Betzenhauser, Matthew J.

Nienaber, Tricia

Wagner, Larry E.

Yule, David I.

Elsevier - Publisher Connector 

An Analysis of the Activity of Two Disease Associated Mutations of the Inositol 1,4,5-Trisphosphate Receptor Type-1 

Tuesday, February 23, 2010 513aIP3 Receptors
2655-Pos
Novel Insights into the Role of Junctate in Calcium Homeostasis: Identifi-
cation of Binding Domain on the InsP3R and Cellular Localization as
Determined by TIRF Microscopy
Susan Treves1,2, Johanna Griesser1, Clara Franzini-Armstrong3,
Michael X. Zhu4, Francesco Zorzato1,2.
1Basel Unoversity Hospital, Basel, Switzerland, 2University of Ferrara,
Ferrara, Italy, 3University of Pennsylvania, Philadelphia, PA, USA,
4Ohio State University, Columbus, OH, USA.
Junctate is a 33 kDa calcium binding protein with a single ER/SR membrane
spanning domain expressed in excitable and non-excitable tissues. It is gener-
ated by alternative splicing from the AßH-J-J locus, which also encodes the en-
zyme aspartyl-ß-hydroxylase, the sarcoplasimc reticulum structural protein
junctin and humbug, a truncated version of aspartyl-ß-hydroxylase, lacking
its catalytic domain, which shares with junctate the high capacity moderate af-
finity Ca2þ binding domain and is over-expressed in a variety of tumours. We
have previously shown that junctate forms a macromolecular complex with the
InsP3R and TRPC3 channels and when transiently over-expressed in HEK293
cells, it induces extensive proliferation of the ER resulting in significantly
larger and more frequent couplings between the ER and the plasma membrane.
In the present work we have mapped the binding domain of the cytoplasmic
NH2 terminus of junctate on the InsP3R and show that it binds to the domain
involved in InsP3 binding. Such a result is supported by the finding that in the
presence of a peptide encompassing the NH2 terminal domain of junctate, the
Bmax for InsP3 binding is significantly higher than that obtained in the pres-
ence of an unrelated peptide. Transmission electron microscopy revealed that
clones stably transfected with junctate-YFP display a significant larger number
of junctions between the ER and the plasma membrane compared to control
HEK293 cells and this effect is enhanced in clones that also over-express
TRCP3. The size and distribution of these punctae however, was not affected
by the addition of agonists mobilizing calcium via InsP3R activation.
2656-Pos
Visualizing Alpha Helices in the Transmembrane Region of IP3R1 Cal-
cium Release Channel by Single Particle Electron Cryomicroscopy
Steven J. Ludtke1, Thao P. Tran2, Que T. Ngo2, Vera Y. Moiseenkova-Bell3,
Joshua T. Maxwell4, Gregory A. Mignery4, Wah Chiu1, Irina I. Serysheva2.
1Baylor College of Medicine, Houston, TX, USA, 2The University of Texas
Health Science Center at Houston Medical School, Houston, TX, USA, 3Case
Western Reserve University School of Medicine, Cleveland, OH, USA,
4Stritch School of Medicine, Loyola University Medical Center, Maywood,
IL, USA.
Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), a 1.3 MDa tetrameric
membrane protein regulates the release of Ca2þ from endoplasmic reticulum
stores into the cytoplasm and plays an essential role in a wide variety of cellular
processes. We determined the structure of IP3R1 in the closed conformation at
~9-A˚ resolution by using single particle electron cryomicroscopy. The channel
protein was solubilized with detergent from rat cerebellar microsomes and pu-
rified by immunoaffinity chromatography. The purified channels were reconsti-
tuted into Ca2þ-loaded lipid vesicles and released Ca2þ in response to nanomo-
lar concentrations of IP3, indicating their functionality. Isolated IP3R1 particles
were embedded in vitreous ice for cryo-EM in the presence of 1 mM EGTA to
drive the channel protein into its closed conformation. The 3D density map of
IP3R1 was generated with 37,231 particles extracted from 929 CCD frames col-
lected on a JEOL2010F electron cryomicroscope. The reconstruction was per-
formed using EMAN software. This structure allows visualizing a number of
alpha-helices in the membrane-spanning region of the IP3R1 channel, including
the inner alpha-helices lining the tapering ion conduction pathway. The molec-
ular architecture of the closed pore is established based on the 9-A˚ cryo-EM
density map of IP3R1 and via computational and bioinformatics approaches.
This research is supported by grants from NIH (R01GM072804, P41RR02250)
and MDA Research Grant 88677.
2657-Pos
Spatial Modeling of IP3 Channel Clusters and Calcium Puffs
Sten Ruediger1, Jian-Wei Shuai2.
1Humboldt University, Berlin, Germany, 2Xiamen University, Xiamen,
China.
Using detailed numerical analysis as well as strongly reduced models we study
release of calcium from clusters of IP3 receptor channels. In the first part we
present hybrid stochastic-deterministic simulations of release from a cluster
of nine channels. We adopt recent advances in imaging of calcium release,
which showed a considerable spatial separation of channels. We find that,due to the separation of channels and the three-dimensional transport of cal-
cium away from the source area, the calcium concentration is generally hetero-
geneous in the cluster area. Based on a Markovian description of channel gating
and a fitting of ligand/channel reactions to single-channel data, we obtain puffs
that strongly resemble recent recordings in neuroblastoma cells. We conclude
that spatial heterogeneity is crucial part of the understanding of puffs. In a sec-
ond part of this work we take up the issue of deriving a reduced model in terms
of a discrete or continuous description of gating variables. We argue that lack of
homogeneity in [Ca2þ] obtained in the detailed simulations obliterates the as-
sumption of mixing of reactants (here ligands and channels) and thus the val-
idity of the law of mass action. Effective reaction kinetics can be derived, how-
ever, by distinguishing concentrations of self-feedback of channels and
coupling to different channels, thus eliminating detailed balance. We infer
a minimal Markovian model as well as a corresponding Langevin model. Im-
portantly, only the Markovian description reproduces calcium puffs, while
a Langevin model wrongly predicts a stationary regime of high inhibition.
The analysis of the Markovian model allows further insight into the functioning
of calcium puffs.
2658-Pos
Role of Agonist-Independent Conformational Transition (AICT) in I P3 R
Cluster Behavior
Divya Swaminathan, Peter Jung.
Ohio University, Athens, OH, USA.
Local intracellular calcium (C a2þ) signals arise due to the release of C a2þ ions
from internal stores into the cytosol through small clusters of inositol-1,4,5-tri-
phosphate (I P3 ) receptors. To explain single I P3 receptor open probability data
from nuclear patch clamp experiments, theoretical simulations have favored the
existence of an agonist-independent conformational transition(AICT) from
closed to an open state. We present results from a computational study wherein
we explore the impact of the proposed agonist independent conformational
transition on the collective release of calcium from I P3 R clusters. A wealth
of experimental data profiles collective cluster release. Our results show that
consistency of cluster release between theory and experiments in fact con-
strains the kinetics of the agonist-independent conformational transition
(AITC) to values which lead to small open probabilities for a single I P3 recep-
tor, inconsistent with nuclear patch clamp experimental data.
2659-Pos
Global Dynamic Conformational Changes in the Suppressor Domain of
IP3 Receptor by Stepwise Binding of the Two Lobes of Calmodulin
Yonghyun Song, Sunmi Kang, Hyeji Yang, Chang Kook Suh,
Sunghyuk Park.
School of Medicine, Inha University, Incheon, Republic of Korea.
The roles of calmodulin (CaM) has been a key point of controversy in the reg-
ulation of inositol-1,4,5-trisphosphate receptor (IP3R). Current views differ in
terms of the involvement of CaM as calcium sensors and calcium’s functions in
the process. To help resolve these issues, we studied the interaction between
CaM and the suppressor domain of IP3R, a key allosteric regulatory domain,
in the absence and presence of calcium. Through NMR binding experiments,
we observed dramatic peak disappearances of the suppressor domain upon in-
teraction with apo-CaM. These data indicated that apo-CaM induces large-
scale dynamic conformational changes in the suppressor domain, most proba-
bly involving partial unfolding and sub-domain rearrangement. Resonance as-
signments of CaM surprisingly revealed that its C-lobe alone can cause these
changes. Subsequent NMR binding experiments showed that calcium allows
the free N-lobe to additionally bind to the suppressor domain, which induces
extra conformational changes in both of the proteins. Our data also suggest
that the extra changes in the suppressor domain are secondary to those in cal-
modulin. Based on these results, we propose that apo-CaM, through its C-lobe,
can prime the allosteric regulation by partially unfolding the suppressor do-
main, which could be propagated to distant sites to open inhibitory calcium
binding sites. Calcium then could bind to the CaM’s N-lobe and the inhibitory
binding sites in IP3R, eliciting additional conformational changes and actual in-
hibition of IP3R. We believe that our results reconcile previous allosteric
models and provide new insights in the mechanism of calcium/CaM-mediated
regulation of IP3R.
2660-Pos
An Analysis of the Activity of Two Disease Associated Mutations of the
Inositol 1,4,5-Trisphosphate Receptor Type-1
Tricia Nienaber, Matthew J. Betzenhauser, Larry E. Wagner, David I. Yule.
University of Rochester, Rochester, NY, USA.
Inositol 1,4,5-trisphosphate receptor type 1 (InsP3R1) is abundantly expressed
in the central nervous system. InsP3R1 functions to release Ca
2þ from the en-
doplasmic reticulum (ER) upon stimulation by inositol 1,4,5- trisphosphate
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
2þ mobilization 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 expressing Muscarinic 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


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An Analysis of the Activity of Two Disease Associated Mutations of the Inositol 1,4,5-Trisphosphate Receptor Type-1

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