Betzenhauser, Matthew J.

Masuda, Wataru

Yule, David I.

Elsevier - Publisher Connector 

514a Tuesday, February 23, 2010(InsP3). Recently, perturbations in the InsP3R1 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 InsP3R-1 and results in an ataxic phenotype in mice. We have created
stable cell lines expressing corresponding mutations in the rodent InsP3R1 gene
in DT40-3KO cells, an unambiguously InsP3R 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 Ca2þ
imaging both mutated InsP3R1 receptors are functional Ca
2þ channels. A
comparison of the activity of the mutated receptors with wild type InsP3R1
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 IP3R, regulating Ca
2þ release and thus serve to
define the spatial and temporal characteristics of the cytosolic Ca2þ signal.
IP3R-associated cGMP kinase substrate (IRAG) has been reported to bind
IP3R type-1 and inhibit Ca
2þmobilization in smooth muscle cells. No informa-
tion is however available as to whether IRAG interacts or has functional effects
on other IP3R family members. In this study, we examined whether IRAG binds
to and regulates Ca2þ release via IP3R type-2 or type-3. cDNA encoding IP3R
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, IP3R 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 IP3R was deleted, failed to interact with IP3R type-1,
but was still capable of binding to PKG1b , an additional cognate binding part-
ner of IRAG. Similarly, IP3R type-2 or IP3R type-3 could be shown to interact
with IRAG-GFP but not IRAGDE12-GFP in COS cells. Next, we investigated
if IRAG regulates IP3-induced Ca-release using DT40-3KO cell lines stably ex-
pressing mammalian IP3R type-2 or type-3 in isolation. In DT40-3KO cells sta-
bly expressing IP3R type-2, and transiently expressingMuscarinic M3 receptor,
IRAG-GFP and PKG1b, cell permeable PKG activators reduced the muscarinic
agonist carbachol (CCh)-induced Ca2þ-release. Ca2þ 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 IP3R type-3. These results indicate that
Ca2þ release through all Inositol 1,4,5 trisphosphate receptors are inhibited by
an interaction with IRAG and PKG1b.
2662-Pos
CaMKII-Mediated Phosphorylation of InsP3R2 at Serine-150 Results in
Decreased Channel Activity
Joshua T. Maxwell, A.S. Aromolaran, Gregory A. Mignery.
Loyola University Medical Center, Maywood, IL, USA.
InsP3 mediated calcium release through the type-2 inositol 1,4,5-trisphosphate
receptor (InsP3R2) 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 InsP3R2 function by direct phosphorylation and results in a dramatic de-
crease in the receptor-channel open probability (Po). The results of this study
show that in planar lipid bilayers the channel activity of InsP3Rs 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 InsP3R2 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 InsP3R2, 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 Po under normal recording conditions in
the absence of CaMKII (2mM InsP3 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 InsP3R2 is phosphorylated by CaMKII and results
in a decrease in the channel’s open probability. The mechanism for the regula-
tion of the InsP3R2 appears to be a consequence of altered affinity for InsP3 atthe 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 Stoelzle1, Andrea Bruggemann1, David Guinot1,
Alison Haythornthwaite1, Michael George1, Cecilia Farre1,
Claudia Haarmann1, Ralf Kettenhofen2, Niels Fertig1.
1Nanion Technologies, Munich, Germany, 2Axiogenesis 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 35C, 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. Puglisi1, Jorge A. Negroni2, Donald M. Bers1.
1University of California, Davis, Davis, CA, USA, 2Universidad 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 Ca2þ 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 Ca2þ 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. Ca2þ 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 Ca2þ current, the
slowly activating delayed rectifier Kþ current, sarcoplasmic reticulum (SR)
Ca2þ pump, SR Ca2þ 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 Ca2þ transient dynamic
range by keeping a low diastolic level while increasing the peak Ca2þ 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 Giri1,2, Bob Eisenberg2, Dirk Gillespie2, Douglas Henderson3,
Dezs}o Boda4.
1University of Illinois at Chicago, Chicago, IL, USA, 2Rush University
Medical Center, Chicago, IL, USA, 3Brigham Young University, Provo,
UT, USA, 4University 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 Ca2þ 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


Regulation of Inositol 1,4,5 Trisphosphate Receptors by InsP3 Receptor-Associated cGMP Kinase Substrate (IRAG) 

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Regulation of Inositol 1,4,5 Trisphosphate Receptors by InsP3 Receptor-Associated cGMP Kinase Substrate (IRAG)

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