From the Banting and Best Department of Medical
Research and § Department of Biochemistry, University of
Toronto, Toronto, Ontario M5G 1L6, Canada and
Department of
Biochemistry, University College Cork, Cork, Ireland
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ABSTRACT |
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Malignant hyperthermia (MH) and
central core disease (CCD) mutations were introduced into full-length
rabbit Ca2+ release channel (RYR1)
cDNA, which was then expressed transiently in HEK-293 cells.
Resting Ca2+ concentrations were higher in HEK-293 cells
expressing homotetrameric CCD mutant RyR1 than in cells expressing
homotetrameric MH mutant RyR1. Cells expressing homotetrameric CCD or
MH mutant RyR1 exhibited lower maximal peak amplitudes of
caffeine-induced Ca2+ release than cells expressing wild
type RyR1, suggesting that MH and CCD mutants might be "leaky." In
cells expressing homotetrameric wild type or mutant RyR1, the amplitude
of 10 mM caffeine-induced Ca2+ release was
correlated significantly with the amplitude of carbachol- or
thapsigargin-induced Ca2+ release, indicating that maximal
drug-induced Ca2+ release depends on the size of the
endoplasmic reticulum Ca2+ store. The content of endogenous
sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b
(SERCA2b), measured by enzyme-linked immunosorbent assay,
45Ca2+ uptake, and confocal microscopy, was
increased in HEK-293 cells expressing wild type or mutant RyR1,
supporting the view that endoplasmic reticulum Ca2+ storage
capacity is increased as a compensatory response to an enhanced
Ca2+ leak. When heterotetrameric (1:1) combinations of
MH/CCD mutant and wild type RyR1 were expressed together with SERCA1 to
enhance Ca2+ reuptake, the amplitude of Ca2+
release in response to low concentrations of caffeine and halothane was
higher than that observed in cells expressing wild type RyR1 and
SERCA1. In Ca2+-free medium, MH/CCD mutants were more
sensitive to caffeine than wild type RyR1, indicating that caffeine
hypersensitivity observed with a variety of MH/CCD mutant RyR1 proteins
is not dependent on extracellular Ca2+ concentration.
Malignant hyperthermia
(MH)1 is an autosomal
dominant muscle disorder in which genetically susceptible individuals
among populations of humans and domestic animals respond to the
administration of potent inhalational anesthetics and depolarizing
skeletal muscle relaxants with high fever and skeletal muscle rigidity
(1, 2). Central core disease (CCD) is a rare, non-progressive myopathy, presenting in infancy and characterized by hypotonia and proximal muscle weakness (1). An important feature of CCD is its close association with MH susceptibility (2).
Although diagnosis of CCD is made on the basis of the lack of oxidative
enzymatic activity in central regions of skeletal muscle fibers (3),
the diagnostic test for MH susceptibility in humans is the North
American caffeine halothane contracture test (4) or its European
counterpart, the in vitro contracture test (5). These tests
are based on the hypersensitivity of contracture of muscle strips,
obtained by biopsy, to caffeine or halothane.
Genetic and biochemical data have supported the view that mutations in
the gene encoding the Ca2+ release channel of skeletal
muscle sarcoplasmic reticulum (RYR1) are a major cause of MH
in swine and humans (6-10). In humans, 12 RYR1 mutations at
10 locations (C35R, G248R, G341R, R552W, R614C, R614L, R2163C, V2168M,
T2206M, G2435R, R2458C, and R2458H) have been linked to MH and 5 mutations at 5 locations (R163C, I403M, Y522S, R2163H, and R2436H) have
been linked to CCD plus MH (11-21). Both MH and CCD mutations are
located in two distinct clusters in the linear sequence of
RYR1 lying between residues 35 and 614 (MH domain 1) and
between residues 2163 and 2458 (MH domain 2).
The fact that MH and CCD mutations are found in the same gene suggests
that different genotypic variants might result in a spectrum of
phenotypic responses of different severity (7, 9, 10). No significant
differences were observed in measurements of caffeine and halothane
sensitivity between MH and CCD mutants when they were expressed in
homozygous state in HEK-293 cells (22). It is not clear whether there
are differences in resting myoplasmic Ca2+ concentrations
in muscle fibers of MH and CCD patients (23-26). Difficulties in
obtaining accurate and reproducible measurements of resting
Ca2+ concentrations in MH or CCD muscle fibers arise from
the possibility that compensation may occur in muscle cells and from
the different methods used by different groups who have studied the
different mutations.
We have attempted to alleviate these problems by transfecting all MH
and CCD mutants into HEK-293 cells, which have a homogenous genetic
background, and using a fluorescent (Fura-2) imaging assay to measure
resting Ca2+ concentrations in these transfected cells. In
an earlier study (22), we showed that 15 MH or CCD (MH/CCD) mutant
homotetramers expressed in HEK-293 cells were more sensitive to
caffeine and halothane than wild type RyR1. In addition, the response
observed for the mutant channels expressed in HEK-293 cells correlated closely with the response observed in contracture tests of muscle samples from humans bearing the same mutations (22). In human MH or CCD
individuals, however, heterozygosity is most common. In the present
study, we have measured resting Ca2+ concentrations
in HEK-293 cells expressing the various mutations, analyzed the
relationship between caffeine-induced Ca2+ release and
intracellular Ca2+ stores, tested for an increase in
sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b
(SERCA2b) synthesis as a compensatory factor in cells expressing
wild type or mutant RyR1, tested the caffeine sensitivity of
heterotetrameric MH or CCD mutants expressed in the presence or absence
of SERCA1, and tested the influence of extracellular Ca2+
concentration on caffeine responses of MH or CCD mutant channels.
Materials--
Enzymes for DNA manipulation were obtained from
Boehringer Mannheim, New England Biolabs, Promega, and Amersham
Pharmacia Biotech. Tissue culture reagents were purchased from Life
Technologies, Inc. Monoclonal antibody 34C (27) was a kind gift from
Dr. Judith Airey (University of Nevada, Reno). Fura-2 acetoxymethyl
ester (Fura-2/AM) and pluronic F-127 were from Molecular Probes.
Caffeine was from Sigma. Halothane was from Fluka. Carbachol and
thapsigargin were from Calbiochem.
[45Ca2+]Cl2 (10-40 mCi/mg
Ca2+) was obtained from Amersham Pharmacia Biotech. All
other chemicals were of reagent (or highest available) grade.
Construction of MH/CCD Mutants in RYR1 Cassettes--
The
cloning, expression, and construction of the full-length rabbit
skeletal muscle ryanodine receptor (RYR1) cDNA cassettes were described previously (22, 28). The construction and expression of
single MH/CCD mutant RYR1 cDNAs were also described
previously (22).
Cell Culture and DNA Transfection--
Culture and transfection
of HEK-293 cells using the calcium phosphate precipitation method of
Chen and Okayama (29) were carried out as described earlier (22). Ten
µg of plasmid DNA were used to transfect 2 × 105
cells/60-mm plate. Control cells were treated in the same way, but with
no DNA or with expression vector DNA only.
Fluorescence Measurements--
Ca2+ photometry and
Ca2+ imaging assays were used to measure Ca2+
concentration changes in transfected HEK-293 cells as described previously (22). A Photon Technologies Inc. micro-fluorimetry system
was used in photometric assays to measure caffeine-induced changes in
Fura-2/AM fluorescence resulting from Ca2+ release through
the different ryanodine receptor proteins expressed in HEK-293 cells.
Dose-response curves were generated and normalized to the maximal
Ca2+ release response observed at 10 mM
caffeine for both the caffeine and the halothane responses.
Procedures for Ca2+ imaging were the same as for the
Ca2+ photometric assay, except that the cells were loaded
with 4 µM Fura-2 AM, 0.02% pluronic F-127 for 45 min and
the emitted fluorescence was fed into a charge-coupled device (CCD)
camera (Photon Technologies Inc. SenSys-KAF 1400) instead of a
photomultiplier tube. Acquired digital images (400 ms/frame) were
analyzed with Image Master 2.0 software (Photon Technologies Inc.). The
340/380 nm ratios from single cells were converted to Ca2+
concentrations according to Grynkiewicz et al. (30):
[Ca2+] = Kd·((R Microsome Preparation, Ca2+ Uptake, and Enzyme-linked
Immunoabsorbent Assay (ELISA)--
Microsomes were prepared and
assayed for Ca2+ transport activity, and data were analyzed
as described previously (31). Aliquots of 50 µl of the microsome
preparation (1 mg/ml) were analyzed by ELISA, as described previously
(32). A primary mouse monoclonal anti-SERCA2 ATPase antibody (IgG2a,
Affinity Bioreagents Inc.) was used to detect SERCA2 in microsomes.
Immunofluorescence Labeling and Laser Scanning Confocal
Microscopy--
HEK-293 cells transfected with RYR1
cDNA were cultured on coverslips. They were fixed for 20 min in
3.7% formaldehyde at room temperature, washed once with PBS, and
permeabilized with 0.2% Triton X-100 in PBS for 10 min. Cells were
then incubated with IgG2a (Affinity Bioreagents Inc.) in 1:100 dilution
for 1 h, washed 3 times with PBS, followed by tetramethylrhodamine
isothiocyanate-conjugated anti-mouse secondary antibody in 1:100
dilution for 1 h, and washed 3 times with PBS. All antibodies were
diluted in PBS containing 0.1% Triton X-100. Coverslips were mounted
on microscope slides in ImmunoFloure Mounting Medium obtained from ICN.
Cells were observed and analyzed with a Nikon Optiphot II microscope,
equipped with epifluorescence illumination, and a Bio-Rad MRC 600 confocal laser scanning microscope system. Immunofluorescent images
were recorded on a zip disk and analyzed using NIH Image 5.19 software.
Statistical Methods--
All data are expressed as mean ± S.E. Linear regression analysis was performed using Origin software
(Microcal Software Ltd., Northampton, MA). An unpaired Student's
t test was used for statistical comparisons of mean values
between samples. A value of p < 0.05 was taken to
indicate statistical significance.
Comparison of Resting Ca2+ Concentration and
Caffeine-induced Ca2+ Release among Wild Type, MH Mutant,
and CCD Mutant RyR1 Proteins--
In this study, we used
Ca2+ photometry and Ca2+ imaging to record the
Ca2+ release properties of a number of RyR1 mutants
expressed in HEK-293 cells. Ca2+ photometry, which is fast
and accurate, provides spatially averaged measurements of the
fluorescence used to monitor Ca2+ release. Ca2+
imaging provides spatially resolved measurements and was used to
acquire images at 2.5 s/frame, permitting us to analyze events in
individual cells in response to caffeine.
We used Ca2+ imaging to detect caffeine-induced
Ca2+ release in HEK-293 cells transiently transfected with
RYR1 cDNA, thereby distinguishing transfected cells from
untransfected cells. Ca2+ photometry indicates that
Ca2+ release is a rare event in vector-transfected or
nontransfected HEK-293 cells which is lost in the background
fluorescence when a cluster of 50 or more cells is analyzed. In single
cell imaging of pcDNA vector-transfected cells, 6 out of 200 cells
responded to 10 mM caffeine, increasing cytosolic
Ca2+ concentrations to an average of 180 ± 54 nM (n = 6) and establishing a background
response rate of about 3%. In wild type or MH/CCD mutant
RYR1-transfected cells, however, 40-60% of isolated cells, where higher DNA transfection was observed, responded to 10 mM caffeine, a rate about 13-20-fold higher than
background. Accordingly, caffeine-responsive cells in our
Ca2+ imaging studies were regarded as
RYR1-transfected cells, and an error rate of 3% was
considered to be acceptable.
Resting cytoplasmic Ca2+ concentrations and 10 mM caffeine-induced Ca2+ release for wild type
and 15 MH/CCD mutant RyR1 proteins were measured in single
RYR1-transfected cells. The resting cytoplasmic Ca2+ concentration in untransfected HEK-293 cells was
97 ± 5 nM. Transfection with wild type RyR1 raised
the resting cytoplasmic Ca2+ concentration to 112 ± 11 nM (Fig. 1A).
The resting cytoplasmic Ca2+ concentration in HEK-293 cells
transfected with each of the 10 MH mutants tested (C36R, G249R, G342R,
R553W, R615C, R615L, R2163C, G2435R, R2458C, and R2458H) also raised
the resting cytoplasmic Ca2+ concentration to values
ranging from 103 ± 7 to 119 ± 7 nM with an
average value of 110 ± 2 nM (Fig. 1A).
There were no significant differences in resting Ca2+
concentrations between wild type and any of the MH mutant forms of
RYR1. The resting Ca2+ concentration (Fig.
1A) for each of the 5 CCD mutants tested (R164C, I404M,
Y523S, R2163H, and R2436H) was higher than for any of the 10 MH mutants
and the average resting Ca2+ concentration for the 5 CCD
mutants, 142 ± 9 nM, was significantly higher
(p < 0.001) than the average resting Ca2+
concentration for the 10 MH mutants. However, when resting
Ca2+ concentrations for individual CCD mutants were
compared with wild type RYR1, significant differences were
found for only 2 of the 5 individual CCD mutants tested, Y523S and
R2163H (p < 0.05 for Y523S and p < 0.001 for R2163H).
Average caffeine-induced Ca2+ release and maximal 340/380
nm ratio change values for MH mutants were 590 ± 107 nM (Fig. 1B) and 0.36 ± 0.03 (Fig.
1C) (n = 10), respectively, and the
comparable values for CCD mutants were 495 ± 60 nM
(Fig. 1B) and 0.28 ± 0.06 (Fig. 1C)
(n = 5). The caffeine-induced Ca2+ release
values for MH mutants was not significantly different from the
comparable values for CCD mutants. It should be noted, however, that
many individual MH and CCD mutants had a significantly lower response
to caffeine, measured by both Ca2+ photometry and
Ca2+ imaging, than wild type RYR1 (Fig. 1).
Maximal 340/380 nm ratio changes were significantly lower for the Y523S
(p < 0.001) and R2163H (p < 0.001)
mutants (Fig. 1B), which had higher resting Ca2+
concentrations (Fig. 1A) than wild type RyR1. These results
are consistent with the view that CCD mutants might be more leaky than
MH mutants, accounting for higher resting Ca2+
concentrations and lower Ca2+-releasable stores, and that
MH mutants might also be more leaky than wild type, accounting for
lower concentrations of Ca2+ in caffeine-releasable stores.
To test whether resting Ca2+ concentrations might determine
caffeine sensitivity and influence maximal caffeine-induced
Ca2+ release, a linear correlation analysis was carried
out. Resting Ca2+ concentrations had no linear correlation
with caffeine ED50 values (r = Relationship among Caffeine-, Carbachol-, and Thapsigargin-induced
Ca2+ Release in Transfected Cells--
To test whether
releasable ER Ca2+ stores were really lower in MH/CCD
mutant-transfected cells, thapsigargin and carbachol were used to gate
Ca2+ release, determined by fluorescence measurement of the
amount of Ca2+ released. Thapsigargin increases
intracellular Ca2+ concentration by irreversible blocking
of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)
molecules. Thapsigargin-induced Ca2+ release, therefore,
represents the rapid, passive depletion of the internal
Ca2+ store (33). Carbachol increases intracellular
Ca2+ concentration by activation of the phospholipase C
pathway through an endogenous muscarinic receptor found in HEK-293
cells, resulting in elevation of intracellular IP3 and
activation of the IP3 receptor (34). Thus low ER
Ca2+ stores would respond with low Ca2+ release
following the application of either of these agents.
Figs. 2, A-C, shows that
there was a close linear correlation for Ca2+ release
induced by any of these three reagents in 27 RYR1
cDNA-transfected cells. These cells were challenged sequentially by
10 mM caffeine (RyR1), 20 µM carbachol
(IP3 receptor), and 1.5 µM thapsigargin (SERCA). In cells transfected with different MH/CCD mutant cDNAs to
form either homotetrameric or heterotetrameric channels,
caffeine-induced Ca2+ release was closely correlated with
thapsigargin-induced Ca2+ release (Fig. 2D).
This close correlation among caffeine-, carbachol-, and
thapsigargin-induced Ca2+ release indicates that maximal
caffeine-induced Ca2+ release reflects the size of the ER
Ca2+ store. The lower maximal caffeine-induced
Ca2+ release in MH/CCD mutants is likely to be due to a
lower Ca2+ store, which, in turn, is likely to reflect
Ca2+ leakage through MH/CCD mutant channels.
Comparison of Ca2+ Stores and SERCA2b Content among
Untransfected and RYR1 cDNA-transfected Cells--
To compare ER
Ca2+ stores among untransfected and RYR1
cDNA-transfected cells, carbachol-induced Ca2+ release
was measured by Ca2+ imaging and was regarded as an
indicator of the size of the Ca2+ store. Fig.
3 shows that carbachol-induced
Ca2+ release in untransfected HEK-293 cells was smaller
than Ca2+ release in cells transfected with wild type
RYR1 but was higher than Ca2+ release in cells
transfected with CCD or MH mutant RYR1. The smaller
carbachol-induced Ca2+ release in cells transfected with
MH/CCD mutants supports the view that these mutants are more leaky than
wild type RyR1.
The enhancement of the Ca2+ store by transfection of wild
type RyR1 led us to test the hypothesis that cells transfected with channels that increase release from ER Ca2+ stores may
overexpress endogenous SERCA2b in an attempt to reestablish Ca2+ homeostasis by lowering elevated resting
Ca2+ concentrations to normal (Fig. 1). To determine
whether cells transfected with wild type or mutant RYR1 had
the capacity to create a larger Ca2+ store, SERCA2b
contents were measured by ELISA (Fig.
4A), and Ca2+
uptake was measured in microsomes extracted from transfected cells
(Fig. 4B). When HEK-293 cells were transfected with wild type RYR1, the SERCA2b content was increased to 119%
(ELISA) or 120% (Ca2+ uptake). Transfection with MH mutant
R615L led to an increase in SERCA2b content to 117% (ELISA) or 120%
(Ca2+ uptake), whereas transfection with CCD mutant Y523S
led to an increase in SERCA2b content to 127% (ELISA) or 123%
(Ca2+ uptake). Although transfection efficiency approaches
50% for non-confluent cells, overall transfection efficiency is only
about 25%. Therefore, the real increase in SERCA2b expression in
transfected cells would be about 4-fold higher, approaching a doubling
of SERCA2b content.
Immunofluorescent labeling and confocal microscopy were used in a third
measurement of the enhancement of SERCA2b content in single cells
transfected with mutant RyR1. Because only mouse anti-RyR1 and mouse
anti-SERCA2 monoclonal antibodies are available, green fluorescent
protein (GFP) vector (CLONTECH) and rabbit RyR1 mutant Y523S were cotransfected into HEK-293 cells in a 1:9 molar ratio. GFP fluorescence in cells was regarded as an indicator of
transfected cells. Immunofluorescent staining of rabbit RyR1, detected
by mouse monoclonal 34C antibody, showed that about 80% of transfected
cells expressed both GFP and RyR1, 17% of transfected cells expressed
GFP alone and 3% of transfected cells expressed RyR1 alone (not
shown). The SERCA2 fluorescence from Y523S-transfected cells, which
were GFP-positive, was increased by 74 ± 4% (n = 45, p < 0.001) when compared with fluorescence from
untransfected cells (not shown), supporting our other measurements
showing that the SERCA2 content of Y523S-transfected cells was nearly
doubled. These results suggest that compensation in the form of
enhanced SERCA2 expression occurs in RYR1 or MH/CCD mutant
cDNA-transfected cells.
Caffeine Responses for Homotetrameric and Heterotetrameric MH/CCD
Mutant Channels and the Effect of Coexpression with SERCA1--
The
maximal caffeine-induced Ca2+ release for most MH/CCD
mutant homotetrameric channels was smaller than for wild type channels (Fig. 1). This is in contrast to clinical observations which show that
muscle fibers from malignant hyperthermia-sensitive individuals contract more strongly in low concentrations of caffeine and halothane than fibers from normal individuals, in line with enhanced
Ca2+ release. Although homotetrameric MH/CCD mutant
channels expressed in HEK-293 cells have been shown to be more
sensitive to caffeine and halothane than wild type RyR1 (22),
heterotetrameric MH/CCD mutant channels are most common in humans. In
order to determine whether heterotetrameric wild type and mutant RyR1
channels would be less leaky, R615L (MH mutant) and Y523S (CCD mutant)
were coexpressed in a 1:1 ratio with wild type RYR1. When HEK-293 cells
expressed heterotetrameric channels, their caffeine sensitivities were
found to be intermediate between those of cells expressing
homotetrameric R615L or Y523S and cells expressing homotetrameric wild
type RYR1 (Fig.
5A). Maximal 340/380 nm ratio
changes (Fig. 5B) and 10 mM caffeine-induced
Ca2+ release (Fig. 5D) in heterotetrameric
MH/CCD mutants were higher than those in homotetrameric MH/CCD mutants,
suggesting that heterozygote channels are less leaky than homozygote
channels.
It is assumed that the activity of SERCA pumps is coordinated with the
activity of RyR Ca2+ release channels in healthy skeletal
muscle to regulate cytosolic Ca2+ concentrations. Thus the
2-fold enhancement of SERCA2 synthesis that we observed in cells
expressing MH/CCD mutants could be explained logically as a
compensatory event. Nevertheless, enhanced endogenous SERCA2b synthesis
was not sufficient to compensate for the higher resting
Ca2+ concentrations attributed to leaky mutant channels
R615L or Y523S (Fig. 1). Since it is unlikely that compensatory SERCA
synthesis had reached equilibrium in these cells over the 48-h period
between transfection and analysis, we attempted to increase SERCA
synthesis at a more rapid rate by transfection with SERCA1 under
conditions where high levels of activity are observed within 48 h
(35). Under these conditions, SERCA1 was functional, since it increased the rate of Ca2+ removal from the cytoplasm following
caffeine-induced Ca2+ release and lowered the elevated base
line of Ca2+ fluorescence resulting from repeated caffeine
stimulation of cells cotransfected with wild type or MH/CCD mutant RyR1
(not shown).
Surprisingly, coexpression of mutants R615L or Y523S with SERCA1 raised
resting Ca2+ concentrations (Fig. 5C). The
coexpression of SERCA1 with Y523S, or with Y523S plus wild type RyR1,
or with R615L, or with R615L plus wild type RyR1 decreased caffeine
sensitivity (Fig. 5A), increased resting Ca2+
concentration (Fig. 5C), and increased maximal
Ca2+ release for both RyR1 mutants (Fig. 5, B
and D). These results suggest that SERCA1 can increase
Ca2+ removal rates and Ca2+ stores but
that increased flux through the mutant channels in the ER, even under
conditions where Ca2+ uptake is greatly increased,
ultimately results in an increase in resting Ca2+ concentrations.
We used Ca2+ imaging to test caffeine-induced
Ca2+ release in MH/CCD mutant heterotetramers. We found
that the heterotetrameric R615L mutant plus SERCA1 and the
heterotetrameric mutant Y523S plus SERCA1 were significantly more
sensitive to low concentrations of caffeine (0.5 and 1 mM)
and halothane (0.1 and 0.25 mM) than wild type RyR1 plus
SERCA1 (not shown). The MH/CCD mutant heterotetramers plus SERCA1
released more Ca2+ when challenged by low concentrations of
caffeine and halothane than wild type RyR1 plus SERCA1 (not shown).
Influence of Extracellular Ca2+ Levels on Caffeine
Sensitivity of Wild Type and MH/CCD Mutant RyR1--
All of the
results described above were obtained from HEK-293 cells incubated in a
medium containing 2 mM Ca2+. To confirm that
caffeine-induced Ca2+ release in
RYR1-transfected cells was caused by Ca2+
release from ER Ca2+ stores, caffeine-induced
Ca2+ release in RYR1-transfected cells was
measured in Ca2+-free medium. Ca2+ release,
induced by caffeine or carbachol and measured by Ca2+
imaging in RYR1-transfected cells, was reduced dramatically, and
resting Ca2+ concentrations in both transfected and
untransfected cells were much lower in a Ca2+-free medium
than in Ca2+-containing medium (not shown). These results
indicate that extracellular Ca2+ has a significant
influence on intracellular Ca2+ concentration.
In early experiments, we found that caffeine-induced Ca2+
release could be abolished by the addition of thapsigargin in
Ca2+-containing medium (not shown), indicating that
caffeine-induced Ca2+ release depends on the ER
Ca2+ store. In later studies, RYR1
cDNA-transfected cells were incubated in Ca2+-free
medium with Fura-2/AM for 30 min, and caffeine-induced Ca2+
release was measured in Ca2+-free medium, then in
Ca2+-containing medium, and, finally, in
Ca2+-free medium (Fig.
6A). In Ca2+-free
medium, caffeine responses were reduced dramatically after the third
caffeine stimulation (Fig. 6A). In
Ca2+-containing medium (not shown), caffeine stimulation of
Ca2+ release could be induced repeatedly. The addition of
Ca2+-containing medium (2 mM Ca2+)
resulted in an increase in resting Ca2+ concentration and
an increase in the amplitude of caffeine-induced Ca2+
release (Fig. 6A). When the cells were returned to
Ca2+-free medium, the caffeine response was reduced
dramatically. Similar results were obtained for carbachol responses in
untransfected cells (not shown). These results confirm that
extracellular Ca2+ concentrations have a profound influence
on cellular Ca2+ homeostasis.
When we measured caffeine dose-response curves for wild type and Y523S
and R615L mutants in Ca2+-free medium, we observed that the
mutant channels were more sensitive than wild type to caffeine (Fig.
6B), just as they were in Ca2+-containing
medium. The caffeine ED50 values for RyR1, Y523S, and R615L
in Ca2+-free media were 8.1, 0.99, and 2.64 mM,
respectively. These values are 2 to 4 times higher than the
ED50 values measured in Ca2+-containing medium.
The lower caffeine ED50 values in Ca2+-free
medium may have been due to the lower resting Ca2+ levels
in Ca2+-free media, suggesting that resting
Ca2+ concentrations may also influence the caffeine
sensitivity of RyR1.
Analysis of RyR1 Mutations in HEK-293 Cells--
In a recent paper
Querfurth et al. (36) reported that an unspecified isoform
of the ryanodine receptor (RyR) is expressed in HEK-293 cells. The
level of endogenous RyR expression measured was very low, since RyR
could only be detected through highly sensitive immunoprecipitation of
radiolabeled cells or by Western blotting of immunoprecipitates. We
could not detect RyR proteins in extracts of our HEK-293 cells by
Western blotting, although we observed huge amounts of transfected RyR
under comparable conditions (22). Although Querfurth et al.
(36) reported cellular immuno-staining, a suitable control for
background staining was not provided. Querfurth et al. (36)
reported increases in intracellular Ca2+ concentration of
nearly 500 nM in 23-33% of their untransfected HEK-293
cells by imaging. We cannot confirm this result under the conditions of
our experiments. If this high level of endogenous RyR activity were
present in our cells, we would, unquestionably, have detected it by
Ca2+ photometry when we measured the combined fluorescence
emission from at least 50 cells in experiments repeated over many years (22). Instead, we measured a negligible change in the background 340/380 fluorescence ratio from Fura-2 in untransfected HEK-293 cells,
but we measured a ratio change in HEK-293 cells transfected with wild
type RyR1 that was greater than 0.8.
In this study, in which we present single cell imaging of
pcDNA-transfected HEK-293 cells, we also measured the background of
endogenous Ca2+ release in untransfected and
vector-transfected cells. We found that 6 out of 200 cells responded to
10 mM caffeine, increasing cytosolic Ca2+
concentrations to an average of 180 ± 54 nM and
establishing our background response rate at 3%. In wild type or
mutant RyR1-transfected cells, 40-60% of cells responded to 10 M caffeine, a rate 13-20-fold higher than background,
increasing cytosolic Ca2+ concentrations well above 500 nM (Fig. 1A). The differences in the results of
our experiments and those of Querfurth et al. (36) are most
likely to be due to the way in which experiments were carried out.
Querfurth et al. (36) could not observe caffeine-induced Ca2+ release when they allowed buffer changes to flow over
the cells (the conditions of our experiments). In order to see
Ca2+ release, they had to interrupt flow and then remove
and replace buffer to achieve instant 15 mM caffeine. Thus
re-perfusion at high caffeine seemed to sensitize the endogenous RyR
activity, even though the resulting spike of Ca2+ release
was actually slower than that which we observed. Even if there were
re-perfusion artifacts in the Ca2+-imaging protocol of
Querfurth et al. (36) that could account for the exaggerated
Ca2+ release attributed to endogenous RyR, they would not
be relevant to our studies, since we used a different protocol that,
maximally, triggered only rare cases of Ca2+ release in
untransfected cells.
Functional Differences in MH and MH/CCD Mutations--
In earlier
publications (7, 9, 10), we proposed that there might be spontaneous
Ca2+ leakage through MH mutant channels but that
compensatory mechanisms would lead to rapid re-establishment of
Ca2+ homeostasis. Thus muscle hypertrophy in MH swine might
result from spontaneous Ca2+ release from an abnormal
Ca2+ release channel leading to spontaneous muscle
contracture. We also proposed that CCD mutations might lead to more
serious spontaneous Ca2+ release, which would disrupt
Ca2+ homeostasis in the core of the cell but not in the
periphery where re-establishment of Ca2+ homeostasis would
be aided by plasma membrane Ca2+-ATPases or
Na+/Ca2+ exchangers in the plasma membrane (9,
10).
In this study, we observed higher resting Ca2+
concentrations in HEK-293 cells transfected with wild type
RYR1, suggesting that even the normal expressed channel
might increase the permeability of the ER Ca2+ store. This
might explain the difficulty that we have encountered in attempts to
obtain a stable HEK-293 cell line expressing RYR1. When the
cells were transfected with RYR1 carrying MH mutations, resting cytosolic Ca2+ concentrations were raised over that
of wild type RyR1-transfected cells in some cases (Fig. 1), but average
values were not significantly different from wild type. Of greater
interest was the observation that the average resting cytosolic
Ca2+ concentration was elevated significantly over wild
type and over MH mutant RyR1 proteins for the five CCD mutant RyR1
proteins expressed in HEK-293 cells. For the individual CCD mutants,
Y523S and R2163H, resting cytosolic Ca2+ concentrations
were elevated significantly over wild type RyR1.
Maximal caffeine-induced Ca2+ release, measured by both
Ca2+ photometry and Ca2+ imaging, was lower in
cells transfected with individual MH/CCD mutants than with wild type
RYR1. We measured the size of the ER Ca2+ store
that was releasable by three different triggers acting through three
different mechanisms as follows: caffeine, which releases
Ca2+ through RyR; thapsigargin, which inhibits SERCA2,
thereby preventing re-uptake of Ca2+ lost through passive
leaks; and carbachol, which releases Ca2+ indirectly
through the IP3 receptor. The close correlation of caffeine-, carbachol-, and thapsigargin-induced Ca2+
release indicates that the maximal caffeine-induced Ca2+
release is proportional to the size of the ER Ca2+ store.
We noted a large variation in amplitude of Ca2+ release in
different cells to the same releasing agents (caffeine, carbachol, and
thapsigargin) (Fig. 2). This variation was not caused by DNA
transfection, because untransfected cells also showed a large variation
in their response to carbachol and thapsigargin. It is more likely that
variation was based on factors such as age or cell cycle stage. Despite
this variation among individual cells, we were able to obtain clear
correlations from measurements of sizes of ER Ca2+ stores
in response to different triggering agents. Accordingly, leakage from
these stores through an abnormal RyR1 would be predicted to lower
the store available for caffeine-induced Ca2+ release
and, at the same time, to increase cytosolic Ca2+
concentrations, as observed in Fig. 1. These observations suggest that
CCD and MH mutant channels are more leaky than wild type RYR1 channels.
Resting cytosolic Ca2+ concentrations were not correlated
with caffeine sensitivity or with maximal caffeine-induced
Ca2+ release. This lack of correlation is consistent with
clinical observations and with our previous results (22), which showed no differences in caffeine and halothane sensitivity between MH and CCD
mutants. The results suggest that resting Ca2+
concentrations do not have a major influence on the caffeine sensitivity of MH/CCD mutants.
Caffeine ED50 values for Ca2+ release through
MH/CCD mutant proteins were linearly correlated with maximal caffeine
responses, and the maximal caffeine-induced Ca2+ release
was also linearly correlated with clinical caffeine thresholds, indicating that higher ER Ca2+ stores inhibit caffeine
responses. In single-channel measurements with rabbit RYR1,
an increase in lumenal Ca2+ concentration, from micromolar
to millimolar, has been shown to decrease single channel activity
(37-39). Since high lumenal Ca2+ lowers channel open
probability, it may raise caffeine ED50. The corollary is
that low lumenal concentrations may increase caffeine ED50.
On the other hand, lower Ca2+ concentration in the
sarcoplasmic reticulum lumen may provide a compensatory mechanism in
MH/CCD skeletal muscle cells. Whether halothane will trigger an MH
reaction may depend not only on the sensitivity of MH/CCD mutants to
halothane but also on the balance between the decreased inhibitory
effect of lumenal Ca2+ concentration and the reduced net
Ca2+ efflux from the sarcoplasmic reticulum. This may
explain why some individuals who carry MH mutations do not show higher
sensitivity to caffeine and halothane.
The observation of lower caffeine-induced Ca2+ release in
HEK-293 cells transfected with MH/CCD mutants is not consistent with clinical observations. However, in vivo, MH individuals
rarely have homozygous MH/CCD mutations, and an increase in the number of SERCA pumps could compensate for enhanced Ca2+ release.
We tested whether MH/CCD mutants had higher caffeine-induced Ca2+ release when they were coexpressed with wild type RyR1
and SERCA1. The caffeine sensitivity of the heterotetrameric MH/CCD
mutants was between that of the homotetrameric MH/CCD mutants and wild type RyR1 (Fig. 2), but maximal 340/380 nm ratio changes in MH/CCD heterotetrameric mutants were higher than those in homotetrameric MH/CCD mutants. RyR1 isolated from pigs heterozygous for the R614C MH
mutation demonstrated intermediate values for the rate of
Ca2+ release and the affinity for
[3H]ryanodine (40-44). Maximal Ca2+ release
responses were similar in heterotetrameric MH/CCD mutants and wild type
homotetramers, but the heterotetrameric MH/CCD mutants plus SERCA1 were
more sensitive to low concentrations of caffeine and halothane than the
cells transfected with wild type RyR1 plus SERCA1 (Fig. 5). Overall,
these results indicate that coexpression of SERCA1 increases the ER
Ca2+ store and that expression of heterotetrameric MH/CCD
mutants reduces the abnormal leak of homotetrameric MH/CCD mutant channels.
Compensation in RYR1-transfected Cells--
Our overall
observations concerning the expression of wild type, MH, and CCD mutant
RyR1 in HEK-293 cells can be interpreted as a coherent pattern of
events. The expression of wild type RyR1 raises the resting
Ca2+ concentration (Fig. 1A), increases the size
of the Ca2+ store (Fig. 3), and increases both SERCA2b
content and activity (Fig. 4). These observations can be explained on
the basis of an increased permeability of the ER Ca2+ store
which is compensated for by Ca2+-induced synthesis of
SERCA2b, with a consequent enlargement of the Ca2+ store.
Despite this attempt at re-establishment of Ca2+
homeostasis, the enhanced flux of Ca2+ through the ER still
results in a higher resting Ca2+ concentration. Expression
of MH or CCD mutant RyR1 channels also raises resting Ca2+
concentrations and enhances synthesis of SERCA2b, leading to a higher
potential for Ca2+ storage. This potential was not
realized, however, since the carbachol-releasable stores for MH and CCD
mutants were seen to be depleted (Fig. 3). This suggests that the
attempt to re-establish Ca2+ homeostasis for MH and CCD
mutants was less successful, possibly because the flux through even
more permeable channels would require even higher synthesis of SERCA2b.
In attempts to determine whether full compensation could ever be
achieved, we coexpressed SERCA1 with homozygous and heterozygous mutant
channels (Fig. 5). We found that coexpression of higher levels of
SERCA1 did not reduce resting Ca2+ concentrations but did
increase caffeine-releasable Ca2+ stores, particularly for
RyR1 heterozygotes (Fig. 5). These experiments provide new insights
into the way in which diseases arising from defects in Ca2+
regulatory proteins progress and are compensated. Clearly compensation is a much more complex process involving Ca2+ regulatory
proteins in the sarcoplasmic reticulum, the plasma membrane, and
mitochondria (7, 9, 10), and the contributions of these systems to
disturbances in Ca2+ homeostasis will have to be
investigated in future studies.
Our current observations provide support for the hypothesis (7, 10)
that MH and CCD mutants have enhanced permeability and that
compensatory mechanisms such as enhanced SERCA synthesis are brought
into play to restore Ca2+ homeostasis. The synthesis of
SERCA1 is enhanced in myoblasts by elevated Ca2+ (45, 46).
Our results confirm that SERCA2b expression is enhanced in transfected
HEK-293 cells and show that the most leaky CCD mutant channel (Y523S)
induces the highest overexpression of endogenous SERCA2 (Fig. 4). The
correlation between higher permeability and enhancement of
Ca2+ stores, however, was not perfect, however. We observed
only slightly higher synthesis of SERCA2b for the MH and CCD mutants
than for wild type RyR1. This may simply reflect the fact that our
observations were made only 48 h after transfection, a period too
short for equilibrium to be established. One of the striking
morphological features of CCD muscle is a proliferation of the
sarcotubular system in the core (47). The development of such cores
occurs over a period of months or even years, so that compensatory
mechanisms may require a long time to reach equilibrium.
In Fig. 7, we illustrate the
Ca2+ concentration changes that we have observed in our
studies of the size of the ER Ca2+ stores and resting
cytosolic Ca2+ concentrations in cells transfected with
wild type, MH, or CCD mutant RyR1. The expression of wild type RyR1
increases resting cytosolic Ca2+ concentrations. In
response to increased cytosolic Ca2+ concentration, the
transfected cells increase SERCA2b expression, increasing the potential
to store more ER Ca2+. This potential is realized as a
higher Ca2+ store for the cells transfected with wild type
RyR1. The MH mutant R615L is more leaky than wild type RyR1, resulting
in an elevated resting Ca2+ concentration and enhanced
SERCA2b synthesis, but the potential for an increased Ca2+
store is not realized because Ca2+ flux out of the store is
higher than Ca2+ flux into the store. A lower ER
Ca2+ store results. For the same reasons, the CCD mutant,
Y523S, being even more leaky, results in higher resting
Ca2+ concentration and an even higher SERCA2b synthesis,
but because Ca2+ fluxes are not balanced, the
Ca2+ store is even lower. The result is that the cells
transfected with a CCD mutant have the highest resting Ca2+
concentration and the lowest ER Ca2+ store. These results
support the view that MH and CCD mutants can result in a spectrum of
phenotypes ranging from muscle hypertrophy, induced by spontaneous
Ca2+ leaks, to muscle atrophy, caused by
Ca2+-induced damage to the core of the muscle cell where
compensatory function is least effective (7, 9, 10).
Influence of Extracellular Ca2+ Concentration on
Intracellular Ca2+ Homeostasis--
Extracellular
Ca2+ concentrations were not considered when we compared
caffeine or halothane sensitivities among wild type, MH, and CCD mutant
RyR1 because we were measuring Kactivation properties of different mutant proteins. It became a concern, however,
when the size of the ER Ca2+ store was measured. Under the
conditions of these experiments, we found that extracellular
Ca2+ has a profound effect on caffeine-induced
Ca2+ release and ER Ca2+ stores (Fig. 6). It is
impossible to obtain an accurate RyR1 caffeine ED50 in a
Ca2+-free medium, because cytosolic Ca2+ is
lost to extracellular spaces after it is released from the ER.
Accordingly, caffeine ED50 measured in
Ca2+-free medium reflected not only the sensitivity of the
RyR1 channel but also the size of the ER Ca2+ store in the
transfected cells. The lower caffeine sensitivity in
Ca2+-free medium than in Ca2+-containing medium
indicates that resting Ca2+ concentrations can influence
RyR1 caffeine sensitivity.
In this study, we have identified at least three factors that influence
the RyR1 caffeine response. These are the sensitivity of RyR1 proteins,
the size of the releasable ER Ca2+ store, and the resting
Ca2+ concentration. Several laboratories have shown that MH
mutants are more sensitive to a variety of channel activators,
including Ca2+, ATP, caffeine, and halothane, than wild
type (9, 22, 38, 40-44) and are less sensitive to Mg2+
(48). Thus the main cause of MH is the hypersensitivity of RyR1 mutant
proteins to very basic stimulus, but the occurrence of MH in humans may
also be influenced by the size of the Ca2+ store and
resting Ca2+ concentration which may be modulated by a
system of regulatory proteins in skeletal muscle.
The present results were obtained with HEK-293 cells transfected with
wild type and MH/CCD mutant RYR1 constructs, and the data
were obtained 48 h after transfection. HEK-293-transfected cells
differ from skeletal muscle cells in that they lack many of the
proteins that may modulate myoplasmic Ca2+ concentrations.
The advantage of the HEK-293 cell expression system is that it permits
the expression of homozygous and heterozygous MH/CCD mutants in a
homogeneous genetic background. This has facilitated the detection of
functional differences between normal and abnormal channels, which may
not be detectable in native skeletal muscle samples. It is difficult or
impossible to obtain substantial quantities of human MH mutant muscle,
and most human samples are from heterozygotes where it is more
difficult to detect small changes in RyR1 function. Moreover,
differences between MH/CCD and normal samples might be too small to be
detected due to the compensatory effects of other Ca2+
regulatory systems unique to skeletal muscle cells.
The absence of potential Ca2+ regulatory systems in HEK-293
cells is a disadvantage that must be weighed against the advantage of
cleaner analysis of the Ca2+ release channel. It would be
of interest, if cell lines could be established, to observe the long
term compensatory effects that are probably missing in transiently
transfected HEK-293 cells. Despite the differences in the assay
systems, it is reasonable to believe that MH/CCD mutants, which are
more leaky in HEK-293 cells, are also more leaky in skeletal muscle cells.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Rmin)/(Rmax
R))·(Sf2/Sb2). The Rmax value of 16 was obtained using 10 µM ionomycin. Rmin was determined
to be 0.4, and the proportionality constant
(Sf2/ Sb2) was determined to be 15.2. A value
of 224 nM was used for the apparent Kd
of Ca2+ binding to Fura-2 (30).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Comparison of resting Ca2+
concentration and caffeine-induced Ca2+ release in HEK-293
cells transfected with wild type and MH/CCD mutant RYR1
constructs. Cells were loaded with 4 µM Fura-2/AM
about 48 h after transfection, placed on the stage of an inverted
microscope, and stimulated with 10 mM caffeine. The changes
in Fura-2 fluorescence, presented as the ratio of fluorescence at
340/380 nm, were recorded with a CCD camera and converted to cytosolic
Ca2+ concentrations. Cells responding to caffeine were
regarded as RYR1-transfected cells, and cells not responding
were regarded as untransfected cells. Resting Ca2+
concentrations (A) and stimulated Ca2+
concentrations (B) in cells transfected with wild type and
MH/CCD mutant RYR1 constructs were measured by
Ca2+ imaging. Maximal 340/380 ratio changes (C)
were measured by Ca2+ photometry.
0.31,
p = 0.24) or with maximal caffeine-induced
Ca2+ release (r =
0.31, p = 0.24). Surprisingly, a linear correlation was observed between
ED50 and maximal caffeine-induced Ca2+ release
(r = 0.63, p < 0.05) and between
ED50 and maximal 340/380 nm ratio change (r = 0.67, p < 0.05, data not shown). A linear correlation analysis was also carried out between maximal
caffeine-induced Ca2+ release for 9 MH/CCD mutants
obtained in this study and caffeine-induced muscle tension or caffeine
threshold obtained through in vitro contracture test in an
earlier study (19). A linear correlation was observed between maximal
caffeine-induced Ca2+ release and caffeine-induced muscle
tension (r =
0.83, p < 0.05) and
between maximal caffeine-induced Ca2+ release and caffeine
threshold (r = 0.83, p < 0.05).
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Fig. 2.
Correlation among drug-induced
Ca2+ release, measured by Ca2+ imaging, in
HEK-293 cells transfected with wild type RYR1
(A-C) and individual MH/CCD mutants
(D). The linear analysis method was used to analyze
the following: the correlation between Ca2+ release induced
by carbachol, an agonist of an endogenous muscarinic receptor which
releases IP3 and acts on an IP3 receptor, and
by caffeine, a drug which acts directly on RyR1 (A); the
correlation between Ca2+ release induced by thapsigargin,
an irreversible SERCA inhibitor, and by caffeine (B); the
correlation between Ca2+ release induced by carbachol and
thapsigargin (C); and the correlation between mean
thapsigargin- and caffeine-induced Ca2+ release values in
MH/CCD mutant homotetramers and heterotetramers (D).
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Fig. 3.
Comparison of carbachol-induced
Ca2+ release in untransfected and transfected HEK-293
cells. Ca2+ release induced by 20 µM
carbachol was measured by Ca2+ imaging and regarded as an
indicator of the size of the ER Ca2+ store.
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Fig. 4.
SERCA2 contents measured by ELISA
(A) and 45Ca2+uptake
(B) in untransfected and transfected HEK-293 cells.
SERCA2 contents in microsomes (1 mg/ml) from untransfected control
cells or transfected HEK-293 cells were measured in both ELISA (50-µl
aliquots) and 45Ca2+ uptake (20-µl aliquots)
assays.
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Fig. 5.
Comparison of resting Ca2+
concentrations and caffeine-induced Ca2+ release in HEK-293
cells transfected with wild type and MH/CCD mutant RYR1
constructs to form homotetrameric or heterotetrameric RyR1 in the
presence or absence of SERCA1. HEK-293 cells were transfected with
wild type or MH/CCD mutant RYR1 cDNA to obtain
homotetrameric RyR1 or cotransfected with wild type and CCD or MH
mutant RYR1 constructs in a 1:1 molar ratio to obtain
heterotetrameric RyR1. Caffeine ED50 (A) and
maximal 340/380 nm fluorescence ratio changes (B) were
measured by Ca2+ photometry. Resting Ca2+
concentrations (C) and 10 mM caffeine-induced
Ca2+ release (D) in cells transfected with the
DNA constructs indicated were measured by Ca2+
imaging.
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Fig. 6.
Ca2+ concentration changes
induced by caffeine in RYR1-transfected cells in
Ca2+-containing and Ca2+-free media
(A) and caffeine dose-response curves for wild type and
MH/CCD mutant RyR1 in Ca2+-free medium
(B). A, trace of the response of
RYR1-transfected cells to caffeine in Ca2+-free
or Ca2+-containing media recorded by Ca2+
photometry; B, dose-response curves were obtained by
Ca2+ photometry of HEK-293 cells transfected with wild type
RYR1 or CCD (Y523S) or MH (R615L) mutant RYR1.
About 48 h after transfection, the cells were loaded with 1 µM Fura-2/AM for 30 min in Ca2+-free media
and stimulated with different concentrations of caffeine. The amplitude
of each peak caffeine response was normalized to the amplitude of the
peak caffeine response. Changes in the fluorescence ratio are presented
as R = (R
Rmin)/(Rmax
Rmin), where R refers to the 340/380
nm fluorescence ratio at each caffeine concentration, and
Rmin and Rmax refer to
the fluorescence ratio under resting conditions and at the highest
response to caffeine. R is plotted as a function of caffeine
concentration.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 7.
A model for changes in resting cytosolic and
ER lumenal Ca2+ concentrations in untransfected HEK-293
cell and cells transfected with wild type or MH or CCD mutant
RYR1. The expression of wild type RyR1 in HEK-293
cells increases resting cytosolic Ca2+ concentrations,
probably by the enhanced permeability of ER Ca2+ stores. In
an effort to compensate for increased Ca2+ release to
restore Ca2+ homeostasis, the HEK-293 cells express more
SERCA2b, increasing the potential for more Ca2+ storage.
Transfection with MH mutant RYR1 increases permeability of the ER
Ca2+ store even more, so that ER Ca2+ stores
begin to be depleted. Resting cytosolic Ca2+ concentrations
are higher, and ER Ca2+ stores are lower in CCD mutant
RYR1-transfected cells, because of the high
permeability of the CCD mutant RyR1 proteins.
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FOOTNOTES |
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* This work was supported in part by grants (to D. H. M.) from the Medical Research Council of Canada, the Muscular Dystrophy Association of Canada, and the Canadian Genetic Diseases Network of Centers of Excellence.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a studentship from the Medical Research Council of Canada.
** To whom correspondence should be addressed: Banting and Best Dept. of Medical Research, University of Toronto, Charles H. Best Institute, 112 College St., Toronto, Ontario, Canada M5G 1L6. Tel.: 416-978-5008; Fax: 416-978-8528; E-mail: david.maclennan{at}utoronto.ca.
The abbreviations used are: MH, malignant hyperthermia; CCD, central core disease; ER, endoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; ELISA, enzyme-linked immunoabsorbent assay; PBS, phosphate-buffered saline; GFP, green fluorescent protein; RyR, ryanodine receptor; IP3, inositol 1,4,5-trisphosphate.
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