cardiac fibers also have diffusion restrictions. This is surprising because rain- bow trout cardiomyocytes are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. However, results from fibers may be affected by incomplete separation of the cells. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by com- paring ADP-kinetics of mitochondrial respiration in permeabilized fibers, per- meabilized isolated cardiomyocytes and isolated mitochondria from rainbow trout heart. We developed a new solution specific for trout cardiomyocytes, where they retained their shape and showed stable steady state respiration rates. The apparent ADP-affinity of permeabilized cardiomyocytes was differ- ent from that of fibers. It was higher, independent of temperature and not in- creased by creatine. However, it was still about ten times lower than in iso- lated mitochondria. This suggests that intracellular diffusion of ADP is indeed restricted in trout cardiomyocytes. The difference between fibers and cardiomyocytes suggest that results from trout cardiac fibers were affected by incomplete separation of the cells. The lack of a creatine effect indicates that trout heart lacks mitochondrial creatine kinase tightly coupled to respira- tion. These results from rainbow trout cardiomyocytes are similar to those from neonatal mammalian cardiomyocytes. Thus, it seems that metabolic reg- ulation is related to cardiac performance. It is likely that rainbow trout can be used as a model animal for further studies of the localization and role of dif- fusion restrictions in low-performance hearts. Next step will be to identify the contribution of mitochondrial outer membrane and cytosolic factors in intra- cellular diffusion restriction. 3823-Pos Novel Method for Investigation of Interactions between Mitochondrial Creatine Kinase and Adenine Nucleotide Translocase Minna Varikmaa 1 , Madis Metsis 2 , Rita Guzun 3 , Tuuli Ka ¨a ¨mbre 1 , Alexei Grichine 3 , Valdur Saks 3,1 . 1 National Institute of Chemical Physics and Biophysics, Tallinn, Estonia, 2 Tallinn University of Technology, Tallinn, Estonia, 3 University of Joseph Fourier, Grenoble, France. The aim of this study was to elaborate fluorescent labeling of mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) to inves- tigate the mechanism of their functional coupling with Fo ¨rster resonance en- ergy transfer (FRET) technique. New alternative fluorescent labeling tech- nique - Fluorescein Arsenical Hairpin (Flash/tetracystein) binder technology was exploited to fluorescently label MtCK. Implementation of fluorescent proteins such as GFP for MtCK fluorescent tagging was excluded because of the functional importance of MtCK C- and N-terminal part and insertion of large fluorescent protein inside the MtCK protein imposes potential risk to interfere the structure, localization and function of the fused protein. Tetra- cysteine motifs were introduced into five different positions in MtCK by mu- tagenesis. Sequentially the recombinant MtCK constructs were expressed in different eukaryotic cells lines and activity of the constructs were determined. The cells were stained with Flash labeling reagent and the expression of tet- racysteine tagged MtCK mutants were visualized ab inito with epifluorescent and confocal microscopy. Improved variant of cyan fluroescent protein Ceru- lean as an appropriate FRET partner for Flash was chosen to fluorescently label ANT. Both N- and C-terminally fused ANT-Cerulean constructs were generated. ANT fusion proteins were expressed in different eukaryotic cell lines and their expression was visualized with epifluorescent and confocal microscopy. Functional constructs of MtCK and ANT-Cerulean were selected for studies of their interaction in cardiomyocytes by applying FRET tech- nique. 3824-Pos VDAC Phosphorylation Regulates Interaction with Tubulin Kely L. Sheldon, Sergey M. Bezrukov, Tatiana K. Rostovtseva. The Natl. Institutes of Health, Bethesda, MD, USA. Mitochondria and mitochondrial bioenergetics are believed to be involved in glycogen synthase kinase (GSK3b)-related cardioprotection. Recently it was suggested that cardioprotection could be achieved through the preservation of mitochondrial binding of hexokinase II (HXKII), or/and through GSK3b phosphorylation of voltage dependent anion channel (VDAC) (Pastorino et al., Cancer Res., 2005; Das et al., Circ. Res., 2008). VDAC, the most abundant channel in the mitochondria outer membrane (MOM), is known to be respon- sible for most of the metabolite and ATP/ADP fluxes across MOM. Recently we have found that dimeric ab-tubulin regulates mitochondrial respiration by directly blocking VDAC and hence, permeability of MOM for ATP/ADP (Ros- tovtseva et al., PNAS, 2008). Here, using mammalian VDAC reconstituted into planar lipid membrane, we show that tubulin-VDAC interaction appears to be very sensitive to the state of VDAC phosphorylation. When VDAC is phos- phorylated in vitro by either GSK3b or protein kinase A (PKA), the on-rate of tubulin binding increases up to 100 times compared with untreated VDAC. Importantly, the basic properties of VDAC, such as single-channel con- ductance, selectivity, and voltage gating, remain almost unaltered after phos- phorylation. Nonspecific alkaline phosphatase and tyrosine kinase inhibitor PP2A dephosphorylate VDAC, which results in decreased tubulin binding. Gel analysis and subsequent phospho-staining confirm that VDAC contains motifs recognized by both GSK3b and PKA. Phosphorylation causes a pro- nounced asymmetry of tubulin binding to VDAC. These findings allow us to point to the tentative GSK3b and PKA serine/threonine phosphorylation sites positioned on the cytosolic loops of VDAC. The results show that VDAC phos- phorylation enhances tubulin-induced VDAC closure and thus could reduce MOM permeability and mitochondria respiration. We suggest that GSK3b car- dioprotective effect is more complex that was initially thought because along with HXKII it involves tubulin as a potent regulator of VDAC and hence, cel- lular respiration. 3825-Pos Free Tubulin and cAMP-Dependent Phosphorylation Modulate Mitochon- drial Membrane Potential in Hepg2 Cells: Possible Role of VDAC Eduardo N. Maldonado, Jyoti R. Patnaik, John J. Lemasters. Medical University of South Carolina, Charleston, SC, USA. BACKGROUND: Conductance of the voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane has been proposed to limit mi- tochondrial metabolism in cancer cells and contribute to the Warburg effect. Since tubulin binding and phosphorylation promote VDAC closure, we hy- pothesized that free tubulin and cAMP-dependent phosphorylation by protein kinase A (PKA) modulate DJ in cancer cells by regulating VDAC-dependent flux of substrates into mitochondria. Our AIM was to modulate VDAC clo- sure and opening in intact cells by increasing and decreasing endogenous free tubulin and by promoting and blocking PKA activation. METHODS: HepG2 human hepatoma cells were incubated in Hank’s solution with 5% CO 2 /air, and DJ was assessed by confocal microscopy of TMRM. Free and polymerized tubulin was determined using a commercial kit. RESULTS: Myxothiazol (10 mM), a respiratory inhibitor, caused only a slight decrease of (TMRM fluorescence), but subsequent addition of oligomycin (10 mg/ml), aF 1 -F 0 -ATPase inhibitor, collapsed DJ nearly completely, showing that in- hibition of both respiration and ATPase are required to collapse DJ. Stabili- zation of microtubules by paclitaxel (10 mM) increased DJ by 60%, whereas disruption by colchicine (10 mM) or nocodazol (10 mM) decreased DJ by 60- 70%. Paclitaxel pretreatment prevented the depolarizing effect of colchicine and nocodazol. Dibutyryl cAMP (1 mM) decreased DJ by 45% whereas H89 (1 mM), a specific inhibitor of PKA, increased DJ by 94% and blocked the effect of dibutyryl cAMP. CONCLUSION: Free tubulin and cAMP/PKA- dependent phosphorylation modulate mitochondrial DJ in HepG2 cells, most likely by regulating VDAC conductance. Up and down regulation of DJ by tu- bulin polymerization/depolarization and PKA dependent phosphorylation/de- phosphorylation is consistent with the hypothesis that VDAC is rate-limiting for mitochondrial metabolism in cancer cells and responsible, at least in part, for the Warburg effect. 3826-Pos Hypothermic Cardioprotection Attenuates Mitochondrial Permeability Transition Pore Opening and Calcium Loading in Isolated Cardiac Mitochondria Johan Haumann 1,2 , James S. Heisner 2 , Age D. Boelens 2 , Mohammed Aldakkak 2 , David F. Stowe 2 , Amadou K.S. Camara 2 . 1 Amsterdam Medical Center, Amsterdam, Netherlands, 2 Medical College of Wisconsin, Milwaukee, WI, USA. Ischemia-reperfusion injury (IRI) is associated with mitochondrial permeabil- ity transition pore (mPTP) opening and impaired mitochondrial respiration. Hypothermia attenuates IRI. We examined mitochondrial function in mito- chondria obtained from isolated hearts subjected to warm or cold ischemia. Guinea pig isolated hearts were perfused at constant pressure with Krebs- Ringer’s solution at 37  C and subjected to 30 min global ischemia at 37  C or 17  C. After 5 min of reperfusion mitochondria were isolated. Mitochon- drial [Ca 2þ ] m , membrane potential (DJ m ), and NADH were measured by spectrophotometry at appropriate wavelengths with indo-1, BCECF, rhoda- mine 123 fluorescent dyes, and autofluorescence, respectively. After energiz- ing with pyruvic acid, 0-100 mM CaCl 2 (0.03-60 mM free [Ca 2þ ] e ) was added followed by 250 mM ADP. Ca 2þ -induced mPTP opening was assessed by col- lapse of DJ m . 10 mM [Ca 2þ ] e resulted in mPTP opening after 37  C IRI, but only at 35 mM [Ca 2þ ] e after 17  C IRI. ADP decreased DJ m and NADH and increased [Ca 2þ ] m in all mitochondria, but the fall in DJ m was greater and the responses to ADP with Ca 2þ overloading were worse after 37  C IRI vs. 17  C IRI. The incidence of no state 4 respiration was 25% with no added Wednesday, February 24, 2010 735a