(InsP 3 ). Recently, perturbations in the InsP 3 R1 receptor have been linked to a human neurodegenerative disorders. The slow, progressive neurological disease, Spinocerebellar Ataxia type15 (SCA15), is inherited through an auto- somal dominant gene and causes degeneration of the cerebellum. A missense mutation P1059L in the regulatory and coupling domain of the receptor (P1073L in mice) has been suggested by linkage analysis to result in SCA15 in humans. A further mutation, Itpr1D18/D18, causes the deletion of 6 amino acids in InsP 3 R-1 and results in an ataxic phenotype in mice. We have created stable cell lines expressing corresponding mutations in the rodent InsP 3 R1 gene in DT40-3KO cells, an unambiguously InsP 3 R null background. Immunoblot- ting confirmed expression of the mutant channels at comparable levels to wild- type. In both "on-nuclear" single channel patch clamp experiments and Ca 2þ imaging both mutated InsP 3 R1 receptors are functional Ca 2þ channels. A comparison of the activity of the mutated receptors with wild type InsP 3 R1 will be presented. 2661-Pos Regulation of Inositol 1,4,5 Trisphosphate Receptors by InsP3 Receptor- Associated cGMP Kinase Substrate (IRAG) Wataru Masuda, Matthew J. Betzenhauser, David I. Yule. University of Rochester, Rochester, NY, USA. Various factors interact with IP 3 R, regulating Ca 2þ release and thus serve to define the spatial and temporal characteristics of the cytosolic Ca 2þ signal. IP 3 R-associated cGMP kinase substrate (IRAG) has been reported to bind IP 3 R type-1 and inhibit Ca 2þ mobilization in smooth muscle cells. No informa- tion is however available as to whether IRAG interacts or has functional effects on other IP 3 R family members. In this study, we examined whether IRAG binds to and regulates Ca 2þ release via IP 3 R type-2 or type-3. cDNA encoding IP 3 R type-1, IRAG-GFP, and protein kinase G1b (PKG1b) were transiently trans- fected into COS cells. Following immunoprecipitation from cell lysates with an anti-GFP antibody, IP 3 R type-1 was detected by immunoblotting. In con- trast, an IRAG-GFP construct (IRAGDE12-GFP) in which 40 amino acids re- quired for binding with IP 3 R was deleted, failed to interact with IP 3 R type-1, but was still capable of binding to PKG1b , an additional cognate binding part- ner of IRAG. Similarly, IP 3 R type-2 or IP 3 R type-3 could be shown to interact with IRAG-GFP but not IRAGDE12-GFP in COS cells. Next, we investigated if IRAG regulates IP 3 -induced Ca-release using DT40-3KO cell lines stably ex- pressing mammalian IP 3 R type-2 or type-3 in isolation. In DT40-3KO cells sta- bly expressing IP 3 R type-2, and transiently expressing Muscarinic M3 receptor, IRAG-GFP and PKG1b, cell permeable PKG activators reduced the muscarinic agonist carbachol (CCh)-induced Ca 2þ -release. Ca 2þ oscillations induced by low concentrations of CCh or by stimulating the endogenous B cell receptor were similarly attenuated. No inhibitory effect was evident in cells expressing IRAGDE12-GFP or in the absence of IRAG-GFP. Similar results were obtained with DT40-3KO cells stably expressing IP 3 R type-3. These results indicate that Ca 2þ release through all Inositol 1,4,5 trisphosphate receptors are inhibited by an interaction with IRAG and PKG1b. 2662-Pos CaMKII-Mediated Phosphorylation of InsP 3 R2 at Serine-150 Results in Decreased Channel Activity Joshua T. Maxwell, A.S. Aromolaran, Gregory A. Mignery. Loyola University Medical Center, Maywood, IL, USA. InsP 3 mediated calcium release through the type-2 inositol 1,4,5-trisphosphate receptor (InsP 3 R2) in cardiac myocytes results in the activation of associated CaMKIId (Bare et al, 2005, JBC; Wu et al, 2006, JCI), enabling the kinase to act on downstream targets, such as histone deacetylases 4 & 5 (HDAC4 & HDAC5) (Zhang et al, 2007, JBC). The CaMKII activity also feedback modu- lates InsP 3 R2 function by direct phosphorylation and results in a dramatic de- crease in the receptor-channel open probability (P o ). The results of this study show that in planar lipid bilayers the channel activity of InsP 3 Rs can be in- hibited by CaMKII-mediated phosphorylation, and that effect can be reversed by addition of protein phosphatases. Furthermore, we have used fragments of the InsP 3 R2 and site-directed mutagenesis to determine that Serine at residue 150 is the CaMKII phosphorylation site responsible for modulation of channel activity. Non-phosphorylatable (S150A) and phospho-mimetic (S150E) muta- tions were constructed in the full-length InsP 3 R2, expressed in COS cells and studied in planar lipid bilayers. Upon treatment with CaMKII, the non-phos- phorylatable channel showed no decrease in activity. Conversely, the phospho- mimetic channel displayed a very low P o under normal recording conditions in the absence of CaMKII (2mM InsP 3 and 250nM [Ca 2þ ] FREE ), thus mimicking a channel that has been phosphorylated by CaMKII. The results of this study show that Serine-150 of the InsP 3 R2 is phosphorylated by CaMKII and results in a decrease in the channel’s open probability. The mechanism for the regula- tion of the InsP 3 R2 appears to be a consequence of altered affinity for InsP 3 at the ligand binding site and/or perturbation of the receptor amino to carboxyl- terminal interaction and is currently being examined. These studies were supported by National Institutes of Health grant PO1HL080101. 2663-Pos Excitement Over Automated Patch Clamp: Action Potentials from Cardiac Myocytes Sonja Stoelzle 1 , Andrea Bruggemann 1 , David Guinot 1 , Alison Haythornthwaite 1 , Michael George 1 , Cecilia Farre 1 , Claudia Haarmann 1 , Ralf Kettenhofen 2 , Niels Fertig 1 . 1 Nanion Technologies, Munich, Germany, 2 Axiogenesis AG, Cologne, Germany. The use of cardiac myocytes is becoming increasingly important for drug safety testing. Unique features of certain planar patch clamp workstations, coupled with ease-of-use and higher data throughput, make these devices ideal tools for ion channel screening and safety testing. Using stem cell derived cardiac myocytes, recordings could be made not only in the voltage-clamp mode but also in the current-clamp mode on a planar patch clamp workstation. This dem- onstrates for the first time parallel current-clamp recordings on a planar patch clamp workstation. Ion channels important in drug discovery, such as hERG and voltage-gated Naþ, Ca2þ and Kþ channels in the voltage-clamp mode from stem cell derived cardiac myocytes will be shown. In addition, action potential recordings in the cur- rent-clamp mode at 35  C, and modu- lation of the action potentials by hERG active compounds, will also be shown. 2664-Pos In Silico Studies of Cardiac Inotropy using a New Model of Force Generation Jose L. Puglisi 1 , Jorge A. Negroni 2 , Donald M. Bers 1 . 1 University of California, Davis, Davis, CA, USA, 2 Universidad Favaloro, Buenos Aires, Argentina. An improved model of force generation was incorporated into a complete math- ematical description of action potential (AP), ionic currents and Ca 2þ transient of the rabbit ventricular myocyte (LabHEART 5.0). This new model repro- duces the main events involved in Excitation-Contraction Coupling, namely the AP (excitation), the shortening (contraction) and the Ca 2þ transient as the link between them. LabHEART 5.0 was able to reproduce isotonic and iso- metric contractions and the classical curves of Force vs. Ca 2þ and Force vs cell length. Cardiac inotropy was investigated by simulating the application of iso- proterenol (ISO). This effect was achieved by altering L-type Ca 2þ current, the slowly activating delayed rectifier K þ current, sarcoplasmic reticulum (SR) Ca 2þ pump, SR Ca 2þ leak, myofilament Ca-sensitivity and cross-bridge cy- cling. The latter modification was essential for replicating the ISO-induced in- crease in force generation/shortening development experimentally observed. AP duration (APD, for 90% of repolarization) adaptation to pacing frequencies was also examined. ISO shortened APD at all frequencies with respect to con- trol and flattened the adaptation curve, thus allowing an APD compatible with short cycle length (up to 5 Hz). ISO also increased the Ca 2þ transient dynamic range by keeping a low diastolic level while increasing the peak Ca 2þ at all the simulated frequencies (0.5 to 9 Hz). This model provides a useful framework to study cardiac inotropy and constitutes a starting point to investigate the electro- mechanical feedback in cardiac performance. The new version LabHEART 5.0 is freely available online at www.labheart.org. Voltage-gated Ca Channels I 2665-Pos Monte Carlo Simulation of Free Energy Components: Energetics of Selec- tive Binding in a Reduced Model of L-Type Ca Channels Janhavi Giri 1,2 , Bob Eisenberg 2 , Dirk Gillespie 2 , Douglas Henderson 3 , Dezs} o Boda 4 . 1 University of Illinois at Chicago, Chicago, IL, USA, 2 Rush University Medical Center, Chicago, IL, USA, 3 Brigham Young University, Provo, UT, USA, 4 University of Pannonia, Veszpre ´m, Hungary. A reduced model of voltage-gated L-type Ca channels is used to study the en- ergetics of selective binding of Ca 2þ versus monovalent and divalent cations. Widom’s particle insertion method is combined with Grand Canonical Monte Carlo simulations to compute the electrostatic and excluded volume compo- nents of the free energy difference between channel and bath. We have shown (in ~ 30 papers) that selectivity of the L-type Ca channel and voltage activated 514a Tuesday, February 23, 2010