reduce or abolish the propensity for store-overload induced Ca release (SOICR) in HEK293 cells. In addition, single channel analysis showed that these muta- tions significantly reduce the response of the channel to activation by cytosolic and luminal Ca. These observations demonstrate, for the first time, that sup- pressed or loss of RyR2 function may be a common mechanism underlying idiopathic ventricular fibrillation, which is opposite to the gain-of-function RyR2 mutations associated with CPVT. Thus, understanding the exact molec- ular defects of disease-causing RyR2 mutations will help to develop novel approaches to the diagnosis and personalized treatment of these lethal cardiac arrhythmias (Supported by CFI, CIHR, AIHS, and LCIA). 573-Pos Board B328 Suppression of Spontaneous CA 2D Release by Cardioprotective Drugs Jingqun Zhang 1 , Chris D. Smith 2 , Qiang Zhou 1 , Jianmin Xiao 3 , Guogen Wu 1 , Alma Nani 1 , Thomas G. Back 2 , Michael Fill 1 , S.R. Wayne Chen 1,3 . 1 Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL, USA, 2 Department of Chemistry, University of Calgary, Calgary, AB, Canada, 3 Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada. Spontaneous sarcoplasmic reticulum (SR) Ca 2þ release in the form of Ca 2þ waves occurs in cardiac cells under conditions of SR Ca 2þ overload. This store-overload induced Ca 2þ release (SOICR) is a well-known cause of delayed afterdepolarization and triggered arrhythmias. Hence, inhibiting SOICR may represent a promising therapeutic strategy for Ca 2þ -triggered arrhythmias. Indeed, we have recently shown that carvedilol, one of the most effective beta-blockers for preventing ventricular tachyarrhythmias and sudden death in heart failure, possesses a novel anti-SOICR activity. To identify more SOICR inhibitors, we searched the DrugBank database (http://www. drugbank.ca) for chemicals with structures similar to carvedilol. We selected a number of hit compounds and assessed their impact on SOICR in HEK293 cells expressing a SOICR-promoting cardiac ryanodine receptor (RyR2) muta- tion (R4496C). We found that curcumin and resveratrol (natural phenols) and bevantolol (a beta blocker and a Ca 2þ channel blocker) suppress SOICR in HEK293 cells with IC50s of 6.750.3, 48.453.6, 64.7511.3mM, respectively. On the other hand, gliclazide, diphenhydramine, crocin, dexrazoxane, and trazodone displayed little or no SOICR inhibition. We also tested several other known cardioprotective drugs. We found that docosahexaenoic acid (DHA), eicosapentaenoic (EPA), arachidonic acid (AA), anandamide, and a novel derivative of anandamide (CS-X-95) inhibit SOICR in HEK293 cells with IC50s of 11.254.2, 5.150.5, 5.550.9, 14.755.1, and 11.550.7 mM, respec- tively, whereas ranolazine and S107 have no significant impact on SOICR. These observations indicate that many cardioprotective drugs exhibit anti-SOICR activity, and that SOICR inhibition may contribute, in part, to their cardioprotective effect. (Supported by NIH) 574-Pos Board B329 Cryo-EM Studies of RyR1 Channel in Detergent-Free Aqueous Environment Olga B. Popova 1 , Guizhen Fan 1 , Mariah R. Baker 1 , Joanita Jakana 2 , Wah Chiu 2 , Steven J. Ludtke 2 , Irina I. Serysheva 1 . 1 Biochemistry and Molecular Biology, UT-Medical School at Houston, Houston, TX, USA, 2 National Center for Macromolecular Imaging, Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA. Skeletal muscle ryanodine receptor (RyR1) is a Ca 2þ release channel in the sarcoplasmic reticulum membrane and plays a key role in excitation- contraction coupling. Obtaining high-resolution 3D structure of RyR1 is a formidable challenge due to its enormous size (~2.3 MDa), dynamic nature and location in the lipid membrane in native state. Detergents are traditionally used to make membrane proteins water soluble and suitable for single-particle cryo-EM. However, detergents tend to destabilize and inactivate membrane proteins. In addition, the presence of detergent in the protein sample reduces surface tension of water making it difficult to control the ice thickness and dis- tribution of channel particles in cryospecimen, and leads to low-contrast in cryo-images. To overcome these difficulties we used amphipathic polymer, am- phipol 8-35 (APol8-35), to substitute for detergent in RyR1 preparations. We tested functionality of RyR1/APol8-35 in a [ 3 H]ryanodine binding assay, which yielded K d and B max values similar to those of the purified RyR1 in the presence of CHAPS, indicating that the high-affinity binding site for ryanodine is re- tained in RyR1/APol8-35. The use of Apol8-35 allowed us to reproducibly obtain ice-embedded specimens of RyR1 for cryo-EM analysis and resulted in improved ice thickness with channel particles uniformly distributed across the holes in the grids. The protein contrast of ice-embedded RyR1/APol8-35 is substantially higher than in detergent preparations using a traditional CCD detector. Furthermore, low-dose images of vitrified RyR1/APol8-35 have been collected on the DE-12 back-thinned DDD camera using JEM3200FSC electron cryomicrosope operated at 300 keV at liquid nitrogen temperature. Preliminary analysis of these cryo-EM images demonstrates image signals that extend beyond ~6A ˚ . Optimal conditions for both sample vitrification and data acquisition were reached in order to achieve a higher res- olution RyR1 structure. Supported by NIH (R21AR063255, R01GM072804, R01GM080139, P41GM103832) and AHA (12GRNT10510002). 575-Pos Board B330 Ligand-Induced Conformational Changes in Tetrameric IP 3 R1 Revealed by Single-Particle Cryo-EM Guizhen Fan 1 , Olga B. Popova 1 , Steven J. Ludtke 2 , Irina I. Serysheva 1 . 1 Biochemistry and Molecular Biology, The University of Texas Medical School, Houston, TX, USA, 2 National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and M, Baylor College of Medicine, Houston, TX, USA. A functional hallmark of inositol 1,4,5-trisphosphate receptors (IP 3 Rs), the main Ca 2þ release channels in the endoplasmic reticulum of virtually all eukaryotic cells, is the coupled interplay between the binding of primary ligands, IP 3 and Ca 2þ , and channel gating. IP 3 Rs are exceptionally large integral membrane proteins, comprising four subunits of over 300 kDa each. A central mechanistic question of IP 3 R function is how IP 3 binding in the N-terminal sequence of the channel protein is communicated to the ion- conduction pore, which is formed close to the C-terminus. Using single- particle cryo-EM, we have performed structural analysis of purified tetrameric IP 3 R1 vitrified in the presence of micromolar Ca 2þ and saturating concentra- tion of adenophostin A (AdA), a structural mimetic of IP 3 , that is a high- affinity, full agonist of IP 3 Rs. Cryo-EM images of IP 3 R1 were recorded on a Gatan 4k x 4k CCD camera in the JEM2010F cryomicroscope operated at low-dose conditions. Using EMAN2, ~40,000 particle images were merged to generate a preliminary 3D map of ligand-bound IP 3 R1 at ~15 A ˚ resolution. A comparison with our recent structure of IP 3 R1, determined without the addi- tion of any channel ligands (Ludtke et al., 2010: Structure 19, 1192-99), reveals structural rearrangements in the cytoplasmic domains of the ligand-bound IP 3 R1 channel. Furthermore, we have performed docking studies of available crystal structures of the ligand-binding domains into reconstructed cryo-EM density maps of the entire IP 3 R1. All together our studies suggest that AdA/ Ca 2þ binding induces conformational changes in the quaternary structure of tetrameric IP 3 R1 that might underlie the long-range allosteric mechanism of agonist-mediated activation of the ion-conducting pore of IP 3 R. Supported by grants from NIH (R21AR063255, R01GM072804, R01GM080139, P41GM103832) and by AHA (12GRNT10510002). 576-Pos Board B331 Coupled Gating of Ryanodine Receptors: Evidence for a Role of Physical RyR-RyR Interactions Paula L. Diaz-Sylvester 1 , Jake T. Neumann 1 , Sidney Fleischer 2 , Julio A. Copello 1 . 1 Pharmacology, SIU-SOM, Springfield, IL, USA, 2 Biological Sciences, Vanderbilt University, Nashville, TN, USA. We studied the synchronous function (coupled gating) of arrays of striated muscle ryanodine receptors (RyRs) reconstituted into planar bilayers. We think that understanding coupled gating would help explain local Ca 2þ sparks and global Ca 2þ transients which arise from the simultaneous activation of groups of RyRs in the sarcoplasmic reticulum. We have previously found that coupled gating of multiple cardiac or skeletal RyRs requires luminal Ca 2þ as current carrier. In principle, this would suggest that Ca 2þ flowing from the open pore of a RyR, could activate neighboring channels (local CICR). However, further analysis showed that coupled events are insensitive to changes in the magnitude of lumen-to-cytosol Ca 2þ flux. This is not compatible with the idea of individual channels modulated by local CICR. As an alternative, we tested the involvement of physical RyR-RyR interactions in the process of coupled gating. We found that coupled gating of RyRs reconstituted in planar bilayers is not significantly affected by agents that disrupt cytoskeletal networks, FKBPs and/or kinases/phosphatases. However, the polycationic amine spermine interfered with coupled RyR gating (suggesting a role for electrostatic interactions). Additionally ryanodol, known to lock open channels in a sub-conductance state, also affected coupled RyRs. Nevertheless, event termination (synchronous channel closures) occurred even during ryanodol substates. This was unexpected because single channels do not transition from the ryanodol/ryanodine substate to the closed state. Overall, our results suggest that modulation of coupled RyRs is different from that expected for individual channels interacting by local CICR. We think that luminal Ca 2þ pro- motes conformational changes in RyRs allowing for cytosolic RyR-RyR Sunday, February 16, 2014 109a