Central Core Disease Mutations Research Articles

Genotype-Phenotype Correlations of the Central Core Disease Mutations in the C-Terminal Region of the RyR1 Channel

Genotype-Phenotype Correlations of the Central Core Disease Mutations in the C-Terminal Region of the RyR1 Channel

Genotype-Phenotype Correlations of Malignant Hyperthermia and Central Core Disease Mutations in the Central Region of the RYR1 Channel.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in some muscle diseases, including malignant hyperthermia (MH) and central core disease (CCD). Over 200 mutations associated with these diseases have been identified, and most mutations accelerate Ca2+ -induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, it remains largely unknown how specific mutations cause different phenotypes. In this study, we investigated the CICR activity of 14 mutations at 10 different positions in the central region of RYR1 (10 MH and four MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging, the mutant channels exhibited an enhanced sensitivity to caffeine, a reduced endoplasmic reticulum Ca2+ content, and an increased resting cytoplasmic Ca2+ level. The three parameters for CICR (Ca2+ sensitivity for activation, Ca2+ sensitivity for inactivation, and attainable maximum activity, i.e., gain) were obtained by [3 H]ryanodine binding and fitting analysis. The mutant channels showed increased gain and Ca2+ sensitivity for activation in a site-specific manner. Genotype-phenotype correlations were explained well by the near-atomic structure of RYR1. Our data suggest that divergent CICR activity may cause various disease phenotypes by specific mutations.

Divergent Activity Profiles of Type 1 Ryanodine Receptor Channels Carrying Malignant Hyperthermia and Central Core Disease Mutations in the Amino-Terminal Region

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in several diseases, including malignant hyperthermia (MH) and central core disease (CCD). Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca2+ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca2+ in the ER and an increase in resting cytoplasmic Ca2+. RyR1 channel activity was quantitatively evaluated by [3H]ryanodine binding and three parameters (sensitivity to activating Ca2+, sensitivity to inactivating Ca2+ and attainable maximum activity, i.e., gain) were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca2+ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca2+ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca2+ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.

Effects of MH and CCD Mutations in the Central Region on RyR1 Channels

Type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum and the major target for muscle diseases, e.g., malignant hyperthermia (MH) and central core disease (CCD). It is widely believed that MH and CCD mutations cause hyperactivation of the Ca2+-induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, it remains unclear how the disease-associated mutations affect CICR. We have recently characterized several disease-associated mutations in the amino-terminal region by live-cell Ca2+ imaging and [3H]ryanodine binding and found that these mutations divergently affect the gain (i.e., peak activity) and the sensitivity to activating Ca2+ of CICR. In this study, we extended this approach to 15 MH and MH/CCD mutations in the central region (1592-2508). The disease-associated mutations increased the gain and the sensitivity to activating Ca2+ in a site-dependent manner. The calculated CICR activity strongly correlated with the ER Ca2+ level, an index of Ca2+ leak. Importantly, the accelerated sensitivity to activating Ca2+ was linked to pathogenesis of CCD. Overall, the effects were similar to those of the amino-terminal mutations. The underlying molecular mechanism will be discussed.

Lobe-Specific Calmodulin Binding to Different Ryanodine Receptor Isoforms

Ryanodine receptors (RyRs) are large ion channels that are responsible for the release of Ca(2+) from the sarcoplasmic/endoplasmic reticulum. Calmodulin (CaM) is a Ca(2+) binding protein that can affect the channel open probability at both high and low Ca(2+) concentrations, shifting the Ca(2+) dependencies of channel opening in an isoform-specific manner. Here we analyze the binding of CaM and its individual domains to three different RyR regions using isothermal titration calorimetry. We compared binding to skeletal muscle (RyR1) and cardiac (RyR2) isoforms, under both Ca(2+)-loaded and Ca(2+)-free conditions. CaM can bind all three regions in both isoforms, but the binding modes differ appreciably in two segments. The results highlight a Ca(2+)/CaM and apoCaM binding site in the C-terminal fifth of the channel. This binding site is the target for malignant hyperthermia and central core disease mutations in RyR1, which affect the energetics and mode of CaM binding.

Effects of Amino-Terminal Disease-Associated Mutations on the CICR Activity of RyR1 Channel

Type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum and plays a pivotal role in excitation-contraction coupling in skeletal muscle. RyR1 is the major target for muscle diseases, e.g., malignant hyperthermia (MH) and central core disease (CCD). To date, over 200 mutations have been identified in the RyR1 gene of MH and CCD patients. It is widely believed that MH and CCD mutations cause hyperactivation of the Ca2+-induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. CICR shows biphasic Ca2+ dependence, thus the activity can be determined by three parameters: sensitivity to activating Ca2+, sensitivity to inactivating Ca2+, and the gain (i.e., peak activity). However, it remains unclear how the disease-associated mutations affects these parameters. In this study, we expressed several RyR1 channels carrying different MH/CCD mutations at amino-terminal region in HEK cells and tested their CICR by live-cell Ca2+ imaging and [3H]ryanodine binding. Our results suggest that the amino-terminal disease-associated mutations divergently affects the parameters of CICR depending on the sites for mutation. The underlying molecular mechanism will be discussed.

C.P.2 In silico analysis of recessive RYR1 mutations identifies novel potential disease mechanisms

Mutations in RYR1 are responsible for malignant hyperthermia (MH) and several forms of congenital myopathy. Dominant mutations that give rise to central core disease (CCD) and MH cluster in three hotspot regions and the disease mechanisms associated with these diseases are well studied. In comparison, little is known about how recessive mutations, which occur throughout the gene, lead to disease. To address this issue, we reviewed the literature to map potentially functional domains within the ryanodine receptor type 1 (RyR1) and then analysed the nature and position of RYR1 mutations in 74 individuals with recessive disease, together with their clinical and histological phenotypes. Hypomorphic mutations, which abolish or markedly reduce RyR1 protein expression, were associated with more severe clinical phenotypes and with histological phenotypes other than CCD, such as multi-minicore disease, centronuclear myopathy and congenital fibre type disproportion. For non-hypomorphic mutations, CCD mutations were enriched in MH/CCD hotspot 3 and non-CCD core mutations in hotspot 2. Clustering of non-hypomorphic mutations in the triadin-binding domain (which lies within hotspot region 3) highlights abnormal triadin-binding as a potentially important disease mechanism. Non-hypomorphic mutations associated with ophthalmoplegia clustered in regions involved in ‘interdomain interactions’ which play roles in stabilising the closed conformation of the RyR1 channel. These results suggest that the variability seen in recessive RYR1 histological phenotypes may stem from differences in disease mechanism, determined in part by the location of non-hypomorphic mutations. Reduced overall RyR1 function, reduced stability of the closed channel conformation and abnormal triadin-RyR1 interactions are highlighted as potentially important disease mechanisms in various recessive RYR1-myopathies.

Calcitonin gene‐related peptide restores disrupted excitation–contraction coupling in myotubes expressing central core disease mutations in RyR1

Central core disease (CCD) is a congenital human myopathy associated with mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), resulting in skeletal muscle weakness and lower limb deformities. The muscle weakness can be at least partially explained by a reduced magnitude of voltage-gated Ca(2+) release (VGCR). To date, only a few studies have focused on identifying potential therapeutic agents for CCD. Therefore, in this work we investigated the potential use of the calcitonin gene related peptide (CGRP) to restore VGCR in myotubes expressing CCD RyR1 mutants. We also examined the influence of CCD mutants on Ca(2+)-dependent processes involved in myogenesis (myoblast fusion and sarcoendoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2) gene expression). C2C12 cells were transfected with cDNAs encoding either wild-type RyR1 or CCD mutants, and then exposed to CGRP (100 nm, 1-4 h). Expression of the I4897T mutant significantly inhibited SERCA2 gene expression and myoblast fusion, whereas the Y523S mutant exerted the opposite effect. Interestingly, both mutants clearly inhibited VGCR (50%), due to a reduction in SR Ca(2+) content. However, no major changes due to CGRP or CCD mutants were observed in I(CaL). Our data suggest that the Y523S mutant results in store depletion via decompensated SR Ca(2+) leak, while the I4897T mutant inhibits SERCA2 gene expression. Remarkably, in both cases CGRP restored VGCR, likely to have been by enhancing phospholamban (PLB) phosphorylation, SERCA activity and SR Ca(2+) content. Taken together, our data show that in the C2C12 model system, changes in excitation-contraction coupling induced by the expression of RyR1 channels bearing CCD mutations Y523S or I4897T can be reversed by CGRP.

Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation–contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1I4898T mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4–6-mo-old IT/+ mice. The sensitivity of the SR Ca2+ release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca2+ permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation.

Understanding The Molecular Defects Of Human MH And CCD

Malignant Hyperthermia (MH) and Central Core Disease (CCD) are skeletal muscle disorders linked to mutations in the skeletal muscle ryanodine receptor (RyR1). Based on their phenotypes, disease-causing RyR1 mutations can be separated into three major groups: MH-only, both MH and CCD (MH/CCD), and CCD-only. The molecular basis for these different genotype-phenotype relationships is largely undefined. We have recently demonstrated that the porcine MH mutation, R615C, increases the sensitivity of the RyR1 channel to luminal Ca2+ activation and reduces the threshold for spontaneous Ca2+ release during store Ca2+ overload, also known as store-overload-induced Ca2+ release (SOICR). To investigate whether human MH and CCD mutations also alter the luminal Ca2+ activation of RyR1 and SOICR, we have generated a number of MH-only, MH/CCD, and CCD-only mutations located in the NH2 terminal, central, and COOH-terminal regions, and established stable, inducible HEK293 cell lines expressing these mutants. Using single cell Ca2+ imaging, we found that MH-only and MH/CCD mutations enhance the propensity for SOICR. On the other hand, some CCD-only mutations suppress or abolish SOICR, but retain caffeine-induced Ca2+ release, while other CCD-only mutants display little or no ryanodine- or caffeine-sensitive channel activity. Single channel studies reveal that, like the R615C MH mutation, MH/CCD mutations markedly sensitize the RyR1 channel to activation by luminal Ca2+. To assess their impact in the context of muscle cells, we are currently establishing stable, inducible mouse skeletal muscle C2C12 cell lines expressing MH and CCD RyR1 mutants. Further studies will address the question of whether altered luminal Ca2+ activation of RyR1 underlies a common defect of human MH and CCD mutations.

Ryanodine Receptor Structure: Progress and Challenges

Ryanodine Receptor Structure: Progress and Challenges

Two central core disease (CCD) deletions in the C-terminal region of RYR1 alter muscle excitation-contraction (EC) coupling by distinct mechanisms

Central core disease (CCD) and malignant hyperthermia (MH) are skeletal muscle disorders that are linked to mutations in the gene that encodes the type 1 ryanodine receptor (RYR1). The RYR1 ion channel plays a central role in excitation-contraction (EC) coupling by releasing Ca(2+) from an internal store. Pathogenic CCD mutations in RYR1 result in changes in the magnitude of Ca(2+) release during EC coupling. CCD has recently been linked to two novel deletions (c.12640_12648delCGCCAGTTC [p.Arg4214_Phe4216del] and c.14779_14784delGTCATC [p.Val4927_Ile4928del]) in the C-terminal region of RYR1. To determine the phenotypic consequences of these mutations and extend our understanding of the pathogenic mechanisms that underlie CCD, we determined functional effects on Ca(2+) release channel activity of analogous deletions (p.Arg4215_Phe4217del and p.Val4926_Ile4927del) engineered into rabbit RYR1 following expression in RYR1-null (dyspedic) myotubes and HEK293 cells. In addition, we assessed effects of the p.Arg4214 Phe4216del mutation on RYR1 function in lymphoblastoid cells obtained from CCD patients heterozygous for the mutation. Here we report that both deletions significantly reduce Ca(2+) release following RYR1 activation, but by different mechanisms. While the p.Arg4214_Phe4216del deletion promotes Ca(2+) depletion from intracellular stores by exhibiting a classic "leaky channel" behavior, the p.Val4927_Ile4928del deletion reduces Ca(2+) release by disrupting Ca(2+) gating and eliminating Ca(2+) permeation through the open channel.

Effects Of Skeletal Muscle Specific Fkbp12 Deficiency On Eccentric Contraction-induced Injury In Mouse Muscle

Central Core Disease (CCD) is an inheritable skeletal muscle disease that results in weakness and motor development delays. Although CCD is known to stem from mutations in the ryanodine receptor (RYR1), the exact etiology of CCD is unknown. PURPOSE To test the hypothesis that removal of FKBP12, a 12-kD binding protein known to bind to RYR1 sites near the locus of some CCD mutations, would induce central core lesions and exacerbate strength deficits in mouse anterior crural muscles after eccentrically biased exercise. METHODS Skeletal muscle specific FKBP12 deficient mice were created using the CreLoxP gene recombination technique with Cre transgene expression under the regulation of the muscle creatine kinase promoter. Anterior crural muscle strength was measured in FKBP12 deficient (n=1 1) and control (n=1 1) mice before, immediately after, and at 3, and 14 d after performance of 150 eccentric contractions in vivo. Isometric force and caffeine sensitivity (1–50 mM) were measured in EDL muscles in vitro. Histological analyses were used to characterize muscle damage and central core lesions in TA muscles. RESULTS Body weight (BW) was 8% lower in FKBP12 deficient mice compared with controls (i.e., Cre, wild-type). Before injury there were no differences between groups in isometric torque as a function of stimulation frequency (20–400 Hz) except at 20 Hz, with peak isometric torque normalized to BW being 15% lower in FKBP12 deficient mice. There was no difference in peak torque normalized to BW on the first eccentric contraction between FKBP12 deficient (204.1±3.5 Nmm/kg) and control (202.0±3.3 Nmm/kg) mice. Relative decreases (43.0±0.9%) in peak eccentric torque over the 150 contractions were similar for both groups. There were no differences in isometric torque deficits between groups immediately after, or at 3 and 14 d after injury. Although peak isometric specific force in the EDL muscle was not different between groups, FKBP12 deficient mice exhibited a greater (20%) response to 50 mM caffeine at 14 d. TA muscles from both groups exhibited cellular damage, however, no evidence of central core lesions was found. CONCLUSIONS Skeletal muscle FKBP12 deficiency does not exacerbate strength deficits or induce central core lesions after eccentric contraction-induced injury in mouse anterior crural muscle. Supported by NIH grant 2 R01 AR41802-11.

Distinct Effects on Ca 2+ Handling Caused by Malignant Hyperthermia and Central Core Disease Mutations in RyR1

Malignant hyperthermia (MH) and central core disease (CCD) are disorders of skeletal muscle Ca 2+ homeostasis that are linked to mutations in the type 1 ryanodine receptor (RyR1). Certain RyR1 mutations result in an MH-selective phenotype (MH-only), whereas others result in a mixed phenotype (MH + CCD). We characterized effects on Ca 2+ handling and excitation-contraction (EC) coupling of MH-only and MH + CCD mutations in RyR1 after expression in skeletal myotubes derived from RyR1-null (dyspedic) mice. Compared to wild-type RyR1-expressing myotubes, MH + CCD- and MH-only-expressing myotubes exhibited voltage-gated Ca 2+ release (VGCR) that activated at more negative potentials and displayed a significantly higher incidence of spontaneous Ca 2+ oscillations. However, maximal VGCR was reduced only for MH + CCD mutants (Y4795C, R2435L, and R2163H) in which spontaneous Ca 2+ oscillations occurred with significantly longer duration (Y4795C and R2435L) or higher frequency (R2163H). Notably, myotubes expressing these MH + CCD mutations in RyR1 exhibited both increased [Ca 2+] i and reduced sarcoplasmic reticulum (SR) Ca 2+ content. We conclude that MH-only mutations modestly increase basal release-channel activity in a manner insufficient to alter net SR Ca 2+ content (“compensated leak”), whereas the mixed MH + CCD phenotype arises from mutations that enhance basal activity to a level sufficient to promote SR Ca 2+ depletion, elevate [Ca 2+] i, and reduce maximal VGCR (“decompensated leak”).

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

Dynamic alterations in myoplasmic Ca 2+ in malignant hyperthermia and central core disease

Ca 2+ ions play a pivotal role in a wide array of cellular processes ranging from fertilization to cell death. In skeletal muscle, a mechanical interaction between plasma membrane dihydropyridine receptors (DHPRs, L-type Ca 2+ channels) and Ca 2+ release channels (ryanodine receptors, RyR1s) of the sarcoplasmic reticulum orchestrates a complex, bi-directional Ca 2+ signaling process that converts electrical impulses in the sarcolemma into myoplasmic Ca 2+ transients during excitation–contraction coupling. Mutations in the genes that encode the two proteins that coordinate this electrochemical conversion process (the DHPR and RyR1) result in a variety of skeletal muscle disorders including malignant hyperthermia (MH), central core disease (CCD), multiminicore disease, nemaline rod myopathy, and hypokalemic periodic paralysis. Although RyR1 and DHPR disease mutations are thought to alter excitability and Ca 2+ homeostasis in skeletal muscle, only recently has research begun to probe the molecular mechanisms by which these genetic defects lead to distinct clinical and histopathological manifestations. This review focuses on recent advances in determining the impact of MH and CCD mutations in RyR1 on muscle Ca 2+ signaling and how these effects contribute to disease-specific aspects of these disorders.

Central core disease mutations R4892W, I4897T and G4898E in the ryanodine receptor isoform 1 reduce the Ca2+ sensitivity and amplitude of Ca2+-dependent Ca2+ release.

Three CCD (central core disease) mutants, R4892W (Arg4892-->), I4897T and G4898E, in the pore region of the skeletal-muscle Ca2+-release channel RyR1 (ryanodine receptor 1) were characterized using a newly developed assay that monitored Ca2+ release in the presence of Ca2+ uptake in microsomes isolated from HEK-293 cells (human embryonic kidney 293 cells), co-expressing each of the three mutants together with SERCA1a (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1a). Both Ca2+ sensitivity and peak amplitude of Ca2+ release were either absent from or sharply decreased in homotetrameric mutants. Co-expression of wild-type RyR1 with mutant RyR1 (heterotetrameric mutants) restored Ca2+ sensitivity partially, in the ratio 1:2, or fully, in the ratio 1:1. Peak amplitude was restored only partially in the ratio 1:2 or 1:1. Reduced amplitude was not correlated with maximum Ca2+ loading or the amount of expressed RyR1 protein. High-affinity [3H]ryanodine binding and caffeine-induced Ca2+ release were also absent from the three homotetrameric mutants. These results indicate that decreased Ca2+ sensitivity is one of the serious defects in these three excitation-contraction uncoupling CCD mutations. In CCD skeletal muscles, where a mixture of wild-type and mutant RyR1 is expressed, these defects are expected to decrease Ca2+-induced Ca2+ release, as well as orthograde Ca2+ release, in response to transverse tubular membrane depolarization.

Génétique des pathologies associées à un dysfonctionnement du complexe de mobilisation calcique du muscle squelettique

Myoplasmic calcium homeostasis is an essential feature of skeletal muscle contraction. The calcium mobilisation complex (CMC) located at the level of the triadic junction plays a major role for the regulation of calcium fluxes between extra-cellular, cytoplasmic and intra-cellular compartments. The ryanodine receptor type I (RYR1), which is located at the level of the terminal cisternae of the sarcoplasmic reticulum is a key component of the CMC. RYR1 allow the release into the myoplasm of the intralumenal stores of calcium. RYR1 interacts with other proteins: DiHydroPyridine Receptor, triadin, calsequestrin, FKBP12, calmodulin. Malignant hyperthermia (MHS) and congenital core myopathies have been associated with a dysfunction of the CMC. MHS is an autosomic dominant pharmacogenetic disease. The MH crisis is induced by exposure of the predisposed patients to halogenated volatile anaesthetics. MHS is characterised by a genetic heterogeneity and two genes, RYR1 and CACNA1S, have been associated so far with the disease. Mutations in the RYR1 gene have been recently associated with heat stroke, a related syndrome. Central Core Disease (CCD) and Multi minicore Disease (MmD) are congenital myopathies presenting with clinical variability and characterized by the presence of specific although heterogeneous muscle histological features: the cores. Clinical boundaries between the two diseases may overlap and the specific diagnosis is often based on the nature of the cores. These diseases show genetic heterogeneity with both autosomic dominant and recessive mode of inheritance and mutations in the SEPN1, RYR1, ACTA1, TPM3 genes have been reported. Mutations associated with MHS were mainly identified into 2 regions of the N-terminal part of RYR1. Functional role of these two domains is still unclear. Mutations responsible for congenital myopathies mainly mapped to the C terminal region of RYR1 that form the transmembrane calcium channel. Functional studies of the RYR1 mutations have shown that MHS mutations were mainly associated with an alteration of the calcium fluxes in response to caffeine or halothane while CCD mutations would result in a leaky RYR1 channel or would alter the Excitation-Contraction coupling at the level of the CMC.

Functional effects of central core disease mutations in the cytoplasmic region of the skeletal muscle ryanodine receptor.

Central core disease (CCD) is a human myopathy that involves a dysregulation in muscle Ca(2)+ homeostasis caused by mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the protein that comprises the calcium release channel of the SR. Although genetic studies have clearly demonstrated linkage between mutations in RyR1 and CCD, the impact of these mutations on release channel function and excitation-contraction coupling in skeletal muscle is unknown. Toward this goal, we have engineered the different CCD mutations found in the NH(2)-terminal region of RyR1 into a rabbit RyR1 cDNA (R164C, I404M, Y523S, R2163H, and R2435H) and characterized the functional effects of these mutations after expression in myotubes derived from RyR1-knockout (dyspedic) mice. Resting Ca(2)+ levels were elevated in dyspedic myotubes expressing four of these mutants (Y523S > R2163H > R2435H R164C > I404M RyR1). A similar rank order was also found for the degree of SR Ca(2)+ depletion assessed using maximal concentrations of caffeine (10 mM) or cyclopiazonic acid (CPA, 30 microM). Although all of the CCD mutants fully restored L-current density, voltage-gated SR Ca(2)+ release was smaller and activated at more negative potentials for myotubes expressing the NH(2)-terminal CCD mutations. The shift in the voltage dependence of SR Ca(2)+ release correlated strongly with changes in resting Ca(2)+, SR Ca(2)+ store depletion, and peak voltage-gated release, indicating that increased release channel activity at negative membrane potentials promotes SR Ca(2)+ leak. Coexpression of wild-type and Y523S RyR1 proteins in dyspedic myotubes resulted in release channels that exhibited an intermediate degree of SR Ca(2)+ leak. These results demonstrate that the NH(2)-terminal CCD mutants enhance release channel sensitivity to activation by voltage in a manner that leads to increased SR Ca(2)+ leak, store depletion, and a reduction in voltage-gated Ca(2)+ release. Two fundamentally distinct cellular mechanisms (leaky channels and EC uncoupling) are proposed to explain how altered release channel function caused by different mutations in RyR1 could result in muscle weakness in CCD.

Ryanodine receptor mutations in malignant hyperthermia and central core disease.

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle that manifests in response to anesthetic triggering agents. Central core disease (CCD) is a myopathy closely associated with MH. Both MH and CCD are primarily disorders of calcium regulation in skeletal muscle. The ryanodine receptor (RYR1) gene encodes the key channel which mediates calcium release in skeletal muscle during excitation-contraction coupling, and mutations in this gene are considered to account for susceptibility to MH (MHS) in more than 50% of cases and in the majority of CCD cases. To date, 22 missense mutations in the 15,117 bp coding region of the RYR1 cDNA have been found to segregate with the MHS trait, while a much smaller number of these mutations is associated with CCD. The majority of RYR1 mutations appear to be clustered in the N-terminal amino acid residues 35-614 (MH/CCD region 1) and the centrally located residues 2163-2458 (MH/CCD region 2). The only mutation identified outside of these regions to date is a single mutation associated with a severe form of CCD in the highly conserved C-terminus of the gene. All of the RYR1 mutations result in amino acid substitutions in the myoplasmic portion of the protein, with the exception of the mutation in the C-terminus, which resides in the lumenal/transmembrane region. Functional analysis shows that MHS and CCD mutations produce RYR1 abnormalities that alter the channel kinetics for calcium inactivation and make the channel hyper- and hyposensitive to activating and inactivating ligands, respectively. The likely deciding factors in determining whether a particular RYR1 mutation results in MHS alone or MHS and CCD are: sensitivity of the RYR1 mutant proteins to agonists; the level of abnormal channel-gating caused by the mutation; the consequential decrease in the size of the releasable calcium store and increase in resting concentration of calcium; and the level of compensation achieved by the muscle with respect to maintaining calcium homeostasis. From a diagnostic point of view, the ultimate goal of development of a simple non-invasive test for routine diagnosis of MHS remains elusive. Attainment of this goal will require further detailed molecular genetic investigations aimed at solving heterogeneity and discordance issues in MHS; new initiatives aimed at identifying modulating factors that influence the penetrance of clinical MH in MHS individuals; and detailed studies aimed at describing the full epidemiological picture of in vitro responses of muscle to agents used in diagnosis of MH susceptibility.