Functional Characterization of a Central Core Disease RyR1 Mutation (p.Y4864H) Associated with Quantitative Defect in RyR1 Protein

Abstract

Background:
Central Core Disease (CCD) is a congenital myopathy often resulting from a mutation in RYR1 gene. Mutations in RyR1 can increase or decrease channel activity, or induce a reduction in the amount of protein. The consequences of a single mutation are sometimes multiple and the analysis of the functional effects is complex.

Objective:
The consequences of the p.Y4864H mutation identified in a CCD patient have been studied regarding both RyR1 function and amount.

Methods:
The amount of RyR1 in human and mouse muscles was evaluated using qRT-PCR and quantitative Western blot, and calcium release was studied using calcium imaging on primary cultures. The results were compared between human and mouse.

Results:
The p.Y4864H mutation induced an alteration of calcium release, and in addition was associated to a reduction in the amount of RyR1 in the patient's muscle. This suggests two possible pathophysiological mechanisms: the alteration of calcium release could result from a modification of the channel properties of RyR1 or from a RyR1 reduction. In order to discriminate between the two hypotheses, we used the heterozygous RyR1 knockout (RyR1+/-) mouse model showing a comparable RyR1 protein reduction. No reduction in calcium release was observed in primary muscle culture from these mice, and no muscle weakness was measured.

Conclusions:
Because the reduction in the amount of RyR1 protein has no functional consequences in the murine model, the muscle weakness observed in the patient is most likely the result of a modification of the calcium channel function of RyR1 due to the p.Y4864H mutation.

Full text

Journal of Neuromuscular Diseases 2 (2015) 421–432
DOI 10.3233/JND-150073
IOS Press
421
Research Report
Functional Characterization of a Central
Core Disease RyR1 Mutation (p.Y4864H)
Associated with Quantitative Defect
in RyR1 Protein
Marine Cacheuxa,b, Ariane Bluma,b, Muriel S´
ebastiena,b, Anne Sophie Woznya,b,c,
Julie Brocarda,b, Kamel Mamchaouid, Vincent Moulyd, Nathalie Roux-Buissona,b,c,
John Rendua,b,c, Nicole Monniera,b,c, Ren´
ee Krivosice, Paul Allenf, Arnaud Lacourg,
Jo¨
el Lunardia,b,c, Julien Faur´
ea,b,cand Isabelle Martya,b,∗
aINSERM U836, Grenoble Institut des Neurosciences, Equipe Muscle et Pathologies, Grenoble, France
bUniversit´eJoseph Fourier, Grenoble, France
cCentre Hospitalier R´egional Universitaire de Grenoble, Hˆopital Michallon, Biochimie G´en´etique
et Mol´eculaire, Grenoble, France
dUMRS974 Inserm, UMR7215 CNRS, Institut de Myologie, GH Piti´eSalp´etri`ere, 47 bd de l’hˆopital, Paris, France
eD´epartement Anesth´esie-R´eanimation, Hˆopital Roger Salengro, CHRU de Lille, Lille, France
fDepartment of Molecular Biosciences, School of Veterinary Medicine, University of California at Davis,
Davis CA, USA
gService de Neurologie, Hˆopital Roger Salengro, CHRU de Lille, Lille, France
Abstract.
Background: Central Core Disease (CCD) is a congenital myopathy often resulting from a mutation in RYR1 gene. Mutations
in RyR1 can increase or decrease channel activity, or induce a reduction in the amount of protein. The consequences of a single
mutation are sometimes multiple and the analysis of the functional effects is complex.
Objective: The consequences of the p.Y4864H mutation identified in a CCD patient have been studied regarding both RyR1
function and amount.
Methods: The amount of RyR1 in human and mouse muscles was evaluated using qRT-PCR and quantitative Western blot, and
calcium release was studied using calcium imaging on primary cultures. The results were compared between human and mouse.
Results: The p.Y4864H mutation induced an alteration of calcium release, and in addition was associated to a reduction in the
amount of RyR1 in the patient’s muscle. This suggests two possible pathophysiological mechanisms: the alteration of calcium
release could result from a modification of the channel properties of RyR1 or from a RyR1 reduction. In order to discriminate
between the two hypotheses, we used the heterozygous RyR1 knockout (RyR1+/–) mouse model showing a comparable RyR1
protein reduction. No reduction in calcium release was observed in primary muscle culture from these mice, and no muscle
weakness was measured.
Conclusions: Because the reduction in the amount of RyR1 protein has no functional consequences in the murine model, the
muscle weakness observed in the patient is most likely the result of a modification of the calcium channel function of RyR1 due
to the p.Y4864H mutation.
Keywords: Ryanodine receptor, Central Core Disease, Malignant Hyperthermia, calcium release
∗Correspondence to: Isabelle Marty, GIN-U836-Eq 4, Bat EJ
Safra – Chemin Fortun´
e Ferrini, 38700 La Tronche – France. Tel.:
+33 4 56 52 05 71; Fax: +33 4 56 52 05 72; E-mail: isabelle.marty@
ujf-grenoble.fr.
ISSN 2214-3599/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial License.

422 M. Cacheux et al. / RyR1 Quantitative Defect
ABBREVIATIONS
CCD central core disease
CmC 4-chloro-m-cresol
CTRL control
He heterozygous
KO knockout
MH Malignant Hyperthermia
MHN MH Negative
MHS MH Susceptible
RyR ryanodine receptor
SERCA SarcoEndoplasmic reticulum Ca2+-ATPase
SR Sarcoplasmic reticulum
INTRODUCTION
The sarcoplasmic reticulum (SR) calcium channel
ryanodine receptor RyR1 is encoded by the RYR1 gene
(MIM#180901). In association with the voltage gated
calcium channel dihydropyridine receptor (DHPR) and
numerous regulating proteins, they form the skele-
tal muscle calcium release complex responsible for
the excitation-contraction coupling process in skeletal
muscle [1].
Mutations in the RYR1 gene have been associated
with congenital myopathies such as Central Core Dis-
ease (CCD; OMIM#117000) [2–4] and Multiminicore
Disease (MmD; OMIM# 255320) [5–7]. CCD has
been named after anatomo-pathological muscle anal-
ysis, and is characterized by the presence of cores in
type 1 muscle fibers, which are large areas of abnormal
myofibrillar architecture with sarcomeric disorganiza-
tion and absence of mitochondria [2, 8]. CCD presents
either an autosomal dominant or a recessive transmis-
sion pattern. Clinical presentations are heterogeneous
ranging from mild phenotype, with moderate hypo-
tonia during early childhood, delayed motor abilities,
and slowly progressive proximal muscle weakness, to
severe phenotype, including fetal akinesia, respiratory
insufficiency at birth and generalized muscle weakness
[9, 10].
Dominantly inherited RYR1 mutations have been
extensively studied to identify the pathophysiological
mechanism. Two mechanisms have been proposed so
far: either a gain of function of RyR1 (hyperactivity)
leading to a calcium leak or a loss of RyR1 function
with impaired calcium conductance [11–14].
It has been observed that recessive mutations in
RYR1 can also result in a reduction in the amount
of protein [15, 16], which leads to muscle weakness
and altered calcium release [17]. In the present work,
we studied the pathological mechanisms leading to a
moderate CCD in which a missense dominant muta-
tion in RyR1 was associated with a 25% decrease in
the amount of protein. Until now the effect of such a
RyR1 reduction has never been explored. Using an ani-
mal model presenting with a similar reduction in the
amount of RyR1, we showed no alteration of muscle
calcium release or of muscle strength. We concluded
that the consequence of this RyR1 mutation in the
patient’s muscle was not related to the reduction in
the amount of protein, but rather to the direct effect
of the mutation on the channel function. These results
provide new insights into the pathogenesis of RYR1-
related myopathies with RyR1 deficiency.
MATERIALS AND METHODS
Ethics statement
Investigations on patient material were performed
after signature of an informed consent according to
the French regulation and have received approval
from the local ethical committee (Comit´
e de Protec-
tion des Personnes-Sud-Est, France). All procedures
using animals were approved by the Institutional Ethics
Committee and followed the guidelines of the National
Research Council Guide for the care and use of labo-
ratory animals.
Muscle biopsy and IVCT
Part of the muscle biopsy realized in vastus
lateralis has been directly processed for in vitro
contracture test (IVCT) according to the Euro-
pean Malignant Hyperthermia Group Guidelines
(http://www.emhg.org/emhg/mh-diagnosis/). Another
part was used for cell culture and Western blot, and the
remaining muscle was frozen in liquid nitrogen cooled
isopentane for histological and mRNA analyses.
Molecular genetic studies
RYR1 mutations screening was performed using
cDNA obtained after reversetranscription of total RNA
extracted from muscle biopsy as previously described
[2]. The cDNA was amplified in overlapping frag-
ments. Sequencing reactions were analyzed on an ABI
3130 DNA Analyzer (Life Technologies, Saint Aubin,
France). The presence of the mutation identified in the
transcript was confirmed in genomic DNA by direct

M. Cacheux et al. / RyR1 Quantitative Defect 423
sequencing of the corresponding exon and intron-exon
junctions.
Mouse lines
RyR1 knock out mice (RyR1–/–), heterozygous
mice (RyR1+/–) and wild type (RyR1+/+) were
obtained by crossing heterozygous Ryr1tm1Alle mice
(http://www.informatics.jax.org/allele/key/637575).
In this mouse line, the insertion of a neomycin selec-
tion cassette in the KPN site at nt840 in exon10 of
the RYR1 gene leads to disruption of gene expression
[27].
Mouse muscle homogenate preparation
Skeletal muscles were collected from the hind
limbs of adult (6-7 months old) male mice. Crude
homogenates were prepared by homogenization in 200
mM sucrose, 20 mM HEPES (pH 7.4), 0.4 mM CaCl2,
200 ␮M phenylmethylsulfonyl fluoride, 1 mM diiso-
propyl fluorophosphate, as described previously [18].
Protein concentration was measured using a modified
Folin assay in presence of SDS.
Quantitative western blot analysis
The amount of RyR1 present in muscle samples
(20–40 ␮g of muscle homogenate) was determined
by quantitative Western blot analysis using antibod-
ies directed against RyR1 [19] and normalized to the
amount of myosin heavy chain as described previ-
ously [16]. Briefly, after electrophoretic separation on
a 5–15% gradient acrylamide gel and electrotrans-
fer to Immobilon P (Biorad, Marnes la Coquette,
France) during 4h at 0.8A to ensure a complete trans-
fer of the loaded proteins, the membrane was incubated
with anti-RyR1 antibodies and then HRP-labelled sec-
ondary antibodies. Variation in protein loading or in
muscle protein content due to heterogeneity of the sam-
ple, e.g. muscle fibrosis, was evaluated as the amount of
myosin in each lane determined by Coomassie staining
of the Immobilon membrane after immunorevelation
(measured as the surface of the myosin band). The total
amount of RyR1 in each experiment (total signal on
the two or three bands) was thus corrected from the
amount of myosin and normalized to the amount of
RyR1 present in the control referred as 100%. Quantifi-
cation was also performed using anti-desmin antibody
(Dakocytomation, Les Ulis, France) for normalization
compared to the amount of desmin, and similar results
were obtained. Signal quantification was performed
using a ChemiDoc XRS apparatus (Biorad, Marnes
la Coquette, France) and the Quantity One software
(Biorad).
q RT-PCR
Human muscle
Total RNA was extracted from frozen muscle biopsy
using TRIzol reagent (Life Technologies, Saint Aubin,
France) as previously described [16]. First strand
cDNA synthesis from 500ng total RNA was realized
using random priming with the High Capacity cDNA
Reverse Transcription kit (Life Technologies, Saint
Aubin, France). Real time quantification of mRNAs
of target gene RYR1 and of reference genes (hRPL27
and ACTB) was performed with Power SYBR Green
PCR Master Mix (Life Technologies, Saint Aubin,
France) using a STEPONEPLUS (Life Technologies,
Saint Aubin, France) detection system. The following
primers were used (5→3): RYR1 pcr1 forward: CAT
GGC TTC GAG ACT CAC AC, RYR1 pcr1 reverse:
CTC CTG ACC CGT GTG TTC T, RYR1 pcr2 for-
ward: GAC TCA CAC GCT GGA GGA G, RYR1 pcr2
reverse: TCC AGA CAT AAG ACT CCT GAC C,
ACTB forward: CTC CTG AGC GCA AGT ACT CC,
ACTB reverse : TGT TTT CTG CGC AAG TTA GG,
hRPL27 forward : CGC AAA GCT GTC ATC GTG,
hRPL27 reverse : GTC ACT TTG CGG GGG TAG.
The following experimental protocol was used: denat-
uration 95◦C for 10 min and 40 cycles of 95◦C for
15 sec and 60◦C for 60 sec. Melting curve analysis
showed specific melting temperatures.
Mouse muscle
Total RNA from skeletal muscle (tibialis anterior)
of 2-month-old WT (RyR1+/+) and heterozygous
(RyR1+/–) mice (3 animals in each group) was
extracted using TRIzol reagent (Life Technologies,
Saint Aubin, France) and PureLink RNA Mini Kit
(Life Technologies, Saint Aubin, France). First strand
cDNA was obtained using oligo(dT) primed reverse
transcription from 500ng of RNA. Real time quantifi-
cation of mRNAs of target gene RYR1 and of reference
gene (GAPDH) was performed by iQ SYBR Green
supermix (BioRad, Marnes la Coquette, France) using
an IQ iCycler (BioRad, Marnes la Coquette, France)
detection system. The following primers were used
(5→3): RYR1, forward - ATG ACC GTA GGG CTC
CTG GCC GTA G, reverse - GGG TCC TCG ATC
TCG TCC CCG A; GAPDH forward - GTA TGA CTC
CAC TCA CGG CAA A, reverse - TTC CCA TTC
TCG GCC TTG. The following experimental protocol

424 M. Cacheux et al. / RyR1 Quantitative Defect
was used: denaturation 95◦C for 3 min and 40 cycles
of 95◦C for 10 sec and 55◦C for 45 sec. Melting curve
analysis showed specific melting temperatures.
Data were analyzed with the comparative thresh-
old cycle (Ct) relative-quantification method. Relative
gene expression was quantified as follows: fold
change = 2−(Ct) where Ct =Cttarget −Ctreference
and (Ct) =Ctsample −Ctcontrol. Ct is the frac-
tional cycle number at which the fluorescence passes
the fixed threshold. The target gene represents RYR1
gene, reference genes are the hRPL27 and ACTB gene
in human and the GAPDH gene in mouse, “sample”
refers to patient or heterozygous mice and “control” to
control human or wild type mice.
Production of human and mouse primary cultures
Human satellite cells were produced from a muscle
biopsy of a 25-year-old donor without neuromuscu-
lar disorder (CTRL cells) and from the biopsy of the
patient (Y4864H cells). These cells were immortalized
and cloned as previously described [20, 21]. Pri-
mary cultures of skeletal muscle from WT, RyR1+/–
and RyR1–/–E19 mouse embryos were produced as
described previously [22].
Cell culture
Immortalized human satellite cells or mouse satel-
lite cells were amplified in proliferation medium
composed of Ham’s F-10 (Life technologies, Saint
Aubin, France) supplemented with 20% FBS (Life
technologies, Saint Aubin, France), 2% Ultroser G
(Pall Biosepra, St Germain en Laye, France) and
2% Penicillin-Streptomycin (Life technologies, Saint
Aubin, France). Differentiation into myotubes was
induced by a shift to differentiation medium: DMEM
(Life technologies, Saint Aubin, France) supplemented
with 2% Heat Inactivated Horse Serum (Life tech-
nologies) and 1% Penicillin-Streptomycin. Human
myotubes were cultured for seven to eight days and
mouse myotubes for two to three days before intracel-
lular calcium measurements.
Intracellular calcium measurements
Changes in intracellular calcium were evaluated
using the calcium-dependent fluorescent dye Fluo-
4 AM (Life Technologies, Saint Aubin, France), as
described previously [23]. Calcium imaging was per-
formed in Krebs buffer (136 mM NaCl, 5 mM KCl, 2
mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4, 6
mM D-Glucose). To obtain a calcium-free Krebs solu-
tion, CaCl2was left out, while 1mM EGTA, 10 ␮M
La3+, and 50 ␮MCd
2+were added. Thapsigargin
(Life technologies, Saint Aubin, France) was diluted at
1␮M in this medium and applied simultaneously with
caffeine (40 mM) as described previously [24, 25]. 4-
chloro-m-cresol (4-CmC) (Sigma-Aldrich) was diluted
at 500 ␮M in Krebs buffer and 140 mM KCl solution
was prepared in 2 mM CaCl2, 1 mM MgCl2,10mM
HEPES, pH 7.4 and 6 mM D-Glucose. Fluorescence
was measured by confocal laser scanning microscopy
using a Leica TCS-SPE microscope in the xyt mode.
Changes in intracellular Ca2+concentrations were pre-
sented as the ratio of fluorescence intensities with
respect to the initial fluorescence intensity prior to drug
addition (F/F0). Data are given as mean ±SEM, and
nrepresents the number of myotubes studied in each
condition.
Evaluation of muscle strength
Muscle strength was evaluated in patients using
manual muscle testing according to the Medical
Research Council scale [26]. Muscle strength was eval-
uated in mice using a hang test as described previously
[18]. Two months old male mice were positioned on
a cross-wired surface turned upside down, and time
before fall (up to 300 s) was measured.
Statistical analysis
Data were pooled over animals or cells within
the same group and are presented as means ±SEM.
Differences between CTRL and Y4864H cells were
assessed using Student’s t-test. Differences between
WT, RyR1+/–(He) cells, and RyR1–/–(KO) cells
were assessed using Student’s t-test with Bonferroni
correction for multiple comparison, and GraphPad
Prism 6 software, assuming significance at p< 0.05.
RESULTS
Clinical and genetic reports
The proband (Figs. 1A, III:3) was a 28-year-
old female who presented with a moderate muscle
weakness since childhood, a slightly delayed motor
development and a stable axial muscle deficiency.
Muscle strength evaluation showed no distal defi-
ciency, no reduction in cervical or hamstring muscles
strength, a slight reduction in pectoralis major and

M. Cacheux et al. / RyR1 Quantitative Defect 425
Fig. 1. Case description. (A). Pedigree of the family. Circles rep-
resent females and squares males. Filled symbols indicate affected
individuals. The proband is indicated by the black arrow. Segrega-
tion of alleles is indicated below each individual. MH= Malignant
Hyperthermia; MHN = MH Negative; MHS = MH Susceptible. (B).
Quantitative analysis of RyR1 expression (protein and mRNA)in the
skeletal muscle of the proband (Individual III.3). Twenty ␮g mus-
cle homogenate from control (CTRL, lane 1) or proband (Y4864H,
lane 2) were loaded on a 4–15% polyacrylamide gel. The amount
of RyR1 protein is expressed as the percentage of RyR1 present in
the control muscle, which relative expression compared to myosin
was set at 100%. The central bars graph presents RyR1 protein mean
amount ±SEM from seven different Western blots. The bar graph
on the right is the Q-RT-PCR analysis of levels of RyR1 mRNA
expressed as a percentage of control (which relative expression
compared to reference genes was set to 100%). The data are pre-
sented as mean ±SEM of 9–12 different amplifications. ∗∗p< 0.01,
∗∗∗∗p< 0.0001, Student’s t-test. (C) Representative Western blot of
different controls (C1-C4), non-affected women between 20 and
23 years.
deltoid muscles strength (level evaluated at 4/5), and
a marked reduction in abdominal muscles strength
(level evaluated at 2/5). Creatine kinase (CK) levels
were normal (75 U/L, Table 1, III:3). A muscle biopsy
Table 1
Analysis of patients
CK IVCT data
Hal 2% Caf 2 mM
III : 3 75 6N 5N
II : 2 168 10N 6N
II : 1 51 <2N <2N
CK levels and IVCT data of the proband (III.3) and two individ-
uals from the family (II.2 and II.1). CK = Creatine Kinase activity
is expressed in international units per liter (IU/L; normal <205).
IVCT = In Vitro Contracture Test. Hal= tension in Newton (N) at
2% halothane, the normal value being <2N. Caf = tension at 2 mM
Caffeine, the normal value being <2N.
demonstrated the presence of central cores using
NADH staining and an in vitro contracture test (IVCT)
indicated susceptibility to Malignant Hyperthermia
(MHS) (Table 1, III:3) because of hypersensitivity of
RyR1 to both caffeine and halotane. The genetic analy-
sis performed on the mRNA extracted from the muscle
biopsy identified the c.14590T>C; p.Tyr4864His vari-
ation at heterozygous state in the RYR1 gene, resulting
in the substitution of the tyrosine in position 4864 by a
histidine (p.Y4864H) in the RyR1 protein. A diagnosis
of CCD was proposed.
The proband’s father (Figs. 1A, II:2) presented with
a late onset moderate muscle weakness, and did not
report any muscle weakness during childhood and
adolescence. A clinical examination at age 60 demon-
strated a mild quadriceps amyotrophy, right scapular
winging and scoliosis. Muscle strength evaluation indi-
cated a slight reduction in biceps, pectoralis major
and hamstring muscles strength (evaluated at 4/5),
and an important axial deficiency (abdominal muscles
2/5, cervical muscles 3/5). His CK levels were nor-
mal (168 U/L, Table 1, II:2). Muscle biopsy displayed
an aspect of congenital myopathy with the presence of
atypical cores using NADH staining. He was also diag-
nosed as being susceptible to Malignant Hyperthermia
(MHS) by IVCT (Table 1, II:2), and a genetic analysis
confirmed that he was carrying the same RYR1 het-
erozygous variant c. 14590T>C; p.Tyr4864His as his
daughter.
The patient’s uncle (II:1) who was tested MH Nega-
tive (MHN) by IVCT did not present with any clinical
sign of myopathy and did not have the variant (Fig. 1A
and Table 1). Therefore, the two individuals of the
family carrying the p.Y4864H mutation were affected
by a moderate CCD associated to MH, suggesting a
dominant transmission.
Using quantitative RT-PCR, the amount of RYR1
transcripts in the muscle of the proband (III.3) was
evaluated at 52.6%±3.2% of the control muscle

426 M. Cacheux et al. / RyR1 Quantitative Defect
(Fig. 1B), pointing to either the absence of tran-
script produced from one of the two RYR1 alleles
or a reduction in both transcripts. The presence of
the c.14590T>C variation at a heterozygous state in
the mRNA transcripts detected by Sanger sequenc-
ing, suggested that most probably both transcripts were
reduced. High Resolution Melting (HMR) analysis on
cDNA of the patient confirmed that 2 transcripts were
present in equivalent amount when amplifying a region
encompassing the c.14590 position (data not shown).
No other variation in the RyR1 mRNA was found to
explain the global transcript reduction.
Using quantitative Western blot, we determined that
the amount of RyR1 present in the muscle of the
proband (Ind. III.3) was 75.3 ±6.4% (n=7)ofthe
amount of RyR1 in a control muscle from a 25 years old
female patient without any muscle disease (Fig. 1B). To
confirm that the amount of RyR1 in age-related control
biopsies do not present similar variation and to validate
our quantitative Western blot analysis, a quantitative
Western blot was performed on four different 20–23
years-old non affected women, with normal physical
activity (Fig. 1C). The quantification of the amount of
RyR1 compared to desmin in 11 blots performed from
these 4 controls result in a relative amount of RyR1 of
100% ±6.3%.
Consequences of the mutation
To determine the physiological effects of the muta-
tion, primary cultures were produced from the muscle
biopsy of the patient, and were immortalized by
double retroviral transduction using telomerase and
Cdk4 [20, 21]. Calcium imaging studies were per-
formed on myotubes produced from the proband’s
immortalized cells (Y4864H cells) or from immor-
talized cells of a volunteer of 25 years with no
muscle disease (CTRL cells), to assess their abil-
ity to release calcium after stimulation (Fig. 2). In
response to the membrane depolarization induced by
addition of 140 mM KCl in presence of extracellu-
lar calcium, Ca2+release was significantly reduced in
Y4864H myotubes (Fig. 2A, white circles) compared
to CTRL (Fig. 2A, black circles) (p< 0.0001). Simi-
larly calcium release induced by a direct stimulation
of RyR1 by 4-CmC (Fig. 2B) was also significantly
decreased (p< 0.0001). The amount of Ca2+in the
SR stores was evaluated after caffeine stimulation
in presence of thapsigargin with or without extra-
cellular calcium (Fig. 2C and D). In all cases, the
maximal amplitude of calcium released was signif-
icantly reduced in Y4864H myotubes compared to
CTRL myotubes (Fig. 2E, p< 0.0001). The area under
the curve (Fig. 2F) reflecting the amount of calcium
released was also significantly reduced except for the
caffeine stimulation in presence of thapsigargin and
extracellular calcium. The latter indicates that the
reduction in the free SR calcium rapidly releasable
upon stimulation could be compensated by an influx
of external calcium (Fig. 2C).
These results could be explained either i) by a
reduction in the amount of calcium stored due to
the p.Y4864H mutation leading to a “leaky” RyR1
channel, as usually observed with MH mutations lead-
ing to RyR1 hypersensitivity or ii) by defects in the
RyR1-DHPR coupling leading to impaired calcium
conductance [11, 12], or iii) by the decreased quantity
of RyR1 protein [17].
RyR1 expression in RyR1+/–heterozygous mice
So far, the effect on calcium release of a small
RyR1 protein decrease such as the one measured for
the patient has not been evaluated. To test this effect,
we used cells from heterozygous mice of a RyR1 KO
model [27]. Muscle homogenates were prepared from
WT and heterozygous RyR1+/–mice to measure the
amount of RyR1 transcript and protein. Only one allele
of the RYR1 gene is expressed in RyR1+/–heterozy-
gous mice, and we first confirmed using quantitative
RT-PCR that the mRNA of RyR1 was reduced by about
50% compared to WT (42.6% ±7% for RyR1+/–mice
compared to 100% ±4.8% for WT, n= 3) (Fig. 3C).
Using quantitative Western blot, the amount of RyR1
at the protein level detected in RyR1+/–mice muscles
was estimated to be 83.4 ±2.6% of WT muscle (n=9)
(Fig. 3A and B).
These results showed that both the amount of RyR1
transcript and protein were decreased in RyR1+/–
heterozygous mice compared to WT mice, although
to a lesser extent at the protein level. The relative
protein amount of RyR1 measured in the patient
with the p.Y4864H mutation compared to control
(75.3 ±6.4%, n= 7) was not statistically different from
the one found in RyR1+/–mice compared to WT mice
(83.4 ±2.6%, n= 9), and the relative amounts of tran-
script were also not different (52.6% ±3.2%, n=12
in the patient and 42.6% ±7%, n= 3 in the mouse).
In both case, the alteration in the amount of RyR1 at
the protein level is milder than the 50% lowering of
transcription. Therefore the RyR1+/–mouse line con-
stitutes a good model to study the effect of such a RyR1
reduction.

M. Cacheux et al. / RyR1 Quantitative Defect 427
Fig. 2. Calcium release in immortalized patient cells. Calcium imaging performed on control CTRL cells (black circle), and on patient’s Y4864H
cells (white circle) differentiated for 7 to 8 days before calcium imaging. (A) Fluorescence variation curves induced by membrane depolarization
(KCl 140 mM) applied during 60 s (black bar) in the presence of 2 mM external calcium, presented as mean (symbols) ±SEM. (B) Fluorescence
variation curves induced by application of 4-Chloro-m-Cresol (CmC) 500 ␮M during 60s (black bar) in the presence of 2 mM external calcium,
presented as mean (symbols) ±SEM. (C) Fluorescence variation curves induced by application of caffeine 40 mM plus thapsigargin 1␮M
(Caf+Thapsigargin) during 60 s (black bar) in the presence of 2 mM external calcium, presented as mean (symbols) ±SEM. (D) Fluorescence
variation curves induced by application of caffeine 40mM plus thapsigargin 1␮M in absence of extracellular calcium (Caf+Thapsigargin - Ca2+)
during 60 s (black bar) in the presence of Cd2+and La3+, presented as mean (symbols) ±SEM. (E) The maximal amplitude of the peak for each
curve is presented in the bar plots, with the number of myotubes analyzed in each bar. ∗∗∗∗p<0.0001, Student’s ttest comparisons between CTRL
and Y4864H cells, for each stimulation. (F) The area under each curve (A.U.) has been calculated for each stimulation, in control myotubes
(black bars) and Y4864H myotubes (white bars) and is presented as mean ±SEM of the number of myotubes indicated in each bar. Statistics :
Student’s t-test of Y4864H myotubes compared to control myotubes ∗∗∗∗p< 0.0001, ∗∗∗ p< 0.001, ns: non significant.

428 M. Cacheux et al. / RyR1 Quantitative Defect
Fig. 3. Expression of RyR1 in heterozygous RyR1+/–mouse muscles. (A) Quantitative Western blot analysis of RyR1 expression in skeletal
muscle homogenates from WT mice (WT) or from heterozygous RyR1+/–mice (He). (B) The relative amount of RyR1 at the protein level
compared to myosin was set to 100% in WT mice. The amount of RyR1 in He mice is presented as mean ±SEM of 9 experiments performed in
3 different mice. ∗∗∗p< 0.001 Student’s t-test between WT and He. (C) Q-RT-PCR analysis of levels of RyR1 mRNA expressed as a percentage
of WT mice (which relative expression compared to GAPDH was set to 100%). The data are presented as mean±SEM of 3 different mice.
∗∗p< 0.01 Student’s t-test between WT and He.
Measure of muscle strength in RyR1+/–mice
To evaluate the overall muscle performance of
RyR1+/–heterozygous mice, a hang test was per-
formed. Two-month-old male mice were allowed to
grip on a cross-wired surface placed upside down. Time
spent hanging on the surface before fall was measured
and no significant difference was observed between
WT (182 ±43 s; n= 7) and RyR1+/–(168 ±36 s;
n= 9) mice (supplementary data, Figure S1). This hang
test thus showed that the decrease in RyR1 protein in
RyR1+/–mice did not modify muscle strength.
Effect of RyR1 reduction on calcium fluxes
To check whether the decrease in the quantity of
RyR1 protein in RyR1+/–mice induced defects on
the calcium release, calcium imaging studies were
performed on RyR1+/+(WT), RyR1+/–(He) and
RyR1–/–(KO) myotubes (Fig. 4). In agreement with
some previous results [28] we observed that, com-
pared to WT (Fig. 4A and B, black circles) and He
myotubes (Fig. 4A and B, gray squares), the calcium
release was greatly depressed in KO myotubes (Fig. 4A
and B, white circle) whether it be after a membrane
depolarization (140mM KCl) or after a direct RyR1
stimulation (500 ␮M 4-CmC). Both stimuli induced
calcium release in WT myotubes (Fig. 4A and B,
black circles) and He myotubes (Fig. 4A and B, gray
squares). The amplitude of the peak in WT and He
myotubes using RyR1 direct stimulation was simi-
lar (Fig. 4C). The peak was significantly increased
in He compared to WT when KCl stimulation was
used (Fig. 4C). These data demonstrated that the 16%
decrease in RyR1 protein observed in He mice did not
impair their ability to release calcium upon stimulation
compared to the WT mice, consistent with the lack of
muscle weakness observed during the hang test.
DISCUSSION
Functional studies of common dominant RYR1
mutations associated with CCD have suggested two
main mechanisms responsible for a disturbed func-
tion of the mutant RyR1 channel, namely either the
presence of a leaky and hyperactive calcium channel
associated with a reduction of SR calcium stores [13],
or an “uncoupled” channel with reduced permeabil-
ity of RyR1 to Ca2+[29]. In the present situation,
the p.Y4864H mutation resulted in the presence of a
calcium channel with altered properties which is asso-
ciated with a reduction of the protein amount. A 50%
reduction in the RyR1 mRNA in the patient biopsy
was also observed, but the mechanisms leading to in
this reduction in the RyR1 mRNA was not identi-
fied. In vitro calcium imaging showed that with all the
stimulations used the amount of calcium released in
Y4864H cells was significantly reduced. A reduction
in the amplitude of the calcium store was observed
using caffeine stimulation in the absence of external

M. Cacheux et al. / RyR1 Quantitative Defect 429
Fig. 4. Calcium release in mouse cells. Calcium imaging performed on WT satellites cells (black circle), on RyR1+/–heterozygous mouse cells
(gray square) and on RyR1–/–KO mouse cells (white circle) differentiated for 2 to 3 days before calcium imaging. (A) Fluorescence variation
curves induced by membrane depolarization (KCl 140 mM) applied during 40 s (black bar) in the presence of 2 mM external calcium, presented
as mean (symbols) ±SEM. (B) Fluorescence variation curves induced by application of 4-Chloro-m-Cresol (CmC) 500 ␮M during 40s (black
bar) in the presence of 2 mM external calcium, presented as mean (symbols) ±SEM. (C) The maximal amplitude of the peak for each curve is
presented in the bar plots, with the number of myotubes analyzed in each bar. ∗∗∗∗p< 0.0001, Student’s ttest followed by Bonferroni correction
for multiple comparison, compared to WT. (D) The area under each curve (A.U.) has been calculated for each stimulation, in WT myotubes
(black bars), He myotubes (gray bars) and KO myotubes (white bars) and is presented as the mean ±SEM of the number of myotubes indicated
in each bar. ∗∗∗∗p< 0.0001, Student’s ttest followed by Bonferroni correction for multiple comparison, compared to WT.
calcium influx and in the absence of calcium re-uptake
in the sarcoplasmic reticulum (caffeine plus thapsigar-
gin, Cd2+and La3+, Fig. 2D). This reduction in the
amount of stored calcium, which could reflect a leaky
calcium channel, was compensated by an influx of
external calcium when present (caffeine stimulation in
presence of thapsigargin with Ca2+, Fig. 2C), leading
to equivalent amounts of calcium released in Y4864H
cells compared to control cells (Fig. 2F). This reduc-
tion in the amount of stored calcium could account for
the muscle weakness observed in the patient, but as it
can be compensated in some conditions, it can only be
a partial explanation. In order to identify another mech-
anism for the muscle weakness observed, we focused
our studies on the impact of the reduction of RyR1
quantity. As RyR1 deficiency has been proposed to
result in disruption of EC coupling [17], we studied
calcium release in a mouse model expressing a nor-
mal RyR1 but with a similar reduction in the amount
of protein. This model was the heterozygous RyR1+/–
mouse line that has only one functional RYR1 allele. We
first confirmed the presence of RyR1 transcript at about
a 50% level of a WT mouse. Noticeably, the RyR1 pro-
tein level was higher, reaching 85% of a WT mouse,
suggesting a post-transcriptional regulation of the pro-
tein amount, identical to the situation in observed
in the patient. In the heterozygous mouse myotubes,
no reduction in the amount of released calcium was
observed either after KCl membrane depolarization
mimicking EC coupling or after direct RyR1 stimu-
lation by CmC. In addition, RyR1+/–mice showed
no muscle weakness. Those results confirmed that this
degree of RyR1 reduction was not sufficient to result in
muscle weakness or to alter calcium release. Therefore,

430 M. Cacheux et al. / RyR1 Quantitative Defect
Fig. 5. Localization of the mutation in the structure of the pro-
tein. The localization of the mutation presented in this study,
p.Y4864H, as well as two close amino acids R4864 and I4898 both
involved in CCD, are reported on the structure of RyR1 recently
proposed from single particle electron cryomicroscopy [31, 32].
These amino acids are in the luminal loop between transmembrane
helixes S5 and S6, which contain the pore helix (P-helix) involved in
pore formation.
muscle weakness and alteration of calcium release
observed in the patient expressing the p.Y4864H muta-
tion is most probably not related to the reduction of
RyR1 amount, but rather to the presence of mutant
monomers into the tetrameric channel. As the mutation
induced a reduction in the amount of calcium stored in
the sarcoplasmic reticulum, and as this channel is most
probably hyperactive as observed by the hypersensitiv-
ity evaluated in IVCT, it could be hypothesized that this
mutation resulted in a hyperactive and leaky calcium
channel. Calcium leak would explain the reduction
in the stored calcium and muscle weakness, and the
hyperactive channel would explain the increased sen-
sitivity observed in IVCT leading to the MHS status of
the patient.
The 4864 position mapped within the last luminal
loop of RyR1 monomer, which is involved in the selec-
tivity pore for calcium permeation (P-loop), and is
close to several other sites already found mutated in
association with Central core Disease. A CCD fam-
ily has previously been described with a mutation at
the same position but leading to the substitution of
tyrosine for cysteine instead of histidine (p.Y4864C)
[30]. The affected patients of this family presented with
a moderate myopathy, not associated to MH. Note-
worthy, another mutation in position 4898 (p.I4898T),
localized only 34 amino acids further away in the
same intracellular loop, resulted in a severely uncou-
pled RyR1 [3]. It can therefore be postulated that the
Y4864 and the I4898 amino acids belong to different
functional domains although they are in close vicinity
along the primary sequence of RyR1. A steric inhi-
bition in the movement of the luminal loop has been
proposed as a consequence of the p.I4898T mutation,
but due to a different outcome on muscle physiology,
cannot account for the effect of p.Y4864H. Look-
ing at the structure of RyR1 recently published [31,
32] (Fig. 5), these two residues are on both side of
the so-called P-loop or pore-helix between transmem-
brane segments S5 and S6. Because of the alteration
of RyR1’s channel function found in the patient one
can hypothesize that the p.Y4864H mutation disrupts
a binding site specific of a modulator of RyR1 func-
tion. Triadin could be such a modulator, as 3 amino
acids in position 4878, 49707 and 49708 have been
shown to be involved in the RyR-triadin interaction
[33], D4878 being very close to Y4864. Nevertheless,
mutation for Ala of this single Asp in position 4878
induced a slight reduction in the RyR-triadin bind-
ing, but no modification in the calcium release [34].
Therefore the effect of a mutation in position 4864
should most probably not be related to an alteration of
the interaction RyR-triadin. Mutation on amino acid
4861 has also been frequently associated with CCD,
often as a neo-mutation [35], with no or only a slight
reduction in the amount of protein [35] and small alter-
ation in calcium release (reduction in the amount of
calcium stored, measured on immortalized B-cells,
[36]). The effects of mutation in position 4861 are
quite similar to those observed for mutation in posi-
tion 4864, and a similar physiopathological mechanism
could be hypothesized for mutations at these two posi-
tions.
Overall, our study reports a CCD case where the
effect of the RyR1 Y4864H mutation was explored.
The reduction of total RyR1 mRNA in the patient mus-
cle could not be explained but the fact that the amount
of RyR1 protein was at 75% of a control suggested the
presence of post transcriptional or epigenetic controls.
This hypothesis is strengthened by the study of mRNA
and protein in a RYR1 KO heterozygous mouse model,
that shows the same level of RyR1 produced with a
50% reduction of mRNA. Our work also showed that
the pathophysiological mechanisms linked to defects
in calcium release in cells of the patient could not be
attributed to a reduction in RyR1 protein levels, but
rather to a direct effect of the p.Y4864H mutation on
the channel properties.

M. Cacheux et al. / RyR1 Quantitative Defect 431
ACKNOWLEDGMENTS
We thank all family members for their contribution
to this study. This work was supported by grants from
the “Association Franc¸aise contre les Myopathies”
(AFM), the “Fondation Daniel Ducoin", the “Institut
National de la Sant´
e et de la Recherche M´
edicale”
(INSERM), and the “Vivier de la Recherche de la
Facult´
edeM
´
edecine de Grenoble “. We thank Dr A.F.
Delmas and Mrs. I. Stix for their help with the muscle
biopsies, and the Myocastor study’s group (MSG) for
fruitful discussions.
SUPPLEMENTARY MATERIAL
Supplementary Figure S1 is available in the
electronic version of this article: http://dx.doi.org/
10.3233/JND-150073.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
REFERENCES
[1] Lanner JT, Georgiou DK, Joshi AD, Hamilton SL. Ryan-
odine receptors: Structure, expression, molecular details, and
function in calcium release. Cold Spring Harb Perspect Biol.
2010;2:a003996.
[2] Monnier N, Romero NB, Lerale J, Landrieu P, Nivoche Y,
Fardeau M, Lunardi J. Familial and sporadic forms of central
core disease are associated with mutations in the C-terminal
domain of the skeletal muscle ryanodine receptor. Hum Mol
Genet. 2001;10:2581-2592.
[3] Lynch PJ, Tong J, Lehane M, Mallet A, Giblin L, Heffron JJ,
Vaughan P, Zafra G, MacLennan DH, McCarthy TV. A muta-
tion in the transmembrane/luminal domain of the ryanodine
receptor is associated with abnormal Ca2+ release channel
function and severe central core disease. Proc Natl Acad Sci
USA. 1999;96:4164-4169.
[4] Davis MR, Haan E, Jungbluth H, Sewry C, North K, Muntoni
F, Kuntzer T, Lamont P, Bankier A, Tomlinson P, S´
anchez A,
WalshP, Nagarajan L, Oley C, Colley A, Gedeon A, Quinlivan
R, Dixon J, James D, M¨
uller CR, Laing NG. Principal muta-
tion hotspot for central core disease and related myopathies
in the C-terminal transmembrane region of the RYR1 gene.
Neuromuscul Disord. 2003;13:151-157.
[5] Ferreiro A, Monnier N, Romero NB, Leroy JP, Bonnemann C,
Haenggeli CA, Straub V, Voss WD, Nivoche Y, Jungbluth H,
Lemainque A, Voit T, Lunardi J, Fardeau M, Guicheney P. A
recessive form of central core disease, transiently presenting
as multi-minicore disease, is associated with a homozygous
mutation in the ryanodine receptor type 1 gene. Ann Neurol.
2002;51:750-759.
[6] Jungbluth H, Muller CR, Halliger-Keller B, Brockington M,
Brown SC, Feng L, Chattopadhyay A, Mercuri E, Manzur
AY, Ferreiro A, Laing NG, Davis MR, Roper HP, Dubowitz
V, Bydder G, Sewry CA, Muntoni F. Autosomal recessive
inheritance of RYR1 mutations in a congenital myopathy with
cores. Neurology. 2002;59:284-287.
[7] Monnier N, Ferreiro A, Marty I, Labarre-Vila A, Mezin P,
Lunardi J. A homozygous splicing mutation causing a deple-
tion of skeletal muscle RYR1 is associated with multi-minicore
disease congenital myopathy with ophthalmoplegia. Hum
Mol Genet. 2003;12:1171-1178.
[8] De Cauwer H, Heytens L, Martin JJ. Workshop report of
the 89th ENMC International Workshop: Central Core Dis-
ease, 19th-20th January 2001, Hilversum, The Netherlands.
Neuromuscul Disord. 2002;12:588-595.
[9] Romero NB, Monnier N, Viollet L, Cortey A, Chevallay M,
Leroy JP, Lunardi J, Fardeau M. Dominant and recessive cen-
tral core disease associated with RYR1 mutations and fetal
akinesia. Brain. 2003;126:2341-2349.
[10] Jungbluth H. Central core disease. Orphanet J Rare Dis.
2007;2:25.
[11] Dirksen RT, Avila G. Altered ryanodine receptor function
in central core disease: Leaky or uncoupled Ca(2+) release
channels? Trends Cardiovasc Med. 2002;12:189-197.
[12] Treves S, Jungbluth H, Muntoni F, Zorzato F. Congenital
muscle disorders with cores: The ryanodine receptor calcium
channel paradigm. Curr Opin Pharmacol. 2008;8:319-326.
[13] Ghassemi F, Vukcevic M, Xu L, Zhou H, Meissner G, Muntoni
F, Jungbluth H, Zorzato F, Treves S. A recessive ryanodine
receptor 1 mutation in a CCD patient increases channel activ-
ity. Cell Calcium. 2009;45:192-197.
[14] Loy RE, Orynbayev M, Xu L, Andronache Z, Apostol S,
Zvaritch E, MacLennan DH, Meissner G, Melzer W, Dirk-
sen RT. Muscle weakness in Ryr1I4895T/WT knock-in mice
as a result of reduced ryanodine receptor Ca2+ ion permeation
and release from the sarcoplasmic reticulum. J Gen Physiol.
2011;137:43-57.
[15] Zhou H, Yamaguchi N, Xu L, Wang Y, Sewry C, Jungbluth H,
Zorzato F, Bertini E, Muntoni F, Meissner G, Treves S. Char-
acterization of recessive RYR1 mutations in core myopathies.
Hum Mol Genet. 2006;15:2791-2803.
[16] Monnier N, Marty I, Faure J, Castiglioni C, Desnuelle C, Sac-
coni S, Estournet B, Ferreiro A, Romero N, Laquerriere A,
Lazaro L, Martin JJ, Morava E, Rossi A, Van der Kooi A,
Verschuuren C, Lunardi J. Null mutations causing depletion
of the type 1 ryanodine receptor (RYR1) are commonly asso-
ciated with recessive structural congenital myopathies with
cores. Hum Mutat. 2008;29:670-678.
[17] Zhou H, Rokach O, Feng L, Munteanu I, Mamchaoui K,
Wilmshurst JM, Sewry C, Manzur AY, Pillay K, Mouly
V, Duchen M, Jungbluth H, Treves S, Muntoni F. RyR1
deficiency in congenital myopathies disrupts excitation-
contraction coupling. Hum Mutat. 2013;34:986-996.
[18] Oddoux S, Brocard J, Schweitzer A, Szentesi P, Giannesini
B, Brocard J, Faur´
e J, Pernet-Gallay K, Bendahan D, Lunardi
J, Csernoch L, Marty I. Triadin deletion induces impaired
skeletal muscle function. J Biol Chem. 2009;284:34918-29.
[19] Marty I, Robert M, Villaz M, Lai Y, De Jongh KS, Cat-
terall WA, Ronjat M. Biochemical evidence for a complex
involving dihydropyridine receptor and ryanodine receptor in
triad junctions of skeletal muscle. Proc Natl Acad Sci USA.
1994;91:2270-2274.
[20] Zhu CH, Mouly V, Cooper RN, Mamchaoui K, Bigot A,
Shay JW, Di Santo JP, Butler-Browne GS, Wright WE.
Cellular senescence in human myoblasts is overcome by
human telomerase reverse transcriptase and cyclin-dependent
kinase 4: Consequences in aging muscle and therapeutic
strategies for muscular dystrophies. Aging Cell. 2007;64:
515-523.

432 M. Cacheux et al. / RyR1 Quantitative Defect
[21] Mamchaoui K, Trollet C, Bigot A, Negroni E, Chaouch S,
Wolff A, Kandalla PK, Marie S, Di Santo J, St Guily JL,
Muntoni F, Kim J, Philippi S, Spuler S, Levy N, Blumen
SC, Voit T, Wright WE, Aamiri A, Butler-Browne G, Mouly
V. Immortalized pathological human myoblasts: Towards a
universal tool for the study of neuromuscular disorders. Skelet
Muscle. 2011;1:1:34.
[22] Marty I, Thevenon D, Scotto C, Groh S, Sainnier S, Robert
M, Grunwald D, Villaz M. Cloning and characterization
of a new isoform of skeletal muscle triadin. J Biol Chem.
2000;275:8206-8212.
[23] Rezgui SS, Vassilopoulos S, Brocard J, Platel JC, Bouron
A, Arnoult C, Oddoux S, Garcia L, De Waard M, Marty
I. Triadin (Trisk 95) overexpression blocks excitation-
contraction coupling in rat skeletal myotubes. J Biol Chem.
2005;280:39302-39308.
[24] Lee EH, Cherednichenko G, Pessah IN, Allen PD. Functional
coupling between TRPC3 and RyR1 regulates the expressions
of key triadic proteins. J Biol Chem. 2006;281:10042-10048.
[25] Shen X, Franzini-Armstrong C, Lopez JR, Jones LR,
Kobayashi YM, Wang Y, Kerrick WG, Caswell AH, Pot-
ter JD, Miller T, Allen PD, Perez CF. Triadins modulate
intracellular Ca(2+) homeostasis but are not essential for
excitation-contraction coupling in skeletal muscle. J Biol
Chem. 2007;282:37864-7874.
[26] Medical Research Council. Aids to the investigation of the
peripheral nervous system. London: Her Majesty’s Stationary
Office; 1943.
[27] Buck ED, Nguyen HT, Pessah IN, Allen PD. Dyspedic mouse
skeletal muscle expresses major elements of the triadic junc-
tion but lacks detectable ryanodine receptor protein and
function. J Biol Chem. 1997;272:7360-7367.
[28] Moore RA, Nguyen H, Galceran J, Pessah IN, Allen PD.
A transgenic myogenic cell line lacking ryanodine receptor
protein for homologous expression studies: Reconstitution
of Ry1R protein and function. J Cell Biol. 1998;140:
843-851.
[29] Avila G, O’Brien JJ, Dirksen RT. Excitation-contraction
uncoupling by a human central core disease mutation in the
ryanodine receptor. Proc Natl Acad SciUSA.2001;98:4215-
4220.
[30] Quinlivan RM, Muller CR, Davis M, Laing NG, Evans GA,
Dwyer J, Dove J, Roberts AP, Sewry CA. Central core disease:
Clinical, pathological, and genetic features. Arch Dis Child.
2003;88:1051-1055.
[31] Zalk R, Clarke OB, des Georges A, Grassucci RA, Reiken S,
Mancia F, Hendrickson WA, Frank J, Marks AR. Structure of
a mammalian ryanodine receptor. Nature. 2015;517:44-49.
[32] Yan Z, Bai XC, Yan C, Wu J, Li Z, Xie T, Peng W, Yin
CC, Li X, Scheres SH, Shi Y, Yan N. Structure of the rabbit
ryanodine receptor RyR1 at near-atomic resolution. Nature.
2015;517:50-55.
[33] Lee JM, Rho SH, Shin DW, Cho C, Park WJ, Eom SH,
Ma J, Kim DH. Negatively charged amino acids within the
intraluminal loop of ryanodine receptor are involved in the
interaction with triadin. J Biol Chem. 2004;279:6994-7000.
[34] Lee EH, Song DW, Lee JM, Meissner G, Allen PD, Kim
DH. Occurrence of atypical Ca2+ transients in triadin-binding
deficient-RYR1 mutants. Biochem Biophys Res Commun.
2006;351:909-914.
[35] Zhou H, Jungbluth H, Sewry CA, Feng L, Bertini E,
Bushby K, Straub V, Roper H, Rose MR, Brockington M,
Kinali M, Manzur A, Robb S, Appleton R, Messina S,
D’Amico A, Quinlivan R, Swash M, M¨
uller CR, Brown S,
Treves S, Muntoni F. Molecular mechanisms and pheno-
typic variation in RYR1-related congenital myopathies. Brain.
2007;130:2024-2036.
[36] Tilgen N, Zorzato F, Halliger-Keller B, Muntoni F, Sewry
C, Palmucci LM, Schneider C, Hauser E, Lehmann-Horn F,
M¨
uller CR, Treves S. Identification of four novel mutations in
the C-terminal membrane spanning domain of the ryanodine
receptor 1: association with central core disease and alteration
of calcium homeostasis. Hum Mol Genet. 2001;10:2879-
2887.