heterogeneous, partitioning in specialized domains called rafts, where modulate signaling pathways. Due to this fundamental role cholesterol levels are highly regulated. Cholesterol distributes to different subcellular compartments by vesicular dependent and independent mechanisms. Compared to plasma membranes, mitochondria are cholesterol-poor organelles, with estimates of 0.5-3% of the total cholesterol pool. While hepatic mitochondrial cholesterol plays an important physiological role such as in the synthesis of bile acids, its accumulation contributes to liver diseases, such as alcoholic (ASH) and non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Mitochondrial choles- terol loading in ASH and NASH models sensitizes hepatocytes to oxidative stress and inflammatory cytokines, contributing to fatty liver disease by a mechanism that involves mitochondrial GSH (mGSH) depletion due to changes in mitochondrial membrane dynamics. mGSH depletion protects cardiolipin from oxidation to peroxidized cardiolipin, which determines mitochondrial membrane permeabilization by proapoptotic bcl-2 family members, such as Bax. Interestingly, mitochondrial cholesterol accumulation also occurs in HCC, which contributes to chemotherapy resistance. However, despite cholesterol loading, HCC cells exhibit unimpaired transport of GSH into mitochondrial matrix due to the overexpression of mGSH carriers, 2-OG and DIC. This maintenance of mGSH prevents cardiolipin peroxidation. Peroxidized cardiolipin, however, over- comes the resistance to mitochondrial membrane permeabilization induced by Bax. These results characterize mitochondrial cholesterol/ peroxidized cardiolipin as a rheostat in cell death regulation. doi:10.1016/j.bbabio.2010.04.358 15L.5 Signalosomes transmit signals from plasma membrane receptors to mitochondria Keith D. Garlid, Caitlin Pesout Portland State University, Department of Biology, USA E-mail: garlid@pdx.edu When an agonist binds to a plasma membrane receptor, a signaling cascade is triggered that targets intracellular organelles. Mitochondria respond by opening the mitochondrial ATP-sensitive K + channel (mitoK ATP ) and producing ROS for further signaling. Cardioprotection against ischemia- reperfusion (IR) injury is a useful experimental model for probing this process, because these signals reduce infarct size by about 70%. Many receptors produce the cardioprotective response, including Gi protein-coupled receptors (adenosine, acet- ylcholine, bradykinin, opioids, and phenylephrine), the Na,K-ATPase (ouabain, digitalis), and the L-type Ca 2+ channel (Ca 2+ ). We have found that the entire signaling cascade is assembled in plasma membrane caveolae, then buds off as a 140 nm signalosome, internalizes, and migrates to mitochondria. The terminal kinases of the cascade phosphorylate a protein on the outer membrane. This leads to activation of an inner membrane PKCε, which opens mitoK ATP by phosphorylation. The signalosomes can be isolated and purified from the perfused heart and displays activity in vitro. This allows us to study a signaling unit in its naturally organized state with preserved functionality. Most signalosomes are functionally active within minutes of receptor activation. Interestingly, the adenosine signalo- some requires an additional step of ROS activation after internalization, and the adenosine receptor remains active in vitro. doi:10.1016/j.bbabio.2010.04.359 15L.6 The gas pedal of brain mitochondria: glutamate supply for OXPHOS is fully regulated by cytosolic Ca 2+ via activation of aralar Frank N. Gellerich 1 , Zemfira Gizatullina 2 , Sonata Trumbekaite 3 , Bernard Korzeniewski 4 , Stefan Vielhaber 2 , Enn Seppet 5 , Aurelius Zimkus 6 , Frank Striggow 1 1 KeyNeurotek Pharmaceuticals AG, ZENIT Technology Park, Magdeburg, Germany 2 Department of Neurology, Otto von Guericke University of Magdeburg, Magdeburg, Germany 3 Institute for Biomedical Research, Kaunas University of Medicine, Kaunas, Lithuania 4 Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland 5 Department of Pathophysiology, Institute of General and Molecular Pathology, University of Tartu, Tartu, Estonia 6 Department of Biochemistry and Biophysics, Faculty of Natural Sciences, Vilnius University, Lithuania E-mail: frank.gellerich@keyneurotek.de The regulation of OXPHOS is still not understood in detail. ADP formed by ATP-consuming enzymes activates OXPHOS but in the heart cytosolic ADP is only insignificantly increased in vivo during elevated work loads [1] and therefore the parallel stimulation of OXPHOS and work load by cytosolic Ca 2+ (Ca 2+ cyt ) was assumed [2]. However, activation of dehydrogenases by matrix Ca 2+ [3] complies only partially with the in vivo findings, therefore we hypothesized that other mechanisms should be responsible for mitochondrial activation by Ca 2+ cyt . We have found recently [4-6] that the glutamate dependent respiration of brain mitochondria can be stimulated by Ca 2+ cyt due to the activation of aralar [7], the glutamate aspartate carrier (S 0.5 = 260 nM Ca 2+ free ). Depending on its initial concentra- tion, Ca 2+ can activate state 3 glu/mal of brain mitochondria up to 120 Abstracts