1606-Pos Board B376 3-D Localization of Subcellular Ca 2D Release Reveals a Cytoskeletal Dependence of RyR Activation Eef Dries, Virginie Bito, Karin Sipido, Niall Macquaide. KULeuven, Leuven, Belgium. In large mammals, many SR Ca 2þ channels (RyRs) are uncoupled from sar- colemma or T-tubules (TTs). Tools for exploring RyR regulation in relation to T-tubule proximity are limited. Here we present a method to measure TT dis- tance and investigate release from coupled and uncoupled RyRs. In isolated pig ventricular myocytes consecutive vertical confocal images were taken af- ter labeling with wheat germ agglutinin-Alexa594. After image processing, the 3-D TT geometry was assessed using Euclidean distance mapping. Whole-cell voltage clamp was subsequently used to elicit Ca 2þ release at 0.5, 1 and 2 Hz and Ca 2þ was recorded in a confocal line scan (0.3 micron pixel-1, 649 Hz). The temporal mid-point of the local Ca 2þ transient upstroke (T F50 ) was used to assess latency of release. Correlation of the T F50 for each site with distance to TT in 3-D yielded a linear relationship for distance be- tween 0.5 and 3 mm from T-tubules. This allowed the use of T F50 to map sub- cellular Ca 2þ release regions as coupled (< 0.5 mm) and uncoupled (> 2 mm). In coupled regions, T F50 occurred within 18 ms, 17 ms and 12 ms, respec- tively for 0.5, 1 and 2 Hz. This resulted in 56% (0.5 Hz), 57% (1 Hz) and 49% (2 Hz) of coupled regions. Spark frequency increased with stimulation frequency in coupled release sites but significantly less so in uncoupled ones. In the presence of the F-actin disrupter Cytochalasin D, this increase in spark frequency was abolished and there was no difference between cou- pled and uncoupled sites. Therefore, the regional discrimination of RyR re- veals a preferential activation of coupled RyRs with frequency, which is dependent on a cytoskeletal interaction. 1607-Pos Board B377 Control of Diastolic Activity of the RyR2 Channel by Luminal Calcium and ATP Barbora Tencerova, Jana Gaburjakova, Marta Gaburjakova, Alexandra Zahradnikova. Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia. The mechanism of activation of the cardiac ryanodine receptor (RyR2) by lu- minal Ca 2þ in the presence of ATP was examined in planar lipid bilayers. The dose response of the RyR2 channel to ATP was characterized at a range of cy- tosolic (100-400 nM) and luminal (0.01 - 53 mM) Ca 2þ concentrations. Lumi- nal Ca 2þ markedly increased the maximal open probability in the presence of ATP (P O max ) and markedly decreased EC 50 for ATP (EC 50 ATP ). Cytosolic Ca 2þ , without substantially activating the RyR2 channel in the absence of ATP, greatly amplified the effects of 1 mM [Ca 2þ ] L on P O max and EC 50 ATP . An allosteric model of RyR2 interaction with ATP showed that the increase in RyR2 open probability by luminal Ca 2þ in the presence of ATP is induced by a decrease of the ATP dissociation constant (K ATP ) at low [Ca 2þ ] L , by an increase in the positive allosteric effect of ATP on channel opening (decrease of f ATP ) at intermediate [Ca 2þ ] L , and by an increase in the stability of the ATP- free RyR2 open state (decrease of K O0 ) at the highest [Ca 2þ ] L . Increasing [Ca 2þ ] C did not affect K ATP but led to a parallel decrease of K O0 and f ATP . These results suggest that the allosteric effect of ATP might be mediated by the energy of the ATP-free open state, i.e., indirectly by the effect of the occu- pancy of the channel by Ca 2þ at the cytosolic and luminal sites on the stability of the open state. The increase of RyR2 open probability at diastolic levels of cytosolic Ca 2þ by elevated luminal Ca 2þ may play a role in the calcium over- load induced Ca 2þ release. Supported by APVV-0721-10, APVV-0628-10, VEGA2/0118/09 and VEGA 2/ 0190/10 1608-Pos Board B378 Calcium Signaling in Myocytes of Injured Myocardium Alexandra Zahradnikova, Jr., Radoslav Janicek, Ivan Zahradnik, Alexandra Zahradnikova. Institute of Molecular Physiology and Genetics, Bratislava, Slovakia. Calcium signaling during cardiac excitation-contraction coupling is compro- mised after myocardial injury (MI). Myocytes of injured myocardium display reduced synchronization of calcium spikes at low calcium current density in- dicating worsened communication between sarcolemmal DHPR and sarco- plasmic reticulum RyR calcium channels [1]. Here we study the activation of Ca-spikes by using strong synchronized calcium triggers. Cardiac myocytes were isolated from left ventricles of young adult male Wistar rats on day 15 after administration of a single high dose of isoproterenol, to model the myo- cardial injury (MI), or a vehicle as the control, and studied by means of the whole cell patch clamp and confocal microscopy techniques. Ca-spikes were activated by a short prepulse to þ60 mV from a holding potential of À50 mV and subsequent step repolarization to negative voltages that induced perfectly synchronized influx of Ca 2þ ions via DHPR channels. Ca-spikes were recorded using 100 mM fluo-3 and 1 mM EGTA at 2 kHz line scan fre- quency. In this arrangement, increase of the calcium influx increased the prob- ability of Ca-spike activation in the same manner in both the control and MI groups. The probability of local Ca 2þ release was not different between the two groups. In the MI group, the amplitude of Ca-spikes was increased and the latency of spikes was abbreviated relative to controls. Both effects could be explained by increased sarcoplasmic reticulum Ca 2þ load revealed by caf- feine application. We conclude that the DHPR-RYR calcium coupling in MI myocytes functions well when the triggering calcium influx is strong, most likely due to sensitizing effect of increased sarcoplasmic reticulum Ca 2þ load on RyR activation by calcium. Supported by grants APVV-0721-10 and VEGA 2/0197/11. References 1. Zahradnikova A Jr, Polakova E, Novotova M, Stankovicova T, Zahradnik I, Zahradnikova A, J Physiol Sci 59(S1): 313, 2009 1609-Pos Board B379 Ryanodine Receptor Recruitment and Construction of Calcium Release Sites in Cardiac Myocytes Alexandra Zahradnikova, Radoslav Janicek, Alexandra Zahradnikova, Jr., Marta Gaburjakova, Ivan Zahradnik. UMFG SAV, Bratislava, Slovakia. Local character of calcium release in cardiac myocytes implies independent re- cruitment of calcium release units by triggering stimuli. Calcium release from individual CRUs also displays quantal character, interpreted either as recruit- ment of small cohorts of independent RYRs (1,2), or as recruitment of small cohorts of RYR clusters with coupled RYRs gating (3). We tested both inter- pretations on published experimental data using a model of virtual calcium re- lease units (vCRUs) consisting of 1-10 clusters that were constructed in accordance with the experimentally observed cluster size distribution (4). If RYR gating was independent, vCRUs consisting of 3 or more clusters provided a good agreement between the model and experimental data (1). If RYR gating was coupled, the calcium release flux of vCRUs did not display quantal struc- ture that would correspond to in situ observations. The model of RYR gating (2) combined with the model of independent RYRs in vCRUs (2) matched the ex- perimentally observed calcium dependence of calcium spark frequency (5) un- der all conditions. However, the Mg2þ-binding parameters of RYRs were in accordance with the single-channel observations (6) only for vCRUs composed of 3 or more clusters. In conclusion, these results favor independent over fully coupled RYR gating in situ, and predict the presence of at least 40 RYRs per cardiac calcium release unit. Supported by grants APVV-0441-09, APVV-0721-10, VEGA 2/0190/10, 2/ 0203/11, and 2/0197/11. References 1. Wang SQ et al., PNAS 101: 3979-3984, 2004 2. Zahradnikova A et al., J. Gen. Physiol. 136: 101-116, 2010 3. Xie W et al., J. Gen. Physiol. 136: 129-133, 2010 4. Baddeley D et al., PNAS 106: 22275-22280, 2009 5. Lukyanenko V, Gyorke S, J Physiol. 521: 575-585, 1999 6. Zahradnikova A et al., Am J Physiol. 285, C1059-1070, 2003 Exocytosis & Endocytosis 1610-Pos Board B380 Properties of the Weibel-Palade Body Fusion Pore Emma A. Cookson 1 , John Dempster 2 , Matthew J. Hannah 3 , Tom Carter 1 . 1 MRC NIMR, London, United Kingdom, 2 University of Strathclyde, Glasgow, United Kingdom, 3 Health Protection Agency, London, United Kingdom. Weibel-Palade bodies (WPBs) are regulated secretory organelles found in endothelial cells (ECs). The major WPB constituent is the haemostatic 316a Monday, February 27, 2012