(Received for publication, October 1, 1996, and in revised form, November 19, 1996)
From the Malignant hyperthermia is an inherited autosomal
disorder of skeletal muscle in which certain volatile anesthetics and
depolarizing muscle relaxants trigger an abnormally high release of
Ca2+ from the intracellular Ca2+ store, the
sarcoplasmic reticulum. In about 50% of cases, malignant hyperthermia
susceptibility is linked to the gene encoding the skeletal muscle
ryanodine receptor/Ca2+ release channel (RYR1). To date,
eight point mutations have been identified in human RYR1. Although
these mutations are thought to lead to an increased caffeine and
halothane sensitivity in the contractile response of skeletal muscle,
their functional consequences have not been investigated on the
molecular level. In the present study, we provide the first functional
characterization of a point mutation located in the central part of
RYR1, Gly2434 Malignant hyperthermia (MH)1 is a
pharmacogenetic skeletal myopathy of humans and swine and is one of the
main causes of death due to anesthesia. Predisposed patients are at
high risk for undergoing a fulminant MH crisis when exposed to certain
volatile anesthetics and depolarizing muscle relaxants commonly used in
anesthesia. A point mutation (Arg615 Taq polymerase was purchased from
Pharmacia (Freiburg, Germany), and AlwNI was from New
England Biolabs (Schwalbach, Germany). A DNA preparation kit was
obtained from MWG-Biotech (Ebersberg, Germany).
(9,21-3H(N))Ryanodine was purchased from DuPont NEN (Bad
Homburg, Germany). Ryanodine was from Calbiochem (Bad Soden, Germany),
and protease inhibitors were from Boehringer (Mannheim, Germany).
Protein molecular mass standard was purchased from Bio-Rad
(München, Germany), and DNA size standard was from MBI Fermentas
(St. Leon-Rot, Germany). All other chemicals were of analytical grade.
Filter membranes for [3H]ryanodine binding were purchased
from Schleicher & Schüll (Dassel, Germany).
Skeletal muscle biopsies
(Musculus vastus lateralis) were taken from a patient who
had suffered from a typical MH crisis and from his relatives for the
test of susceptibility to MH. DNA was extracted from anticoagulated
blood of individuals from this pedigree found to be heterozygous for
the RYR1 Gly2434 Genomic DNA was
isolated from 10 ml of blood from MHN and MHS individuals using a DNA
preparation kit (MWG-Biotech). For analysis of mutation G7300A
predicting the Gly2434 to Arg substitution, flanking
primers as designed from the published sequence (14) (Ex45RyR sense
5 MHS muscle samples obtained by
biopsy from the M. vastus lateralis were collected from five
individuals carrying the RYR1 mutation Gly2434 SR vesicles (at a
protein concentration of 400 µg/ml) were incubated with indicated
concentrations of [3H]ryanodine in a medium containing
100 mM KCl, 100 µM EGTA, 20 mM
Na-PIPES, 200 µM Pefabloc, pH 6.8, for 3 h at
37 °C. Varying concentrations of Ca2+, calmodulin,
caffeine, and 4-chloro-m-cresol were added to the incubation
medium as indicated in the figure legends. Unbound ryanodine was
separated from protein-bound ryanodine by filtration of protein
aliquots (14 µg) through Schleicher & Schüll GF51 filters
pre-soaked in 1% polyethylenimine. Filters were washed three times
with ice-cold buffer solution as described above. Radioactivity
remaining with the filters was measured by liquid scintillation
counting. Specific binding was calculated as the difference of total
and nonspecific binding determined in the presence of a 1000-fold
excess of unlabeled ryanodine. Experiments were carried out in
duplicate.
Protein samples were
denatured in Laemmli buffer at 95 °C for 3 min and separated in
3.5-15% gradient SDS/polyacrylamide minigels. Gels were stained with
Coomassie Brilliant Blue. DNA fragments were separated in linear 10%
polyacrylamide gels. Gels were stained with ethidium bromide.
Free concentrations of
Ca2+ were calculated using the computer program and binding
constants described in Ref. 18. Dose-response curves were fitted using
nonlinear curve-fitting routines based on the Marquardt-Levenberg
algorithm. The data represent the means ± S.D. of two different
MHN and MHS SR preparations.
Human skeletal muscle specimens were obtained from patients who
underwent muscle biopsy for the test of susceptibility to MH. For this
purpose, the caffeine and halothane sensitivity of dissected fiber
bundles of biopsied muscle were tested according to the European IVCT
protocol. MHS samples were collected from individuals of one pedigree
heterozygous for the RYR1 Gly2434
Fig. 2 shows a Coomassie-stained SDS/polyacrylamide gel
that was loaded with aliquots of SR vesicles of MHN and MHS muscle samples. The overall gel pattern was not different for MHN and MHS
vesicles. RYR1s were separated as single high molecular mass bands of
an estimated size of about 450 kDa with no detectable degradation
products.
The [3H]ryanodine affinity of isolated SR vesicles from
MHN and MHS muscle was determined in the presence of an activating Ca2+ concentration of 10 µM. Scatchard
analysis revealed a single class of high affinity binding sites for
both tissues (Fig. 3). The affinity of MHN vesicles
(Kd = 47.0 ± 3.7 nM,
n = 4) was approximately 1.5-fold lower compared with
MHS vesicles (Kd = 31.7 ± 3.9 nM,
n = 2). No significant differences between MHN and MHS
vesicles were found for the maximal activation of
[3H]ryanodine binding (for
Bmax(MHN), 1.37 ± 0.12 pmol/mg protein (n = 4) versus 1.44 ± 0.05 pmol/mg
protein (n = 2) for
Bmax(MHS)).
The binding of [3H]ryanodine is greatly influenced by
ligands of RYR1 that activate or inhibit SR Ca2+ release
(20-22). Because the amount of MHS muscle was very limited (<2.2 g),
further analysis was restricted to the investigation of modulators for
which an abnormal sensitivity has been observed in MHS
(Arg615 Fig. 4 shows the dependence of high affinity
[3H]ryanodine binding on cytoplasmic Ca2+
concentration. Ca2+ activated [3H]ryanodine
binding more potently in MHS vesicles, but the maximum binding was
reached at 10 µM Ca2+ for both vesicle types.
Whereas the threshold of activation for MHN vesicles was around 1 µM Ca2+ (pCa = 6), binding to MHS
vesicles was distinctly activated by this Ca2+
concentration. The largest differences for activating Ca2+
concentrations were found between a pCa of 5.0 and 5.5 and for inhibiting concentrations between a pCa of 4.3 and 3.5. Higher concentrations inhibited binding to both vesicle types to almost the
same extent. Binding to MHN vesicles was half-maximally activated at
3.6 µM, whereas the EC50 for binding to MHS
vesicles was 3-fold lower (1.2 µM). In parallel, MHS
vesicles were approximately 2-fold less sensitive for inhibiting
Ca2+ concentrations (for MHN, IC50 = 135 µM (n = 6), and for MHS, IC50 = 282 µM (n = 3)).
Calmodulin (CaM) inhibits SR Ca2+ release when the release
channel (RYR1) is previously activated by Ca2+ (24-30).
For the experiments described here, [3H]ryanodine binding
was initially activated by 10 µM Ca2+. CaM
inhibited Ca2+-activated binding in a
concentration-dependent manner (Fig. 5). Contrary to the porcine Arg615
In the following experiments, the effects of the two exogenous RYR1
activators, caffeine and 4-chloro-m-cresol, which are used
for the diagnosis of MH, were investigated. Experiments were carried
out in the presence of a free Ca2+ concentration of 0.1 µM, which is below the threshold of activation for both
vesicle types.
Similar to Ca2+, caffeine stimulated
[3H]ryanodine binding to a higher level to MHS than to
MHN vesicles (Fig. 6, left). Major differences between MHN and MHS vesicles were observed for
concentrations starting at 5 mM. The stimulatory effect,
however, was weak compared with caffeine-activated
[3H]ryanodine binding to porcine SR vesicles (Table
I). The calculated EC50 values were about
24.9 mM for MHN and 9.5 mM for MHS
vesicles.
Comparison of the functional effects of the porcine Arg615 Department of Applied Physiology,
Arg. Using high affinity
[3H]ryanodine binding as the experimental approach, we
show that this mutation enhances the sensitivity of RYR1 to activating
concentrations of Ca2+ and to the exogenous and
diagnostically used ligands caffeine and 4-chloro-m-cresol.
In parallel, the sensitivity to inhibiting concentrations of
Ca2+ and calmodulin was reduced, transferring the mutant
Ca2+ release channel into a hyperexcitable state.
Cys) in the
skeletal muscle ryanodine receptor (RYR1), which functions as the
sarcoplasmic reticulum (SR) Ca2+ release channel, has been
linked to porcine stress syndrome, an equivalent to human MH (1).
Contrary to porcine stress syndrome, human MH is a genetically
heterogeneous skeletal muscle disorder. Based on genetic linkage
studies, three MH loci are known. The first has been mapped to
chromosome 19q12-13.2 encompassing the gene that in homology with the
animal model encodes the RYR1 (2, 3), the second has been mapped to
chromosome 7q including the gene for the
2/
subunit
of the skeletal muscle dihydropyridine receptor (4), and the third has
been mapped to chromosome 3q13.1 (5). To date, mutations have only been
identified in RYR1 that count for approximately 50% of human MH cases
(reviewed in Ref. 6). MH mutations seem to cluster in two areas of the
RYR1 sequence. Six mutations have been localized in the N-terminal
sequence of RYR1 containing a homologous mutation to that identified in
porcine MH-susceptible (MHS) muscle. Two further mutations have been
found in the central amino acid sequence. In vitro, all
these mutations induce a hypersensitivity of biopsied muscle to the
contracture-triggering agents caffeine and halothane. This enhanced
sensitivity is exploited in the diagnostic in vitro
contracture test (IVCT). According to the European test protocol (7),
dissected muscle fiber bundles are exposed to increasing concentrations
of caffeine and halothane. Individuals are classified MHS if the
sensitivity is increased for both compounds. Ca2+ release
from human SR vesicles obtained from MHN and MHS muscle samples has
been studied in a few approches (8-13). In these experiments, however,
MHS muscle samples were collected from genetically nonclassified material. Thus, the observed effects could not be addressed to a single
human RYR1 mutation. In the present study, we characterized the
functional effects of a human RYR1 mutation that is located in the
central part of RYR1, Gly2434
Arg. (The numbering of
amino acids follows the corrected sequence data for the human RYR1
according to Ref. 14.) Our data provide the first definitive evidence
that a centrally located mutation is causative for the hypersensitivity
of SR Ca2+ release in MHS muscle. Part of this work has
been submitted in abstract form (15).
Materials
Arg mutation. For control, muscle
specimens were obtained from individuals who had undergone muscle
biopsy for exclusion of MH susceptibility. Muscle samples were tested
according to the protocol of the European Malignant Hyperthermia Group
(7). All procedures were in accordance with the Helsinki convention and
were approved by the Ethics Commission of the University of Ulm.
-TTCCCTGCAGCTTTGGTG-3
and Ex45RyR reverse 5
-GGGTCTCACATGCATCTC-3
)
were used to amplify a 128-bp fragment. PCR was carried out with 50 ng
of genomic DNA and 30 pmol primers each in a total volume of 50 µl.
The PCR reaction contained 50 mM KCl, 20 mM
Tris-HCl, pH 8.4, 2.5 mM MgCl2, 0.01% gelatin,
200 µM of each dNTP, and 1 unit of Taq
polymerase. PCR amplification conditions were 94 °C for 4 min
followed by 35 cycles of 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 30 s. The presence or absence of the mutation
was detected by polyacrylamide gel electrophoresis of products obtained
by digestion of PCR products with AlwNI.
Arg. The
patients consisted of five males, varying in age from 34 to 69 years.
For control, muscle samples were obtained from 28 individuals who were
classified MHN according to the European IVCT protocol (7). Biopsied
muscle specimens were immediately frozen and stored in liquid nitrogen
until use. A microsomal SR fraction was isolated as described
previously (16). Isolated membranes were resuspended in 0.3 M sucrose, 10 mM K-PIPES, pH 6.8, rapidly
frozen in liquid nitrogen and stored at
70 °C. To prevent
proteolysis, the following protease inhibitors were included in various
purification steps: 200 µM Pefabloc
(4-(2-aminoethyl)benzolsulfonyl-fluoride), 100 nM
aprotinin, 1 µM leupeptin, 1 µM pepstatin
A, and 1 mM benzamidine. Protein concentration was
determined according to the method described in Ref. 17 using bovine
serum albumin as a standard.
Arg mutation. To
investigate the presence of the mutation in this pedigree, the 128-bp
region spanning the G7300A mutation was amplified and subjected to
restriction enzyme analysis. The base exchange results in the creation
of an AlwNI restriction site (19). Digestion of
PCR-amplified DNA fragments resulted in MHS individuals in two
additional bands of 100 and 28 bp (Fig. 1). Fig. 1 shows
that the mutation segregated with MH. All patients who were classified
MHS in IVCT carried the point mutation in RYR1. To investigate the
functional consequences of this mutation at the molecular level, a
microsomal SR fraction was isolated from MHS muscle and as control from
MHN samples and utilized for high affinity [3H]ryanodine
binding.
Fig. 1.
Pedigree of an MH family heterozygous for the
RYR1 Gly2434 Arg mutation with corresponding
polyacrylamide gel. A 10% polyacrylamide gel was loaded with
fragments of PCR-amplified genomic DNA following digestion with
AlwNI. The gel was stained with ethidium bromide. The upper
and lower part was cut out for demonstration. PCR products of MHN
individuals show a nondigested PCR product of 128 bp. In MHS patients'
DNA, cleavage of the normal 128-bp PCR fragment generates two
additional bands of 100 and 28 bp. The results of the IVCT test are
indicated by filled (MHS) and open (MHN) symbols.
A question mark denotes untested individuals. Skeletal
muscle biopsies of individuals labeled with an asterisk were
taken for SR preparation. Circles, females;
squares, males.
[View Larger Version of this Image (31K GIF file)]
Fig. 2.
Electrophoretic analysis of SR vesicles from
MHN and MHS skeletal muscle. Aliquots (60 µg) of MHN and MHS SR
vesicles were separated on a 3.5-15% SDS/polyacrylamide gel. The gel
was stained with Coomassie Brilliant Blue. Molecular mass standards (migration indicated on the left-hand side) were myosin (200 kDa), -galactosidase (116 kDa), phosphorylase B (97 kDa),
bovine serum albumin (66 kDa), ovalbumin (45 kDa), and trypsin
inhibitor (21 kDa). The arrow indicates the position of
RYR1.
[View Larger Version of this Image (69K GIF file)]
Fig. 3.
Specific binding of
[3H]ryanodine to MHN and MHS skeletal muscle SR
vesicles. Binding was carried out in 0.1 M KCl, 10 µM Ca2+, pH 6.8, and the indicated
concentrations of [3H]ryanodine as described under
"Experimental Procedures." Right, corresponding
Scatchard plots. The symbols represent the means of one representative
experiment carried out in duplicate.
[View Larger Version of this Image (34K GIF file)]
Cys) porcine muscle (reviewed in Ref. 23).
Fig. 4.
Dependence of high affinity
[3H]ryanodine binding on cytosolic Ca2+.
Ca2+ dependence of [3H]ryanodine binding in
the presence of 12 nM [3H]ryanodine and
indicated concentrations of free Ca2+. Data were fitted
acoording to the following equation: B = Bmax × {[Ca]n1/(k1 + [Ca]n1) [Ca]n2/(k2 + [Ca]n2)}, where B
corresponds to bound [3H]ryanodine and
Bmax corresponds to maximally bound
[3H]ryanodine, [Ca] is free Ca2+
concentration, k1 and k2
are the binding constants for the Ca2+ activating and
inhibitory sites, and n1 and
n2 are the corresponding Hill coefficients.
Resulting half-maximal activating and inhibiting concentrations were:
MHN, EC50 = 3.55 µM, IC50 = 135 µM (n = 6); MHS, EC50 = 1.20 µM, IC50 = 282 µM
(n = 3), where n represents the number of
experiments carried out in duplicate. Data points labeled with
asterisks are significantly different on the
p < 0.05 level (Student's t test).
[View Larger Version of this Image (20K GIF file)]
Cys mutation (30), the
human Gly2434
Arg mutation resulted in a loss of
sensitivity for CaM. In MHN vesicles, CaM inhibited binding to about
30% of control, whereas binding to MHS vesicles was only reduced to
50%. Significant differences were found in the presence of CaM
concentrations greater than 0.1 µM.
Fig. 5.
Inhibition of [3H]ryanodine
binding by CaM. [3H]Ryanodine binding was performed
in the presence of 12 nM [3H]ryanodine at a
pCa of 5. The data were normalized to the amount of
[3H]ryanodine bound in the absence of CaM. Data points
represent the means ± S.D. of five experiments of MHN SR vesicles
and four experiments for MHS vesicles. An asterisk indicates
significant differences between corresponding data points at
p < 0.05 (Student's t test).
[View Larger Version of this Image (21K GIF file)]
Fig. 6.
Activation of [3H]ryanodine
binding by caffeine and 4-CmC. The activating effect of caffeine
(left) and 4-CmC (right) was investigated in the
presence of 0.1 µM Ca2+ and 12 nM
[3H]ryanodine. Data were fitted according to the Hill
equation: EC50/napp (caffeine): MHN,
24.9 mM/1.8 (n = 3), and MHS, 9.5 mM/1.8 (n = 3);
EC50/napp (4-CmC): MHN, 535 µM/1.7 (n = 8), and MHS, 190 µM/1.6 (n = 2). The data are derived from
n experiments performed in duplicate. If no error
bars (S.D.) are shown, they are encompassed within the
symbol.
[View Larger Version of this Image (16K GIF file)]
Cys with the human Gly2434
Arg mutation
Cys and
Gly2434
Arg mutation.
Arg615
Cys (porcine
RYR1)a
Gly2434
Arg (human RYR1)
High
affinity [3H]ryanodine binding
MHN,
Kd = 43.3 nM
MHN,
Kd = 47.0 nM
MHS,
Kd = 11.1 nM
MHS,
Kd = 31.7 nM
Ca2+ dependence
MHN, EC50 = 2.60 µM
MHN, EC50 = 3.55 µM
MHS, EC50 = 0.96 µM
MHS, EC50 = 1.20 µM
MHN, IC50 = 270 µM
MHN, IC50 = 135 µM
MHS, IC50 = 560 µM
MHS, IC50 = 282 µM
Caffeine dependence
MHN, EC50 = 10.7 mM
MHN, EC50 = 24.9 mM
MHS,
EC50 = 3.7 mM
MHS, EC50 = 9.5 mM
4-Chloro-m-cresol dependence
MHN,
EC50 = 395 µM
MHN, EC50 = 535 µM
MHS, EC50 = 193 µM
MHS,
EC50 = 190 µM
a
Data were taken from Ref. 16.
It has recently been shown that 4-chloro-m-cresol (4-CmC) is a potent and specific activator of RYR1 that can be used as a diagnostic tool to distinguish between MHN and MHS muscle (16, 31-33). Comparable with caffeine, 4-CmC stimulated [3H]ryanodine binding to MHS SR with higher affinity than to MHN SR (Fig. 6, right). Contrary to caffeine, the absolute level of activation was about 2-fold higher, and the resulting EC50 values were approximately 20-fold lower. MHS vesicles were about 3-fold more sensitive (EC50 = 190 µM (n = 3)) compared with MHN (EC50 = 535 µM (n = 8)). The largest differences in activation were observed at 4-CmC concentrations between 200 and 500 µM.
The sarcoplasmic reticulum (SR) is the major element in skeletal
muscle that regulates the release and uptake of myoplasmic Ca2+. SR Ca2+ release is mediated by the high
molecular weight ligand-gated Ca2+ release channel (RYR1),
which is biochemically characterized by its high affinity for the plant
alkaloid ryanodine (recent reviews in Refs. 34 and 35). The protein
complex comprises four identical subunits each consisting of about 5000 amino acids as deduced by cloning and sequencing of the cDNA (36,
37). Hydropathy plots suggested 4 (rabbit RYR1) to 10 (human RYR1) hydrophobic segments, the ion pore forming segments in the C-terminal part, comprising about 10-20% of the receptor molecule. The remainder of the protein has been assigned to the cytoplasmic side of the SR
membrane. Human RYR1 mutations have been linked to two skeletal muscle
diseases, malignant hyperthermia and central core disease (CCD) (6,
38-40). Both disorders have been associated with an abnormally high
release of Ca2+ from SR, which is probably due to an
altered function of the mutant Ca2+ release channel. Six of
these mutations (Arg163 Cys (MH, CCD),
Gly248
Arg (MH), Gly341
Arg (MH),
Ile403
Met (MH, CCD), Tyr522
Ser (MH,
CCD), and Arg614
Cys (MH)) have been identified in the
N-terminal part of the receptor and two (Gly2434
Arg
(MH) and Arg2435
His (MH, CCD)) in the central part.
Although the functional effects of a RYR1 mutation in the N-terminal
part of the receptor (Arg614
Cys) have been studied in
detail in the corresponding animal model in porcine skeletal muscle
(23), the functional consequences of genetically defined human
mutations and subsequently of a mutation located in the central part of
the amino acid sequence of RYR1 has not yet been investigated.
An A for G7300 transition in the RYR1 gene leads to the replacement of
a conserved Gly by an Arg at position 2434 in the amino acid sequence.
This mutation has been identified in four Caucasian (19) and four
Canadian pedigrees (41). Comparing the presence or the absence of the
mutation in the pedigree investigated in the present study with the
results of the in vitro contracture test (Fig. 1) revealed
that the Gly2434 Arg mutation precisely segregates with
the MHS phenotype.
Because the amount of MHS muscle sample was very limited (<2.2 g), we used high affinity [3H]ryanodine binding to isolated SR vesicles to study the effect of this mutation on SR Ca2+ release. Binding of [3H]ryanodine reflects the functional state of the SR Ca2+ release channel because ligands that have been shown to activate or inhibit the Ca2+ release channel modulate [3H]ryanodine binding in a similar way (20-22). Using this highly reproducible functional approach, we were able to investigate the effects of some major endogenous and pharmacological ligands of RYR1.
Table I compares the sensitivities of various SR modulators on
[3H]ryanodine binding to porcine and human SR vesicles of
MHN and MHS muscle samples carrying the point mutations
Arg615 Cys and Gly2434
Arg,
respectively. The data are derived from experiments that were carried
out under exactly the same conditions for both tissues (16) and that
have been described as optimal for visualizing differences in
[3H]ryanodine binding to MHS and MHS porcine SR
vesicles (42). In contrast to the material obtained from humans who
were heterozygous for the Gly2434
Arg mutation, porcine
muscle was obtained from pigs that were homozygous for the
Arg615
Cys mutation. [3H]Ryanodine
binding to SR vesicles derived from porcine muscle heterozygous for the
mutation have not been characterized in detail.
Porcine MHS SR vesicles exerted an approximately 4-fold higher affinity for [3H]ryanodine compared with MHN vesicles. This difference is less pronounced for the human mutation. The smaller difference could be either explained by the different locations of the mutations in the amino acid sequence, or it could be due to the fact that the human muscle is heterozygous for the mutation. A heterozygous RYR1 mutation should result in five different populations of RYR1 tetramers: homotetramers consisting of only normal or mutant subunits, respectively, and heterotetramers consisting of one to three mutant subunits. Single channel measurements with heterozygous porcine Ca2+ release channels showed different classes of activities that might be associated to different populations of tetramers (43). In the functional assay used here, we measured the averaged effects of different tetramer populations. The observed lower [3H]ryanodine affinity of the heterozygous human MHS SR vesicles compared with the homozygous pig vesicles may be due to a contribution of heterotetramers exhibiting lower affinity.
Ca2+ activated [3H]ryanodine binding to both porcine and human vesicles in a typically biphasic manner. Compared with porcine vesicles, both human MHN and MHS vesicles were less sensitive to activating and more sensitive to inhibiting Ca2+ concentrations. We also observed a distinct lower sensitivity of human SR vesicles for caffeine, whereas 4-CmC activated binding to porcine and human SR in a similar concentration range. In all cases, however, both the human and the porcine mutation induced a similar shift in the EC50 values to lower and, for inhibiting Ca2+, to higher concentrations.
Differences in the functional consequences of both mutations were
observed for the inhibitory effect of CaM in that the mutant human RYR1
was less sensitive to inhibiting CaM concentrations compared with the
MHN receptor (Fig. 5). A similar tendency has also been described for
the mutant porcine receptor (30). These differences in inhibition,
however, were not found to be significant. The distinct lower
sensitivity of the human MHS receptor can be explained by the close
vicinity of CaM binding sites to the Gly2434 Arg
mutation (30, 44, 45). The mutant porcine receptor has been found more
sensitive to activating CaM concentrations in the absence of
Ca2+ (30). We also investigated this effect on the human
mutation. CaM activation of [3H]ryanodine binding to
human SR vesicles in the absence on Ca2+, however, was so
low that it was not possible to visualize differences in activation
between MHN and MHS vesicles.
In conclusion, our data show that the porcine Arg615 Cys and the human Gly2434
Arg mutation induce similar
shifts in sensitivities of RYR1 toward some major endogenous ligands
and to compounds that are utilized in the diagnosis of MH. It might be
tempting to speculate that in the three-dimensional conformation the
N-terminal and central part of RYR1 are in close vicinity. The
mutations in this area may be acting in a similar manner in
transferring the Ca2+ release channel into a hypersensitive
state.