Iino, Masamitsu

Kanemaru, kazunori

Kurebayashi, Nagomi

Murayama, Takashi

Oyamada, Hideto

Suzuki, Junji

Takemori, Shigeru

Yamazawa, Toshiko

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110a Sunday, February 16, 2014interactions of electrostatic nature that induce coupled events (Supported by
NIH R01 GM078665 and AHA-MWA 12180038).
577-Pos Board B332
Phosphorylation of Maurocalcine Strongly Modifies its Effect on Type 1
Ryanodine Receptor
Wei Feng1, Michel Ronjat2, Yao Dong1, Eloi Bahembera2, Michel De
Waard2, Isaac N. Pessah1.
1University of California, Davis, Davis, CA, USA, 2LabEx Ion Channel
Science and Therapeutics, Grenoble Institut des Neurosciences Inserm U836,
UJF, Grenoble, France.
Maurocalcine (MCa) is a 33-amino acid peptide isolated from the venom of
Scorpio maurus palmatus, a Tunisian scorpion. It possesses efficient cell pene-
tration properties and has been shown to strongly modify ryanodine receptor
(RyR1) channel behavior by increasing open probability and inducing a
long-lasting subconductance state. The amino acid residue threonine at position
26 belongs to a putative phosphorylation site within MCa sequence. We inves-
tigated the effect of a) T26 phosphorylation (MCa T26Phospho) by protein
kinase A and b) replacement of T26 by glutamic acid residue to mimic phos-
phorylation on the effect of MCa on RyR1 properties. Using [3H]ryanodine
([3H]Ry) binding and single channel voltage-clamp measurements, we show
that both MCa T26Phospho and MCa T26E analogs almost completely lose
the ability to induce RyR1 activation. Neither MCa T26Phospho nor MCa
T26E (up to 2mM) increase [3H]rya binding. Small increase (~1.65-fold activa-
tion; p<0.01) was observed with high concentrations (10mM) of either MCa
T26Phospho or MCa T26E. Single channel measurements revealed that neither
MCa T26E nor MCa T26Phospho (up to 2mM) were able to induce the charac-
teristic long-lasting subconductance state of RyR1 observed in presence of
wild-type MCa. MCa T26Phospho altered RyR1 channel gating in a time-
dependent biphasic manner with initial activation of Po followed by almost
complete inactivation. These effects of MCa T26Phospho could be reversed
by 200nM wt MCa. At concentrations R2mM, MCa T26E enhances Po~3
fold by shortening tc without altering to. These results show that single
phosphorylation of MCa modifies RyR1 activity.1P01 AR52354.
578-Pos Board B333
Effects of Amino-Terminal Disease-Associated Mutations on the CICR
Activity of RyR1 Channel
Takashi Murayama1, Nagomi Kurebayashi1, Toshiko Yamazawa2,
Hideto Oyamada3, Shigeru Takemori2, Katsuji Oguchi3, Takashi Sakurai1.
1Juntendo University School of Medicine, Tokyo, Japan, 2Jikei University
School of Medicine, Tokyo, Japan, 3Showa University School of Medicine,
Tokyo, Japan.
Type 1 ryanodine receptor (RyR1) is a Ca2þ release channel in the sarco-
plasmic reticulum and plays a pivotal role in excitation-contraction coupling
in skeletal muscle. RyR1 is the major target for muscle diseases, e.g., malignant
hyperthermia (MH) and central core disease (CCD). To date, over 200 muta-
tions have been identified in the RyR1 gene of MH and CCD patients. It is
widely believed that MH and CCD mutations cause hyperactivation of the
Ca2þ-induced Ca2þ release (CICR), resulting in abnormal Ca2þ homeostasis
in skeletal muscle. CICR shows biphasic Ca2þ dependence, thus the activity
can be determined by three parameters: sensitivity to activating Ca2þ, sensi-
tivity to inactivating Ca2þ, and the gain (i.e., peak activity). However, it
remains unclear how the disease-associated mutations affects these parameters.
In this study, we expressed several RyR1 channels carrying different MH/CCD
mutations at amino-terminal region in HEK cells and tested their CICR by
live-cell Ca2þ imaging and [3H]ryanodine binding. Our results suggest that
the amino-terminal disease-associated mutations divergently affects the param-
eters of CICR depending on the sites for mutation. The underlying molecular
mechanism will be discussed.
579-Pos Board B334
Functional Analysis of Ryanodine Receptor Carrying Malignant
Hyperthermia Associated Mutations
Toshiko Yamazawa1, Takashi Murayama2, Hideto Oyamada3, Junji Suzuki4,
kazunori Kanemaru4, Nagomi Kurebayashi2, Shigeru Takemori1,
Masamitsu Iino4.
1The Jikei University School of Medicine, Tokyo, Japan, 2Department of
Pharmacology, Juntendo University School of Medicine, Tokyo, Japan,
3Department of Pharmacology, School of Medicine, Showa University,
Tokyo, Japan, 4Department of Pharmacology, Graduate School of Medicine,
The University of Tokyo, Tokyo, Japan.
Ryanodine receptors, located in the sarcoplasmic/endoplasmic reticulum
(SR/ER) membrane, are required for intracellular Ca2þ release that is involved
in a wide range of cellular functions. Malignant hyperthermia (MH) is a phar-
macogenetical complication of general anesthesia resulting from abnormalCa2þ-induced Ca2þ release (CICR) via the type 1 ryanodine receptor (RyR1)
in skeletal muscles. The typical symptoms include a rapid increase in body
temperature and induction of a hypermetabolic state with skeletal muscle rigid-
ity. More than 200 mutations in the RyR1 gene have been reported in MH
patients. Most of those mutations have been found in three ‘‘hot spots’’ regions
of RyR1. However, there were only a few experimental results confirming
those mutations being responsible for the increment of the CICR sensitivities.
We improved the method for making MH mutants in the cDNA of RyR1. We
characterized the functional mutations on RyR1 in non-muscle cells, specif-
ically HEK293 cells with tetracycline-regulated RyR1 expression. Rabbit
RyR1 channels carrying corresponding mutations were expressed in HEK293
cells for functional assay. HEK293 cells were loaded at room temperature
with fura-2 AM in physiological salt solution. Fluorescence images were
acquired using an inverted microscope equipped with a objective, a cooled
CCD camera and a polychromatic illumination system. We characterized the
functional mutations on RyR1 in HEK293 cells. It was found that disease-
associated mutations of the RyR1 resulted in enhanced Ca2þ release activity,
therefore these mutations would be responsible for the MH incidence. These
results suggest that exploration of the functional mutations of RyR1 is probably
effective in preventive diagnosis of patients associated with MH disease.
580-Pos Board B335
Malignant Hyperthermia Associated Mutations in S2-S3 Loop of Type 1
Ryanodine Receptor Calcium Channel Alter Calcium Dependent
Inactivation
Angela C. Gomez, Timothy Holford, Naohiro Yamaguchi.
Medical University of South Carolina, Charleston, SC, USA.
Skeletal and cardiac ryanodine receptors (RyRs) are ~65% similar in their
primary sequences, though differences in their regulation by physiological mol-
ecules have been observed. Skeletal RyR (RyR1) is inhibited by millimolar
Ca2þ with ~10 fold higher affinity than cardiac isoform (RyR2). Using
RyR1/RyR2 chimera channels and [3H]ryanodine binding measurements, we
found that two distinct regions are involved in isoform-specific Ca2þ-depen-
dent inactivation. One region includes two EF hand Ca2þ binding motifs
(RyR1 amino acids 4081-4092 and 4116-4127) and the other contains the sec-
ond transmembrane segment (S2). The results suggest a possible cytoplasmic
domain interaction between these two regions (or involving the flanking
regions of S2). Human disease associated mutations have been identified in
S2-S3 cytoplasmic loop of RyR1. We found that G4733E and R4736W malig-
nant hyperthermia associated mutations reduced affinity for Ca2þ dependent
inactivation of the channels by 5-6 fold (IC50: 6.750.6 mM (G4733E) and
5.550.2 mM (R4736W) vs 1.150.1 mM (wild type RyR1)), whereas muta-
tions in S4-S5 cytoplasmic loop (T4825I and H4832Y) reduced affinity by
2-3 fold. We also found that the activities of G4733E- and R4736W-RyR1 mu-
tants are suppressed at 10-100 mMCa2þ, and the suppressions are relieved by 1
mMMg2þ, which was observed in recombinant wild type RyR2 (Chugun et al.,
(2007) Am. J. Physiol. Cell Physiol.292, C535) but not in wild type RyR1.
Taken together, G4733E and R4736W mutations in S2-S3 loop confer
RyR2-type Ca2þ dependent inactivation and Mg2þ activation on RyR1. The
S2-S3 cytoplasmic loop may play a key role for domain interaction involved
in isoform-specific Ca2þ-dependent inactivation of RyRs. Supported by NIH
(R03AR061030), AHA (10SDG3500001), and NSF (EPS0903795).
581-Pos Board B336
Ryanodine Receptor Interaction with FKBP12 is Modulated by the RyR
N-Terminus Repeat Region
Polly Marino, F. Anthony Lai, Lynda Blayney.
School of Medicine, Cardiff Univ, Cardiff, United Kingdom.
The ryanodine receptor (RyR) is an ion channel involved in the release of Ca2þ
from intracellular stores (sarcoplasmic reticulum) in muscle cells. RyR plays a
pivotal role in excitation-contraction coupling and is modulated by several
accessory proteins in vivo, including the immunophilin FK506 binding protein
12 (FKBP12) which, our previous studies demonstrate, binds to RyR with
extremely high affinity. The FKBP12 binding site within the RyR sequence
has not been definitively identified and a number of candidate regions have
been proposed. A central domain phosphorylation region of RyR (CDR) has
been crystallised (Sharma, P. et al, FEBS (2013)288, 903-914; Yuchi, Z.
et al., Structure (2012) 20, 1210-1211). This domain has considerable sequence
and structural homology to its repeat region at the N-terminus (NTR) and a
pseudo atomic model of the two repeat regions, docked onto a topology model
of RyR, positions NTR adjacent to the previously proposed FKBP12 binding
domain and suggests a potential interaction (Zhu, L. et al, JBC (2013) 288,
903-914). The RyR1 interaction with FKBP12 was tested using [3H]ryanodine
binding to measure the Po of the channel of solubilised pig RyR preparations
and microsomal preparations. A dose response to FKBP12 reduced activity


Functional Analysis of Ryanodine Receptor Carrying Malignant Hyperthermia Associated Mutations 

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Functional Analysis of Ryanodine Receptor Carrying Malignant Hyperthermia Associated Mutations

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