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(Received for publication, July 17, 1997)
From the ABSTRACT Malignant hyperthermia (MH) and central
core disease (CCD) are autosomal dominant disorders of skeletal muscle
in which a potentially fatal hypermetabolic crisis can be triggered by
commonly used anesthetic agents. To date, 17 mutations in the human
RYR1 gene encoding the Ca2+ release channel of
skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) have
been associated with MH and/or CCD. Although many of these mutations
have been linked to MH and/or CCD, with high lod (log of the odds
favoring linkage versus nonlinkage) scores, others have been found in
single, small families. Independent biochemical evidence for a causal
role for these mutations in MH is available for only two mutants.
Mutations corresponding to the human MH mutations were made in a
full-length rabbit RYR1 cDNA, and wild type and mutant
cDNAs were transfected into HEK-293 cells. After about 48 h,
intact cells were loaded with the fluorescent Ca2+
indicator, fura-2, and intracellular Ca2+ release, induced
by caffeine or halothane, was measured by photometry. Ca2+
release in cells expressing MH or CCD mutant ryanodine receptors was
invariably significantly more sensitive to low concentrations of
caffeine and halothane than Ca2+ release in cells
expressing wild type receptors or receptors mutated in other regions of
the molecule. Linear regression analysis showed that there is a strong
correlation (r = 0.95, p < 0.001) between caffeine sensitivity of different RYR1 mutants
measured by the cellular Ca2+ photometry assay and by the
clinical in vitro caffeine halothane contracture test
(IVCT). The correlation was weaker, however, for halothane
(r = 0.49, p > 0.05). Abnormal
sensitivity in the Ca2+ photometry assay provides
supporting evidence for a causal role in MH for each of 15 single amino
acid mutations in the ryanodine receptor. The study demonstrates the
usefulness of the cellular Ca2+ photometry assay in the
assessment of the sensitivity to caffeine and halothane of specific
ryanodine receptor mutants.
INTRODUCTION 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
(1-3). Central core disease (CCD) is a rare, non-progressive myopathy, presenting in infancy and characterized by hypotonia and proximal muscle weakness (4). An important feature of CCD is its close association with MH susceptibility (5).
Although diagnosis of CCD is made on the basis of the lack of oxidative
enzymatic activity in central regions of skeletal muscle fibers (6),
the only accepted diagnostic test for MH susceptibility in humans is
the North American caffeine halothane contracture test (CHCT) (7) or
its European counterpart, the in vitro contracture test
(IVCT) (8). These tests are based on the hypersensitivity of
contracture, induced in muscle strips obtained by biopsy, in the
presence of caffeine or halothane.
Early genetic linkage studies showed that a single amino acid mutation,
Arg615 To date, 17 point mutations have been described in human
RYR1. Amino acid residue 4 (Gly4) in the rabbit
RYR1 sequence, expressed in this study, is deleted in the
human RYR1 sequence, accounting for discrepancies in
numbering of these mutations in the two species. Twelve mutations have
been linked to human MH only (C35R, G248R, G341R, R552W, R614C, R614L, R2163C, V2168M, T2206M, G2435R, R2458C and R2458H), and five have been
linked to human CCD plus MH (R163C, I403M, Y522S, R2163H, and R2436H)
in several human families (11, 18-25,
27).3 These mutations clearly
fall in two distinct clusters in the linear sequence of RYR1
lying between residues 35 and 614 and between residues 2163 and
2458.
Of these mutations, only the human R614C mutation has been
characterized thoroughly in the form of the corresponding R615C mutation in swine. In this case, gating has been confirmed to be more
sensitive to caffeine and halothane than the normal Ca2+
release channel in tests utilizing vesicles (28), single channel assays
(29, 30), and cellular Ca2+ photometry (31, 32). The G2434R
mutation has been shown to have higher than normal
[3H]ryanodine binding in human MHS muscle, an indication
of its greater open probability (33). For the remainder of these
mutations, there is no biochemical evidence to support their causal
role in MH susceptibility. Strong linkage data are available for
several of the mutations, but others, such as the G248R mutation, found in the RYR1 gene in a single MH family of five members (11), or the I403M mutation, found in two members of a single, small CCD
pedigree (20), could represent rare polymorphisms rather than causal
mutations. A method of demonstrating defects in these and other
mutations that would associate them in a functional way with MH and/or
CCD is an important goal.
In this study, we have introduced each of 15 known MH mutations into a
rabbit RYR1 cDNA, expressed each mutation in HEK-293 cells, and measured the intracellular responses of wild type and mutated channels to caffeine and halothane. The caffeine and halothane sensitivities of RYR1 mutants, measured in the
Ca2+ photometry assay and by the clinical IVCT, were
compared. The study shows that each mutant protein is more sensitive to
the Ca2+-releasing effects of caffeine and halothane than
the wild type channel. A strong correlation was observed between the
caffeine sensitivities measured in the two assays, but a weaker
correlation was observed for halothane sensitivities.
EXPERIMENTAL PROCEDURES Enzymes for DNA manipulation were obtained from
Boehringer Mannheim, New England Biolabs, Promega, and Pharmacia
Biotech Inc. Tissue culture reagents were purchased from Life
Technologies, Inc. Monoclonal antibody 34C (34) 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, and ryanodine was obtained from AgriSystems
International (Windy Gap, PA). Halothane was from Fluka. All other
reagents were of reagent (or highest available) grade.
The cloning and expression
of the full-length rabbit skeletal muscle ryanodine receptor
(RYR1) cDNA has been described previously (35-37).
Inasmuch as all of our mutations are confirmed by sequence analysis, it
was necessary to develop a series of "cassettes" that can be
removed from full-length RYR1 for mutagenesis and sequencing
using standard procedures (38). As the first step, we divided
RYR1 cDNA into 11 cassettes by utilization of five endogenous unique restriction endonuclease sites and the introduction of five new unique sites at intervals of about 1500 bases, as shown in
Fig. 1B. The five endogenous restriction endonuclease sites
are marked above the long horizontal bar in Fig.
1B, and the five restriction endonuclease sites introduced
into RYR1 cDNA by site-directed mutagenesis are marked
beneath the long horizontal bar. The 5
In the introduction of the new restriction endonuclease sites by
mutagenesis, the HpaI (1686)/HpaI (7155) fragment
from RYR1 cDNA (pBS-RYR1) was subcloned into
pBS1 to yield plasmid pBS1-H. Two restriction endonuclease sites,
BstBI (3798) and MluI (5355), were introduced
into pBS1-H to yield pBS-HBM. The XhoI
(6600)/XhoI (12317) fragment from pBS-RYR1 was
subcloned into Bluescript SK II(+) to yield pBS-X. Two restriction
endonuclease sites, SpeI (6822) and AvrII (9804),
were then introduced into pBS-X to yield pBS-XSA. The NdeI
(11290)/ClaI (14427) fragment from pBS-RYR1 was
subcloned into pBS1 to yield pBS1-NC, and then the NdeI
(11290)/SacII (13015) fragment from pBS1-NC was subcloned
into pBS1 to yield pBS-NS. A restriction endonuclease site,
NheI (12675), was introduced into pBS1-NS to yield pBS1-NSN.
The three fragments from pBS1-HBM, pBS-XSA and pBS1-NSN, with five
newly introduced restriction endonuclease sites, were subcloned back
into pBS-RYR1 to yield pBS-RYR1c in the
Bluescript KS vector. The 11 cassettes were subcloned from pBS-RYR1c into pBluescript KS(+), pBS1, and pBS2 to yield
pBS-RYR1cs1-11 as follows: XbaI
(Bluescript)/SalI (677), SalI/Bsu36I
(2349), Bsu36I/BstBI (3798),
BstBI/MluI (5355),
MluI/SpeI (6822), SpeI/SplI (8461), SplI/AvrII (9804),
AvrII/NdeI (11290),
NdeI/NheI (12675), NheI/ClaI (14427), and
ClaI/HindIII (Bluescript). The full-length RYR1 cDNA, with introduced restriction endonuclease
sites (pBS-RYR1c) (Fig. 1B), could not be cloned
into the PMT2 vector, which has EcoRI, XbaI,
SalI, and EcoRI cloning sites. Accordingly,
PBS-RYR1c was digested with HindIII at a unique
site, blunt-ended, and digested with XbaI to obtain a
full-length XbaI-blunt fragment. This was cloned into the
mammalian expression vector pMT2 (a gift of Dr. R. Kaufman, Genetics
Institute, Boston, MA) after blunt-ending the SalI site and
digesting the XbaI site. Some mutant clones were introduced
directly into the XbaI-HindIII site of an
expression vector pcDNA3( Short fragments were
removed from pBS-RYR1cs1, pBS-RYR1cs2, or
pBS-RYR1cs6, as indicated by the small bar in
Fig. 1C, and ligated into the polylinker region of
Bluescript, pBS1, or pBS2 for site-specific mutagenesis by
oligonucleotide-directed mutagenesis (39). The integrity of the mutated
segment of the cDNA insert was checked by sequencing the entire
insert by the dideoxynucleotide chain termination method of Sanger
et al. (40). Fragments containing mutations were subcloned
back into their original position in pKS-RYR1 or pKS-RYRc
vectors for expression in HEK-293 cells. Mutations R164C, G249R, G342R,
I404M, Y523S, and R615C were subcloned into the original
pMT2-RYR1 vector, and mutations G2435R and R2436H were
subcloned into the pMT-RYR1c vector. Mutations C36R, R553W, R615L, R2163C, R2163H, R2458C, or R2458H were subcloned into
pcDNA3( HEK-293 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
0.1 mM minimal Eagle's medium nonessential amino acids, 4 mM L-glutamine, 100 units of penicillin/ml, 100 mg of streptomycin/ml, 4.5 g of glucose/liter, and 10% fetal calf serum, at 37 °C under 5% CO2. DNA transfection was
carried out by the calcium phosphate precipitation method as described
by Chen and Okayama (41). 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.
Immunocytochemical staining was
carried out as described by Chen et al. (36). Monoclonal
antibody 34C (a kind gift from Dr. Judith Airey) and alkaline
phosphatase-conjugated anti-mouse IgG were used to detect expressed
ryanodine receptor protein in HEK-293 cells. 5-Bromo-4-chloro-3-indolyl
phosphate and p-nitrotetrazolium chloride blue (Sigma) were
used in the development of the color reaction.
Immunoblotting was performed on total
proteins collected from one 60-mm plate of whole cells using detergent
solution, separated by sodium dodecyl sulfate-slab gel electrophoresis
(42), and transferred to nitrocellulose (43). The blots were incubated first with monoclonal antibody 34C at a dilution of 1:500 and then with
horseradish peroxidase-conjugated anti-mouse secondary IgG at a
dilution of 1:5000 in a solution containing 10 mM Tris-HCl, pH 7.5, 1% BSA, 150 mM NaCl, and 0.1% Tween-20). Finally,
the blots were incubated with the SuperSignal ultra chemiluminescent substrate (Pierce) and exposed to a BioMax film (Eastman Kodak Co.).
A Photon Technologies Inc. (PTI)
microfluorimetry system was used to measure the effect of caffeine on
Ca2+ release by the different ryanodine receptor proteins
expressed in HEK-293 cells. Briefly, cell culture and DNA transfection
were carried out as described above. Growth medium was replaced 20 h after transfection, and Ca2+ photometry was carried out
on intact cells about 48 h after transfection (44). The cells were
loaded with 1 µM fura-2 AM, 0.02% pluronic F-127 for 30 min at pH 7.4, and room temperature in a physiological medium (KRH)
containing, in mM: NaCl, 125; KCl, 5; MgSO4
1.2; KH2PO4, 1.2; CaCl2, 2;
glucose, 6; Hepes-NaOH buffer, 25 (45). The glass coverslips were
placed in a holder on the stage of an inverted Diaphot microscope
(Zeiss Canada). The samples were excited alternately at 340 nm and 380 nm using a dual monochromator. The emitted fluorescence was filtered
through a 510-nm filter, before feeding into the photomultiplier tube.
Averaged light intensities over excitation periods of 0.05 s at
each of the two wavelengths were used to calculate the 340/380 nm ratio
using Felix (PTI) software. Ca2+ transients are presented
as the ratio of fura-2 emission with no attempt to calculate
[Ca2+]i. Bathing solutions containing incremental
levels of caffeine or halothane were added on one side of the stage and aspirated on the opposite side. Each solution was then washed out over
a period of a few minutes before addition of the next increment of
releasing buffer. Dose-response curves were generated and normalized to
the maximal release response observed at 10 mM caffeine for
both the caffeine and the halothane responses.
The standard European IVCT protocol (8) was used in
all diagnoses provided. The laboratory of origin of these tests and their detailed analysis is provided by Manning et
al.3
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.
RESULTS The
cloning, sequencing, and functional expression of RYR1
cDNA have been reported previously (35-37). Fig.
1A shows the approximate positions of 15 mutations in the human ryanodine receptor protein which
have been linked to MH (11, 18-25, 27, 46).3 Fig.
1B shows the location of these 15 mutations in
RYR1 cDNA, relative to the 11 cassettes that were
created in the cDNA to facilitate excision and mutagenesis. Fig.
1C shows the subcloned fragments that were used for
site-directed mutagenesis. The figure illustrates the way in which the
cDNA was reduced in size for mutant construction and rebuilt to
form full-length cDNA.
The transient expression of a fully functional RYR1 cDNA
in HEK-293 cells and characterization of the product have been
described (37). We used this transient expression system in this study. Fig. 2 shows immunoblotting of
representative regions of expression vector-transfected
(A) and RYR1 cDNA-transfected (B)
HEK-293 cells stained with a mouse monoclonal antibody (34C) against
the ryanodine receptor. The epitope for this antibody lies between
amino acids Asn2756 and Glu2803 near the middle
of the ryanodine receptor. There was no obvious difference in
transfection efficiency between wild type and MH mutant RYR1
cDNA-transfected cells. Immunostaining was used to obtain an
estimate of the transfection efficiency. In regions containing about
10-50 cells around the edges of islands of confluent cells, 60 ± 10% (n = 10) of the cells stained positively for
ryanodine receptor protein. In subsequent experiments, similar regions
were chosen for cellular Ca2+ photometry.
Western blotting after SDS-PAGE of whole cell extracts of transfected
and non-transfected HEK-293 cells (Fig. 2C) showed that there were no obvious differences in the levels of expression or of
molecular size between wild type and mutant ryanodine receptors.
In control experiments, HEK-293 cells were transfected with different
amounts of RYR1 cDNA (e.g. 1 µg of
RYR1 cDNA plus 3 µg of vector or 4 µg of
RYR1 cDNA/35-mm plate). The expression of
RYR1 protein, monitored by Western blotting, differed, but no significant difference was observed in transfection efficiency, monitored by immunostaining, or in caffeine response, monitored by the
cellular Ca2+ photometry, indicating that transfection
efficiency was not improved by higher cDNA levels and that the
number of expressed channels required for a full caffeine response in
an individual cell was reached at low cDNA concentrations.
Fig.
3A shows representative
fluorometric responses to incremental additions of caffeine, and Fig.
3B shows representative fluorometric response to incremental
doses of halothane for vector-transfected HEK-293 cells and for cells
transfected with the wild type Ca2+ release channel, the MH
mutant channel G2435R and the CCD mutant channel R2436H. Traces from
representative experiments in which fura-2 fluorescence from about 50 HEK-293 cells were monitored after the addition of increasing
concentrations of caffeine are shown in Fig. 3A. Under
resting conditions, there was no significant difference in the
fluorescence ratio measured in vector-transfected HEK-293 cells
and in RYR1 cDNA-transfected HEK-293 cells.
Caffeine from 1 to 10 mM caused a small increase in
base-line fluorescence in vector-transfected HEK-293 cells, but no
clear [Ca2+]i transients were observed. Peak
heights in the fluorescence ratio measuring Ca2+ release in
HEK-293 cells transfected with the G2435R and R2436H mutant cDNAs
indicated that these mutants were sensitive to lower concentrations of
caffeine (0.25-1 mM) and halothane (0.1-0.5 mM) than HEK-293 cells transfected with wild type
RYR1 cDNA. Removal of caffeine by washout allowed the
Ca2+ concentration to return to near resting levels over a
period of about 2 min. Peak heights were measured for each increment in
caffeine and halothane concentration and normalized to the peak height
for maximal Ca2+ release in the case of caffeine, or to the
response obtained with 10 mM caffeine in the case of
halothane. Caffeine ED50 values, calculated from the
dose-response curves generated in this way for wild type, G2435R, and
R2436H mutant channels, are presented in Fig. 3C. They were
1.4, 0.84, and 0.89 mM, respectively.
The traces in Fig. 3B show that vector-transfected HEK-293
cells did not respond to halothane concentrations below 5 mM, but did respond weakly to 10 mM halothane.
In HEK-293 cells transfected with wild type channels, weak responses
were observed at 0.1 and 0.25 mM halothane, but cells
transfected with MH mutant channels (G2435R) and CCD mutant channels
(R2436H) gave robust responses to 0.1 and 0.25 mM
halothane. Inasmuch as vector-transfected HEK-293 cells all responded
to 10 mM halothane, it was not possible to determine a
maximal response to halothane in RYR1-transfected cells.
Accordingly, the effects of halothane were normalized to the maximal
response to caffeine, which was achieved at 10 mM caffeine.
The ED50 values for halothane activation of wild type, G2435R, and R2436H channels, presented in Fig. 3D, were
0.56, 0.28, and 0.29 mM, respectively.
In Fig. 4A, mean
ED50 values for caffeine activation of Ca2+
release are presented for wild type and 15 mutant Ca2+
release channel proteins expressed in HEK-293 cells. In Fig. 4B, mean ED50 values are presented for halothane
activation of Ca2+ release through the same channels. Of
the 15 mutations linked to MH, Ca2+ release in 14 was
significantly more sensitive to lower concentrations of caffeine (Fig.
4A) and halothane (Fig. 4B) than was the wild type channel, when analyzed by cellular Ca2+ photometry.
The exception was the mutant C36R.
IVCT data for nine
different RYR1 mutations have been collected,3
making it possible to compare our test results with the IVCT
data for the nine different mutations (Fig. 4). Correlations between caffeine and halothane responses measured by cellular Ca2+
photometry and by IVCT were subjected to linear analysis. There was a
correlation (coefficient of correlation = 0.79, p < 0.05) between ED50 values for caffeine and halothane,
measured by cellular Ca2+ photometry (Fig. 4E),
but there was no clear correlation between the caffeine and halothane
threshold measured by IVCT (Fig. 4F). This result,
indicating that a muscle biopsy from a MHS individual might have a
strong response to caffeine but not to halothane, must be considered
cautiously, however, because a single data point, the IVCT halothane
response to the R2458C mutation, is an outlier. This mutation was
relatively insensitive to caffeine in both assays and to halothane in
the cellular Ca2+ photometry assay, but was highly
sensitive to halothane in the IVCT (Fig. 4). There was a strong
correlation (r = 0.95, p < 0.001) between the IVCT caffeine threshold and the caffeine ED50,
measured in the cellular Ca2+ photometry assay (Fig.
4G), but not between the IVCT halothane threshold and
halothane ED50 (Fig. 4H), measured by cellular
Ca2+ photometry.
DISCUSSION In this study, we demonstrate the use of HEK-293 cells for the
functional expression and analysis of wild type and MH or CCD mutant
Ca2+ release channels. In our earliest studies, we
encountered difficulty in the characterization of the expressed
Arg615 The single channel properties of the recombinant Ca2+
release channel, expressed in HEK-293 cells under the exact conditions used in this study, have been shown, in extensive analyses, to be
virtually identical to the native channel (37). Although endogenous
forms of Ca2+-induced Ca2+ release channels,
sensitive to ryanodine, have been reported in HEK-293 cells (47), we
have been unable to detect any endogenous skeletal muscle ryanodine
receptor in HEK-293 cells using several ryanodine receptor-specific
antibodies, including 34C, in immunostaining and immunoblotting of
HEK-293 cell extracts (37). We have, however, noted the occasional
burst of Ca2+ release while imaging single HEK-293 cells.
Accordingly, it was important to carry out photometry with a large
number of HEK-293 cells so that spurious bursts of Ca2+
release were minimized by averaging with unresponsive cells. The larger
signal also facilitated the detection of a response to the addition of
very low concentrations of caffeine or halothane to
RYR1-transfected cells. Dose-response curves were readily
generated from the continuous photometric recording.
Among the problems that we have considered in the refinement of this
assay system was the question of whether differences in transfection
efficiency might affect our results. Optimal transfection conditions
were determined on the basis of measurement of maximal transfection
efficiency and photometric response. If photometric measurements were
carried out on different regions of the same coverslip, peak caffeine
and halothane responses did vary from one region of the slide to
another, on occasion up to 3-fold. However, ED50 values for
caffeine and halothane responses did not change, because curves from
which ED50 values were derived were calculated after
normalization to maximal release values obtained with 10 mM
caffeine in the same field.
The question of whether the volatility of halothane could affect
dose-response results was considered. Liquid halothane, diluted to 500 mM in dimethylsulfoxide (DMSO), was diluted further in aqueous assay solutions to achieve the specific concentrations of
halothane used in cellular Ca2+ photometry. The same
diluted halothane solution was tested at 40-min intervals on HEK-293
cells transfected with RYR1 cDNA. Inasmuch as no changes
were observed in the amplitude of the halothane-induced contracture, we
conclude that evaporation of halothane is not a serious problem in our
cellular Ca2+ photometry assay.
In other, unpublished experiments involving mutations unrelated to MH
or CCD, in other regions of RYR1, we have not observed any
increased sensitivity to caffeine and
halothane.4
We have analyzed the ligand gating characteristics of 15 mutations
created in rabbit skeletal muscle ryanodine receptor, which are
associated with MH or CCD in the corresponding human Ca2+
release channel. Our data demonstrate that 14 of the mutant ryanodine receptor proteins respond significantly to abnormally low
concentrations of caffeine and halothane. The abnormal Ca2+
transients in transfected HEK-293 cells are most likely to be due to
intrinsic differences in the gating properties of the mutated RYR1.
Comparison of the caffeine responses for the various RYR1
mutations obtained by cellular Ca2+ photometry and by the
muscle biopsy IVCT shows that there was an excellent correlation
between both responses (Fig. 4). The correlation is surprisingly good,
because the caffeine data were obtained from different laboratories
across Europe over a period of several years.3 The close
correlation that we observed demonstrates that the cellular
Ca2+ photometry system described should be considered a
valuable predictor of RYR1 MH mutations.
In the cellular Ca2+ photometry assay, a significant
correlation was observed between sensitivity to caffeine and
sensitivity to halothane (Fig. 4E). However, the correlation
between the halothane response in the cellular Ca2+
photometry assay and the halothane response in the IVCT was not significant (Fig. 4H). This is not surprising, inasmuch as a
poor correlation also exists between the caffeine and halothane
responses obtained for the IVCT (Fig. 4F).
The poor correlation between the halothane responses in the cellular
Ca2+ photometry assay and in the IVCT could result from the
different nature of the cellular and muscle biopsy tests.
Alternatively, such results could arise from experimental deviation in
halothane responses in the IVCT. There are three main points that argue in favor of the latter explanation. (i) A good correlation was observed
between the halothane and caffeine response in the cellular assay but
not in the IVCT; (ii) in the IVCT, there was a poor correlation between
halothane threshold and tension values3; and (iii) the
incidence of people responding abnormally to halothane, but not to
caffeine, in the IVCT (MHE/h) differs across different centers in
Europe. Thus, it is possible that the halothane response of different
RYR1 mutations may be more accurately measured in the
cellular assay than in the IVCT.
The IVCT responses shown were collected from heterozygous individuals,
whereas the cellular assay system would be expected to mimic a
homozygous response. Interestingly, the ED50 values for
caffeine and halothane are generally half the threshold values for
caffeine and halothane in the IVCT. This further supports the notion
that the cellular assay gives a good representation of the IVCT
response. The results of this study add to a growing body of
biochemical and genetic evidence that mutations in the RYR1
gene are causal of malignant hyperthermia.
A serious problem in the diagnosis of MH by in vitro
contracture testing is that the tests can attain close to 100%
sensitivity, but with no more than 93% specificity (17).2
The 10-20% false positives and the few false negatives that are observed with the test are not likely to be due to technical error. It
is much more likely that they arise because the contracture test
results record the interplay of multiple gene products in the muscle
strips which are assayed. Moreover, it is clear that muscle cells have
the capacity to compensate for genetic changes that might otherwise be
lethal (16, 26, 48). Accordingly, CHCT results in a family will reflect
not only the effect of the RYR1 mutation causing MH, but the
different genetic background of each individual, the level of
"compensation" achieved in that individual, gender differences
(more MH reactions are recorded in males than in females) and age
differences (MH susceptibility peaks in the second to fourth decades)
(1).
Because of these diagnostic difficulties, it is important to consider
the possibility that the CHCT should be replaced, wherever possible, by
DNA based tests for MH. Such tests would easily achieve 100% accuracy.
One hindrance, however, has been to define which RYR1
mutations are, in fact, causal of MH and which are merely polymorphisms. It is clear now that linkage data between
RYR1 mutations and MH will not be tight because of the
limitations of the IVCT
(15).5 Accordingly, we
propose that an alternative is to test each MH mutation, as they are
discovered, for their sensitivity to caffeine and halothane (and
possibly 4-chloro-m-cresol and ryanodine), in the context of
the common genetic background provided by HEK-293 cells. If any
mutation is shown to result in higher sensitivity than wild type to the
Ca2+ releasing effects of caffeine and halothane, then any
individual carrying such a mutation should be considered to be
MH-susceptible and treated accordingly during administration of
anesthetics.
FOOTNOTES ACKNOWLEDGEMENTS We thank Dr. Wayne Chen and Peng Leong for
helpful advice and discussion, Claire Bartlett for technical
assistance, and Stella de Leon for the synthesis of oligonucleotide
primers.
REFERENCES
Volume 272, Number 42,
Issue of October 17, 1997
pp. 26332-26339
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Caffeine and Halothane Sensitivity of Intracellular
Ca2+ Release Is Altered by 15 Calcium Release Channel
(Ryanodine Receptor) Mutations Associated with Malignant Hyperthermia
and/or Central Core Disease*
§¶,
, and§§§
Banting and Best Department of Medical
Research, University of Toronto, Charles H. Best Institute, Toronto,
Ontario M5G 1L6, Canada, the § Department of Biochemistry,
University of Toronto, Medical Sciences Building, Toronto, Ontario M6S
1A1, Canada, the ** Division of Cell Biology, The Research Institute,
Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, and the
Department of Biochemistry, University
College, Cork, Ireland
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Cys (R615C), in the skeletal muscle ryanodine
receptor gene (RYR1) is tightly linked to MH in swine (8,
9). Further genetic and biochemical data have supported the view that
this mutation is causal of MH in swine and humans (2). The
corresponding mutation (R614C) has been linked to MH in some human
families (11, 12), but in other families, discordance has been reported (13, 14). Discordance raises questions concerning the causal nature of
such mutations, but other reasons for discordance include the
possibility that two MH mutations might be segregating in one family or
that the in vitro contracture test for MH susceptibility fails to provide a phenotypic diagnosis of MH with sufficient accuracy
for genetic analysis (15). Analysis of the CHCT as a diagnostic test
for MH indicates that it can achieve 97% sensitivity and 79%
specificity,2 whereas the
IVCT can achieve 99% sensitivity and 93.6% specificity (17). The IVCT
protocol permits an equivocal diagnosis (MHE) if the muscle biopsy
responds to either caffeine or halothane, but not both.
XbaI site and the 3 HindIII site were unique
sites introduced into the clone earlier (36) (Fig. 1B). To
work with these cassettes, two new vectors, pBS1 and pBS2, were also
constructed by the introduction of additional restriction endonuclease
sites into the multiple cloning site between BamHI and
Sma I in pBluescript II KS(+) (Stratagene). The multiple
cloning site in pBS1 contains BamHI-BglII-NdeI-StuI-AatII-BspMII-HpaI-SmaI
and, in pBS2, contains BamHI-NdeI-MluI-Avr
II-BstBI-SplI-Bsu36I-NheI-SmaI
restriction endonuclease sites.
Fig. 1.
Construction of mutations associated with MH
or CCD in rabbit skeletal muscle RYR1 cDNA.
A, the linear amino acid sequence of the ryanodine receptor
is indicated by a solid horizontal line in which the
NH2 and COOH termini and amino acid numbers are marked. The
approximate positions in which the MH mutations are clustered are
depicted as boxes above the horizontal line.
B, the construction of 11 DNA cassettes of about 1500 base
pairs each, used for mutagenesis of RYR1 cDNA. Five
unique restriction sites found in the cDNA are indicated
above the horizontal line, and five more unique restriction endonuclease sites built into the cDNA are indicated below the line. The 5 XbaI site and
the 3 HindIII site are also unique. C, cassettes
used for mutant constructs were derived by further cleavage of the
large cassettes as indicated. After mutagenesis, the small,
mutant-containing cassettes were sequenced and built back into a
full-length cDNA.
[View Larger Version of this Image (27K GIF file)]
) (Invitrogen, San Diego, CA).
) vector. The expression of wild type cDNA in all
these vectors yielded virtually identical expression levels and
activities.
Fig. 2.
Expression of the wild type and 15 mutant
skeletal muscle RYR1 cDNAs in HEK-293 cells.
HEK-293 cells transfected with vector alone (A) or RYR1
cDNA (B) were subjected to immunocytochemical staining
for ryanodine receptor expression. Cells were fixed and permeabilized
about 48 h after transfection. Expressed ryanodine receptors were
detected by immunochemical staining using monoclonal antibody 34C and
secondary alkaline-phosphate-conjugated anti-mouse IgG. C,
immunoblots of HEK-293 cell extracts 48 h after transfection with
wild type and MH mutant constructs. Samples of whole cell extracts
containing about 100 µg of protein were separated by 7.5%
SDS-polyacrylamine gel electrophoresis and transferred to nitrocellulose. The samples were probed with monoclonal antibody 34C
and secondary horseradish peroxidase-conjugated anti-mouse IgG.
Microsomes extracted from rabbit skeletal sarcoplasmic reticulum were
used as a positive control.
[View Larger Version of this Image (104K GIF file)]
Fig. 3.
Triggering of Ca2+ release by
caffeine (A and C) and halothane (B
and D) into the cytoplasm of HEK-293 cells transfected with
vector alone or with wild type or mutant RYR1
cDNAs. Traces of the response of transfected HEK-293 cells to
caffeine (A) and halothane (B), and corresponding
response curves for incremental doses of caffeine (C) and
halothane (D) are shown. HEK-293 cells were transfected with
wild type, G2435R (MH), or R2436H (MH + CCD) RYR1
constructs. The cells were loaded with 1 µm Fura-2-AM about 48 h
after transfection, placed on the stage of an inverted microscope, and
stimulated with different concentrations of caffeine (A) or
halothane (B). The changes in fura-2 fluorescence, which are
recorded as the ratio of fluorescence at 340/380 nm, indicate changes
in cytosolic Ca2+ concentrations. To compare the extent of
Ca2+ release from normal and mutant Ca2+
release channels, the amplitudes of initial peak responses were normalized to the maximum amplitude of the peak response in the fura-2
fluorescence ratio caused by 10 mM caffeine. Halothane responses were also normalized to the 10 mM caffeine
response. In C and D, changes in the fluorescence
ratio are presented as 
R = (R Rmin)(Rmax Rmin), where R refers to the 340/380
nm fluorescence ratio of fura-2 at each caffeine concentration and Rmin and Rmax refer to
the fluorescence ratio under resting conditions and at the highest
response to caffeine (in the case of caffeine) or to the 10 mM caffeine response (in the case of halothane). R is plotted as a function of caffeine or halothane
concentration. ED50 refers to the concentration of caffeine
or halothane required to reach the half-maximal response. All data are
expressed as mean ± S.D.
[View Larger Version of this Image (33K GIF file)]
Fig. 4.
Comparisons and correlations of the effect of
caffeine and halothane on wild type and MH or CCD mutant
Ca2+ release channels. With the exception of mutant
C36R, all MH mutants were significantly more sensitive to caffeine
(mean ED50 = 0.81 ± 0.15 mM) than wild
type RYR1 (ED50 = 1.4 ± 0.26 mM) (A), and all MH mutants were significantly
more sensitive to halothane (mean ED50 = 0.30 ± 0.10 mM) than wild type RYR1 (ED50 = 0.74 ± 0.20 mM) (B). Comparison of the
threshold concentration of caffeine (C) or halothane
(D) for activation of muscle contracture in the IVCT shows
that nine mutant RYR1 were more sensitive to caffeine (mean
threshold = 1.2 ± 0.4 mM) and halothane (mean
threshold = 0.83 ± 0.25% v/v) than wild type
RYR1. The linear analysis method was used to analyze the
correlation between the effects of caffeine and halothane obtained by
the cellular Ca2+ photometry assay (E) and the
IVCT (F) and to analyze the correlation between the effect
of caffeine (G) and halothane (H) obtained by the
two assays. One vertical dashed line indicates the mean value for wild type, and the other indicates the mean value for all
mutants. All data are expressed as mean ± S.E.
[View Larger Version of this Image (30K GIF file)]
Cys RYR1 mutant using cellular
Ca2+ photometry, because of a high background of
Ca2+ release in several different cell lines. This problem
was later solved by Otsu et al. (31), who used C2C12 cells
as host and demonstrated that the mutant Ca2+ release
channel is more sensitive than wild type to both caffeine and
halothane, and by Treves et al. (32), who used COS-1 cells to show that the mutant Ca2+ release channel is more
sensitive to 4-chloro-m-cresol than wild type.
¶
Supported by a studentship from the Medical Research Council
of Canada.
Supported by a postdoctoral fellowship from the Heart and
Stroke Foundation of Ontario. Current address: Dept. of Pharmacology, School of Medicine, Showa University, Tokyo 142, Japan.
§§
To whom correspondence should be addressed. Tel.: 416-978-5008;
Fax: 416-978-8528; E-mail: david.maclennan{at}utoronto.ca.
1
The abbreviations used are: MH, malignant
hyperthermia; CCD, central core disease; IVCT, in vitro
caffeine halothane contracture test; CHCT, caffeine halothane
contracture test.
2
Allen, G. C., Larach, M. G., and Kunselman, A. R., (1997) Anesthesiology, in press.
3
B. M. Manning, K. A. Quane, H. Ørding, A. Urwyler, V. Tegazzin, M. Lehane, J. O. O'Halloran, E. Hartung, L. M. Giblin, P. J. Lynch, P. Vaughan, K. Censier,
D. Bendixen, G. Comi, L. Heytens, K. Monsieurs, T. Fagerlund, W. Wolz,
J. J. A. Heffron, C. R. Müller, and T. V. McCarthy, submitted for publication.
4
J. Tong and D. H. MacLennan, unpublished
results.
5
J. C. P. Loke, and D. H. MacLennan, Am. J. Med., in press.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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