Interestingly, despite full triad targeting, b 3 was unable to restore considerable charge movement (Q max , 2.53 5 0.50 nC/mF) in contrast to the other b iso- forms (Q max , 8.86 5 0.93 to 9.94 5 2.06 nC/mF) upon expression in relaxed myotubes. Systematic exchanges of variable regions and conserved domains of b 1a with corresponding b 3 sequences revealed significantly reduced Q max restoration with SH3 and C-terminal chimeras (Q max , 4.02 5 0.28 and 5.57 5 0.74 nC/mF, respectively). In contrast, b 1a /b 3 chimeras with the N-terminus, HOOK and GK domain exchanged showed complete restoration of charge movement. Together, our data suggest an essential role of the conserved SH3 domain and the variable C-terminus of b 1a in the induction of the voltage-sensing function of the DHPRa 1S in skeletal muscle EC coupling. Grants: FWF-DK-W1101-B12, FWF-P23299-B09 3200-Pos Board B305 Troponin T3 Regulates Calcium Channel Beta1a Subunit Nuclear Trans- location in Skeletal Muscle Tan Zhang, Jackson Taylor, Zhongmin Wang, Osvaldo Delbono. Voltage-dependent calcium channel (VDCC) beta subunits (CaVb) are criti- cal for CaVa subunit membrane expression and gating properties. While CaVb interacts with a variety of other molecular partners, nonchannel func- tions of skeletal muscle CaVb1a have not been reported. In a yeast two hybrid (Y2H) screening of the mouse tibialis anterior (TA) muscle cDNA library, troponin T3 (TNNT3), specific to fast skeletal muscle, was identified using full-length CaVb1a as the bait. Further studies revealed that TNNT3, but not the slow skeletal muscle TNNT1, co-immunoprecipitated with CaVb1a in the mouse skeletal muscle cell line C2C12 and mouse skeletal muscle. Consistently, both CaVb1a and TNNT3 were highly expressed in mouse fast, but not slow, skeletal muscle. Surprisingly, when DsRed-tagged TNNT3 and YFP tagged CaVb1a (CaVb1a -YFP) were transiently co- expressed in C2C12 cells or the mouse flexor digitorum brevis (FDB) muscle, both showed a punctuate distribution pattern in the cytoplasm and nucleus. In comparison, CaVb1a-YFP alone localized uniformly in the cytoplasm. Fur- ther truncation analysis revealed that TNNT3 C-terminus (aa 161-244) local- ized exclusively in FDB nuclei and enriched CaVb1a -YFP accordingly. In contrast, the TNNT3 middle region (aa 55-160) localized only in the cyto- plasm, while the N-terminus (aa 1-54) was found in both cytoplasm and nu- cleus. Note that the N-terminus could also recruit CaVb1a-YFP in the nucleus. Y2H assay verified that the TNNT3 C-terminus has the strongest in- teraction with CaVb1a. Immunoblotting with an antibody targeting the TNNT3 N-terminus detected an increased TNNT3 fragment in the aging mouse skeletal muscle. We conclude that TNNT3 interacts with CaVb1a and regulates its nuclear translocation mainly through its TNNT3 C- or N-ter- minus. These findings strongly support a transcription-regulation function for CaVb1a and TNNT3. 3201-Pos Board B306 ATP Sensitivity and IP 3 -Dependent Calcium Transients Which Regulate Gene Expression in Adult Muscle Fibers are Altered in Mdx Mice Denisse Valladares, Mariana Casas, Reinaldo Figueroa, Alejandro Leyton, Sonja Buvinic, Enrique Jaimovich. ATP has been shown to be released from muscles during exercise and after te- tanic electrical stimulation (ES) and induces IP 3 dependent slow calcium tran- sients involved in gene expression in myotubes. The aim of this study was to characterize these signals in adult fibers and to establish differences between normal and mdx mouse. Experiments were performed in cultured isolated muscle fibers obtained from normal and mdx adult mice. The release of ATP induced by ES was measured using luciferin-luciferase and for calcium release studies the fibers were loaded with fluo-3. The location and expression of the nucleotide receptors were deter- mined by immunofluorescence and western blot. Slow, post tetanic calcium signals were partly inhibited by pharmacological agents targeting pannexin-1 (a pathway for ATP release), purinergic receptors and extracel- lular ATP. These signals had slower decay constants in mdx fibers. ES induced sig- nificant ATP release in normal fibers but this release was not detected in mdx fibers. The cellular location of ATP receptors was similar in both fiber types but their level of expression was higher in mdx fibers. Pannexin-1 expression was significantly augmented in mdx fibers. External ATP induced Ca 2þ signals in both, normal and mdx muscle fibers, but the sensitivity to ATP was much higher in mdx fibers. This study demonstrates that ATP signaling is profoundly altered in mdx fibers and could be implicated in the calcium disturbance described in DMD; a com- pensatory mechanism may be involved in the reduced ATP extrusion apparent in dystrophic cells. FONDAP 15010006, AFM 14562, FONDECYT 1080120, CONICYT PhD fellowship (DV) 3202-Pos Board B307 The Role of Ryanodine Receptor Phosphorylation in Skeletal Muscle Excitation-Contraction Coupling Matthew J. Betzenhauser, Daniel C. Andersson, Steven Reiken, Andrew R. Marks. Activation of the type 1 ryanodine receptor (RyR1) during skeletal muscle excitation-contraction (EC) coupling allows the release of stored Ca 2þ required for muscle contraction. While RyR1 is a well-known substrate of protein kinase A (PKA), the physiological significance of this phosphorylation event is poorly defined. PKA is known to increase the open probability of the channel in lipid bilayers by inducing phosphorylation of a specific residue (S2844). Knock-in mice harboring phosphorylation site deficient (RyR1-S2844A) and phosphomi- metic (RyR1-S2844D) mutations were generated in order to probe the conse- quence of this phosphorylation event in skeletal muscle EC coupling. Specific force measurements were performed on extensor digitorum longus (EDL) muscles and intracellular Ca 2þ release was examined in isolated flexor digitorum brevis (FDB) muscle fibers using confocal microscopy. While iso- proterenol (ISO) enhanced both Ca 2þ release and muscle force in wild type (WT) mice, the positive effects of ISO were abrogated in samples from RyR1-S2844A mice. This demonstrates the enhancing effects of RyR1 phos- phorylation during adrenergic stimulation in skeletal muscle. While transient phosphorylation of RyR1 exerts positive effects, chronic phosphorylation, as occurs in animal heart failure models, is thought to be detrimental to muscle function. We tested this hypothesis by measuring muscle force production and intracellular Ca 2þ in RyR1-S2844D mice. At 3-months of age, muscle function and Ca 2þ release were similar to WT controls. However, muscle force production and intracellular Ca 2þ release were both blunted by 6-months of age suggesting that a pathological Ca 2þ leak induced by the S2844D mutation im- pairs muscle function. 3203-Pos Board B308 Nox2 Dependent Modulation of Skeletal Muscle EC Coupling George G. Rodney, Guoli Shi. Generation of reactive oxygen species (ROS) under physiological conditions is required for normal force production in skeletal muscle. However, high levels of ROS promote contractile dysfunction, resulting in muscle weakness and fa- tigue. Our recent studies suggest that sub-cellular site-specific ROS production governs the beneficial vs. damaging effects of ROS. NADPH oxidase (Nox2) is an enzyme complex that generates ROS. In this study we investigated the role of Nox2 in skeletal muscle EC coupling. Using sub-cellular site-specific re- dox probes we show that cytosolic glutathione redox potential becomes more ox- idized during contractile activity while mitochondrial redox potential does not change. We show that increased contractile activity promotes Nox2 dependent ROS production. Interestingly, we have found that the non-tyrosine kinase Src is activated in response to increased contractile activity. These data support a hy- pothesis in which Src acts as a redox switch to activate Nox2. Increased ROS pro- duction during strenuous exercise would decrease sarcolemmal Ca 2þ influx and decrease sarcoplasmic reticulum refilling, which could contribute to the develop- ment of fatigue. As trials of general antioxidant therapy to combat increased ROS production have not been convincingly beneficial, understanding the sub-cellular signaling pathways by which oxidants influence muscle function will allow for the development of targeted therapeutic interventions to combat the deleterious effects of sustained contractile activity as well as skeletal muscle diseases. 3204-Pos Board B309 Pathological RyR1 Mutations to Identify RyR1 Functional Domains Marine Cacheux, Julien Faure ´, Julie Brocard, Nicole Monnier, Joe ¨l Lunardi, Isabelle Marty. Muscle contraction is achieved when an efficient excitation signal at the plasma membrane triggers intracellular calcium release. This process called ‘‘excita- tion-contraction (E-C) coupling’’ relies on a multimolecular protein complex, the calcium release complex. This complex, spanning the plasma membrane and the sarcoplasmic reticulum (SR), is organized around the calcium channel of the SR, the ryanodine receptor (RyR1). Mutations in RyR1 lead to a number of muscle diseases: Central core disease (CCD), Multi mini core disease (MmD), centronuclear myopathy, Malignant hy- perthermia¼ Nevertheless, the functional consequences of each mutation are largely unknown because of the lack of information on the functional domains in RyR1. For the present study, we use the mutations identified in RyR1 to de- cipher functional regions in RyR1. We have identified more than 200 RyR1 mu- tations in the course of clinical diagnosis. We have recently shown that RyR1 interacts with caveolin-3, via the transmembrane part of the channel (Vassilopou- los et al, 2010, Biochemistry, 49, 6130). Caveolin-3, which is a structural protein involved in intracellular trafficking, could therefore regulate RyR1 target- ing. We are now searching for mutations in RyR1 which could affect the 592a Wednesday, March 9, 2011