Functional Defects in Six Ryanodine Receptor Isoform-1 (RyR1) Mutations Associated with Malignant Hyperthermia and Their Impact on Skeletal Excitation-Contraction Coupling*

Tianzhong Yang {ddagger}, Tram Anh Ta §, Isaac N. Pessah § and Paul D. Allen

From the Brigham and Women's Hospital, Boston, Massachusetts 02115 and the §Department of Molecular Biosciences, University of California, Davis, California 95616

Received for publication, March 3, 2003 , and in revised form, April 14, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that segregates with >60 mutations within the MHS-1 locus on chromosome 19 coding for ryanodine receptor type 1 (RyR1). Although some MHRyR1s have been shown to enhance sensitivity to caffeine and halothane when expressed in non-muscle cells, their influence on EC coupling can only be studied in skeletal myotubes. We therefore expressed WTRyR1, six of the most common human MHRyR1s (R163C, G341R, R614C, R2163C, V2168M, and R2458H), and a newly identified C-terminal mutation (T4826I) in dyspedic myotubes to study their functional defects and how they influence EC coupling. Myotubes expressing any MHRyR1 were significantly more sensitive to stimulation by caffeine and 4-CmC than those expressing WTRyR1. The hypersensitivity of MH myotubes extended to K+ depolarization. MH myotubes responded to direct channel activators with maximum Ca2+ amplitudes consistently smaller than WT myotubes, whereas the amplitude of their responses to depolarization were consistently larger than WT myotubes. The magnitudes of responses attainable from myotubes expressing MHRyR1s are therefore related to the nature of the stimulus rather than size of the Ca2+ store. The functional changes of MHRyR1s were directly analyzed using [3H]ryanodine binding analysis of isolated myotube membranes. Although none of the MHRyR1s examined significantly altered EC50 for Ca2+ activation, many failed to be completely inhibited by a low Ca2+ (≤100 nM), and all were significantly more responsive to caffeine than WTRyR1 at Ca2+ concentrations that approximate those in resting myotubes. All seven mutations had diminished sensitivity to inhibition by Ca2+ and Mg2+. Using a homologous expression system, our study demonstrates for the first time that these 7 MH mutations are all both necessary and sufficient to induce MH-related phenotypes. Decreased sensitivity to Ca2+ and Mg2+ inhibition and inability of MHRyR1s to be fully inactivated at [Ca2+]i typical of normal myotubes at rest are key defects that contribute to the initiation of MH episodes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Malignant hyperthermia (MH)1 is a rare potentially fatal pharmacological disorder of skeletal muscle that can be triggered by commonly used volatile anesthetic agents and depolarizing muscle relaxants. It is clinically characterized by masseter spasm, tachycardia, increased end-tidal CO2, lactic acidosis, and hyperthermia, and if untreated progresses to death. The ryanodine receptor isoform-1 gene (ryr1) on chromosome 19q13.1 clearly represents a primary molecular locus for MH in humans, termed the MHS-1 locus, as mutations in ryr1 have been linked to more than 50% of all MH families and most central core disease (CCD) families (1). The ryr1 gene codes for a large conductance channel (RyR1) essential for release of SR Ca2+ during skeletal muscle excitation contraction (EC) coupling (2, 3). Molecular genetic studies have shown that RyR1 mutations R615C and R614C co-segregate with porcine and human MH, respectively (4, 5). Functional analysis of skeletal muscle expressing either of these analogous mutations has revealed that a causative defect in MH is hypersensitive gating of the Ca2+ release channel. However abnormalities in SR Ca2+ release function have also been indicated even in the absence of RyR1 mutations, suggesting other loci, possibly in genes coding for RyR1 accessory proteins, may be involved in producing a common MH phenotype. To date, about 60 missense and deletion mutations (6) have been associated with an abnormal in vitro contracture test (CHCT/IVCT) and/or clinical MH or CCD. CCD is a non-progressive autosomal dominant myopathy that is characterized by hypotonia and mild proximal weakness affecting mainly the lower limbs. However the relationship between MH and CCD is not clear. Interestingly, all known MH- and CCD-related mutations found in the ryr1 gene are located in one of three "hot spots." The first hot spot is in the N-terminal region clustered between amino acid residues 35 and 614 (MH/CCD region 1); the second between amino acid residues 2163 and 2458 (MH/CCD region 2); and the third in the C-terminal transmembrane region, between amino acid residues 4643 and 4898.

Functional analysis of individual MH mutations have been performed in several ways to demonstrate the effects of mutations on the physiological function of MHRyR1, and to attempt to confirm the causal role of each mutation in the development of MH. As a physiological test, the routinely used diagnostic procedure for MH, the in vitro contracture test (IVCT), is not able to prove that a given mutation in the RyR1 gene is a cause of MH, as it remains possible that the MH phenotype is the result of alterations of both the MHRyR1 and other protein components of the sarcotubular membranes. The same confounding factors exist with the analysis of Ca2+ transients in myotubes or cultured primary muscle cells derived from carriers of a MH mutation (7, 8). To unambiguously define the direct consequence of individual MH mutations on myotube function, a homologous expression system is necessary in which the only variable is the desired RyR1 mutation. Initial studies using this approach were performed by Otsu et al. (9) using cultured C2C12 myoblast cells transfected with WTRyR1 or MHRyR1 R615C cDNA. C2C12 myotubes expressing R615C were indeed found to have significantly higher sensitivity to caffeine challenges than wild type, confirming IVCT and genetic linkage studies that this mutation is sufficient to cause MH. However, this model has not been used by others to study additional MH mutations because these cells also constitutively express all three types of wild-type mouse RyRs (RyR1, RyR2, and RyR3) and these native RyRs interfere with detailed analysis and confound interpretation of the results.

To date, functional analysis of most human MH mutations addressing molecular details has been limited to heterologous non-muscle expression systems such as COS-7 and HEK-293 cells (1013). Results from these heterologous expression systems have consistently shown an increased sensitivity of the mutant RyR1 to exogenous agonists including caffeine, halothane, and 4-chloro-m-cresol (4-CmC) (10, 11). Expression of 15 RyR1 MH/CCD mutants in HEK-293 cells suggested that these cells had smaller endoplasmic reticulum calcium stores, and reduced maximal caffeine-stimulated calcium release compared with cells expressing wild-type RyR1 (14). However, this interpretation cannot necessarily be extended to skeletal muscle where the SR stores are larger and more efficiently filled and where RyR1 makes structural and functional interactions with the {alpha}1s-DHPR ({alpha}1s subunit of dihydropyridine receptor) and several other proteins in the t-tubule/SR junction that are absent in non-muscle cells (15).

The present study extends the molecular and functional characterization of MH mutations to 1B5 skeletal myotubes that express key triadic proteins, such as skeletal triadin, calsequestrin, FKBP-12, sarcoplasmic reticulum Ca2+-ATPase 1, and {alpha}1s-DHPR, but do not constitutively express any RyR protein isoform (16). 1B5 dyspedic myotubes were transduced with the "porcine mutation" R615C, the newly identified the T4826I mutation in the C-terminal region (17), and five of the most common human MH mutations (R163C, G341R, R2163C, V2168M, and R2458H) (18) that together account for about 25% of MH families. Several new findings were made with myotubes expressing any of the 7 MHRyR1s. 1) Heightened sensitivity to K+ depolarization-induced Ca2+ release, whose maximal amplitude was also significantly greater than WTRyR1. 2) Heightened sensitivity to caffeine and 4-CmC, whose maximum amplitude was significantly smaller than WTRyR1. 3) Analysis of [3H]ryanodine binding to membranes vesicles isolated from transduced myotubes revealed that none of the MHRyR1s exhibited altered EC50 to activation by Ca2+ compared with WTRyR1. 4) The MH phenotype seen in myotubes was related to a significant diminution in the inhibitory potency toward both Ca2+ and Mg2+, and unlike WTRyR1, many of the MH mutations failed to be completely inactivated in the presence of a "resting" level (100 nM) of Ca2+. Interestingly T4826I exhibited the strongest phenotype among the seven mutations, supporting the hypothesis that mutations within the putative transmembrane pore region, are most severely compromised to negative regulation by Ca2+ and Mg2+.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Construction of MH Mutants in RyR1 cDNA—Wild-type rabbit RyR1 cDNA was cloned in the HSV-1 amplicon vector pHSVprPUC (gift of Dr. Howard Federoff, University of Rochester, Rochester, NY). Quick-ChangeTM site-directed mutagenesis kit (Stratagene) was used to introduce the mutations corresponding to human MH mutations into small fragments (HindIII (at the 5'-end of RyR1 cDNA in pHSVprPUC)-SalI(1–546) for R164C; KpnI-BsrGI-(870–1229) for G342R; KpnI-AgeI-(870–4934) for R615C; XhoI-NcoI-(6466–7502) for R2163C, V2168M, and R2458H; and ClaI-XbaI-(14312–15230) at the 3'-end of RyR1 cDNA for T4825I) isolated from the full-length rabbit RyR1 cDNA. (R164C, G342R, R615C, R2163C, V2168M, R2458H, and T4825I in rabbit RyR1 are corresponding to human R163C, G341R, R614C, R2163C, V2168M, R2458H, and T4826I, respectively) After sequencing the PCR products to verify the presence of the specific mutations and lack of any random mutations, the fragments were put back into the full-length RyR1 cDNA.

HSV-1 Virion Production—All mutated and WT RyRs were packaged into HSV1 virions using a helper virus-free packaging system (19, 20), which were then used to transduce dyspedic 1B5 myotubes.

Cell Culture and Infection—1B5 cells (RyR-1, RyR-2, and RyR-3 null) were cultured on Matrigel (BD Bioscience) coated plates (96-well plates (Opticlear® COSTAR 3614) for imaging and 10-cm dishes for membrane preparations) in Dulbecco's modified Eagle's medium, 20% fetal bovine serum, 100 units/ml streptomycin sulfate, 100 units/ml penicillin-G in 5% CO2. After reaching 60–70% confluence, cells were induced to differentiate into myotubes for 5 days by changing the growth medium to Dulbecco's modified Eagle's medium containing 2% heat-inactivated horse serum, 100 µg/ml streptomycin sulfate, and 100 µg/ml penicillin-G in 20% CO2. Wells or plates containing differentiated myotubes were infected with virion particles containing mutated/wt RyR1 cDNAs at an MOI of 0.5 for 2 h, and then cultured for 48 h prior to imaging or cell harvest.

Calcium Imaging—Stock concentrations of caffeine and 4-CmC solutions were prepared in imaging buffer (125 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 6 mM glucose, and 25 mM HEPES, pH 7.4). In KCl-containing solutions the concentration of NaCl was adjusted as necessary to maintain the total ionic strength (KCl + NaCl) equal to 130 mM.

Differentiated 1B5 myotubes were loaded with 5 µM Fluo-4AM (Molecular Probes Inc., Eugene, OR) at 37 °C, for 20 min in imaging buffer. The cells were then washed three times with imaging buffer and transferred to a Nikon Diaphot microscope and Fluo-4 was excited at 494 nm using Multivalve Perfusion System (Automate Scientific Inc., Oakland, CA). Fluorescence emission was measured at 516 nm using a x40 quartz objective. Data were collected with an intensified 12-bit digital intensified CCD (Stanford Photonics, Stanford, CA) from regions consisting of 10–20 individual cells and analyzed using QED software (QED, Pittsburgh, PA). A dose-response curve for a single agent was performed in any given well to compare the response to any given agent of the cells expressing the different MH mutations to that of cells expressing WTRyR1 protein. Because low concentrations of these reagents can cause brief Ca2+ transients having peak amplitudes nearly as large as those reached by higher concentrations, but have a much smaller total Ca2+ release, we used the average fluorescence of the calcium transient (area under the curve) to compare responses. Individual response amplitudes were calculated in the following way: the cumulative fluorescence during the 10 s challenge (Ar) minus the average baseline fluorescence for the 10 s immediately previous to the challenge (Ab), was then divided by Ab, and multiplied by 100. In order to compare different experiments individual response amplitudes were normalized to the maximum fluorescence obtained in the same cell at the highest concentration of the reagent that was being tested (20 mM caffeine, 500 µM 4-CmC, and 60 mM KCl, respectively). Nonlinear regression with Sigmoidal dose-response analysis was performed using Prism version 3.02. (GraphPad Software, San Diego, CA) Data are presented as means ± S.E.

Membrane Preparation and Immunoblotting—Crude membrane preparations were made 36 h after infection of differentiated 1B5 myotubes with the appropriate amplicon virions at an MOI of 0.5. Myotubes were harvested in harvest buffer (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, and 0.6 mM EDTA, pH 7.2) from 10–15 10-cm plates and centrifuged for 10 min at 250 x g. The pellet was resuspended in buffer consisting of 250 mM sucrose, 10 mM HEPES pH 7.4, supplemented with 1 mM EDTA, 10 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 5 µg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride, and then homogenized using a Polytron cell disrupter (Brinkmann Instruments, Westbury, NY). The whole cell homogenates were centrifuged for 20 min at 1,500 x g, and the supernatants were collected and re-centrifuged for 60 min at 100,000 x g at 4 °C. The membrane pellets were finally resuspended in 250 mM sucrose, 20 mM HEPES, pH 7.4, frozen in liquid N2, and stored at –80 °C. SDS-polyacrylamide gel electrophoresis was performed on proteins from the crude homogenates. Immunoblots were incubated with monoclonal antibody 34C (Airey and Sutko, ISHB, University of Iowa) that recognizes both RyR-1 and RyR-3, and then incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody. Immunoreactive proteins were revealed with SuperSignal ultra chemiluminescent substrate (Pierce).

[3H]Ryanodine Binding Assays—Specific binding of 1 nM [3H]ryanodine to RyR1 (25 µg of protein) was assayed in buffer containing 250 mM KCl, 100 nM CaCl2, 20 mM HEPES, pH 7.4 (Buffer A). Nonspecific binding of [3H]ryanodine was determined by an addition of 1000-fold of unlabeled ryanodine. For calcium activation/inhibition curves, the concentrations of free Ca2+ were obtained by adding the appropriate amount of EGTA in Buffer A based on calculations and stability constants derived from the computer software Bound and Determined (B. A. D.). The influence of caffeine and Mg2+ on equilibrium binding of [3H]ryanodine (1 nM) was evaluated for each RyR1 preparation by additions from a 100x stock to Buffer A. All reaction mixtures were equilibrated at 37 °C for 3 h. Binding reactions were quenched by rapid filtration through Whatman GF/B glass fiber filters, using a Brandel cell harvester. The filters were washed twice with 3 ml of ice-cold harvest buffer containing 20 mM Tris base, 250 mM KCl, 15 mM NaCl, 50 µM CaCl2, pH 7.1 and were soaked in 5 ml of scintillation mixture overnight. The radioactivity on each filter disk was measured using a scintillation counter (Beckman, model LS 6000IC).

Specific [3H]ryanodine binding was determined by subtracting the nonspecific binding from the total binding. The effect of each MH mutation on the [3H]ryanodine binding was analyzed by obtaining sigmoidal curves using ORIGINTM Technical Graphics and Data Analysis Software (Microcal, Northampton, MA). The IC50 and EC50 values and their respective S.D. were calculated directly from linear curve fitting of all the data points. Hill coefficients and statistical data were obtained from these data.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein Expression—A Western blot of membrane preparations from 1B5 myotubes expressing all of the RyR1s studied is shown in Fig. 1. Although the total amount of any RyR expressed varied slightly from preparation to preparation, there were no significant differences in either the levels of expression or molecular size between MHRyR1s and WTRyR1.



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FIG. 1.
Expression of WTRyR1 and MHRyR1 in 1B5 myotubes. 20 µg of protein from the crude homogenates of differentiated 1B5 myotubes with the amplicon virions carrying WTRyR1 and MHRyR1 cDNAs prepared 36 h after infection were subjected to SDS/PAGE (6% gels), transferred to nitrocellulose filters, and probed with monoclonal antibody 34C and horseradish-peroxidase-conjugated goat anti-mouse secondary antibody. The variation in the total amount of any RyR expressed differed from preparation to preparation due to differences in viral titer that led to differences in the number of cells transduced by the virus in each preparation.

 

Myotubes Expressing MHRyR1s Have Heightened Sensitivity to Depolarization and Direct Channel Agonists—Dyspedic 1B5 myotubes (RyR-null) did not respond when challenged with depolarizing concentrations of K+ as high as 80 mM (data not shown). Myotubes transduced with WTRyR1 responded to K+ ≥30 mM with a significant Ca2+ transient whose primary origin was release from SR stores, whereas ≤20 mM K+ was sufficient to elicit a Ca2+ transient in myotubes expressing any of the MHRyR1s tested (Fig. 2A). Statistical curve-fitting of the dose-response relationships, followed by Tukey's multiple comparison test, revealed that EC50 values for K+-induced responses for myotubes expressing all MH mutations tested were significantly lower (p < 0.05) than those expressing WTRyR1 (Fig. 2B). No significant differences in sensitivity to K+ depolarization were detected among myotubes expressing the seven MHRyR1s tested.



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FIG. 2.
Ca2+ release stimulated by KCl in dyspedic myotubes transfected with WTRyR1 and MHRyR1 cDNAs. A, representative traces of Ca2+ responses to incremental doses of KCl intransfected dyspedic myotubes. B, corresponding sigmoidal dose-response curves for WTRyR1 and MHRyR1s. Calcium imaging, data calculation, and sigmoidal dose-response analysis were performed as described under "Experimental Procedures." EC50 values for all the constructs are presented in the inset. *, EC50 significantly lower than WTRyR1, p < 0.05 (Tukey's multiple comparison test). Data in B are presented as means ± S.E.

 

The heightened sensitivity of myotubes expressing MHRyR1s extended to the direct channel activators caffeine and 4-CmC as well. Caffeine challenges (≤40 mM, 30 s) failed to mobilize Ca2+ release from SR from intact dyspedic myotubes (data not shown). Myotubes transduced with WTRyR1 showed a threshold response to ≥ 2 mM caffeine, whereas myotubes expressing RyR1 bearing any of the MH mutations tested responded to ≤1 mM caffeine (Fig. 3A). Sigmoidal curve fitting revealed the EC50 values of all MHRyR1s for caffeine-induced Ca2+ release were significantly lower than myotubes expressing WTRyR1 (p < 0.05; Fig. 3B). No significant differences in sensitivity to caffeine were observed among the seven MH mutations tested.



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FIG. 3.
Ca2+ release stimulated by caffeine in dyspedic myotubes transfected with WTRyR1 and MHRyR1 cDNAs. A, representative traces of Ca2+ responses to incremental doses of caffeine in transfected dyspedic myotubes. B, corresponding sigmoidal dose-response curves for WTRyR1 and MHRyR1s. Calcium imaging, data calculation, and sigmoidal dose-response analysis were performed as shown under "Experimental Procedures." EC50 values for all the constructs are presented in the inset. *, EC50 significantly lower than WTRyR1, p < 0.05 (Tukey's multiple comparison test). Data in B are presented as means ± S.E.

 

Similarly 4-CmC failed to mobilize Ca2+ release in dyspedic myotubes (≤1 mM; data not shown), whereas myotubes transduced with WTRyR1 responded with a sustained rise in intracellular Ca2+ with an average threshold of 100 µM 4-CmC (Fig. 4A). In contrast, myotubes expressing any MHRyR1 tested had an average 5-fold lower threshold for eliciting responses to 4-CmC (threshold of 20 µM; Fig. 4A). The EC50 values for the 4-CmC responses of all the MH myotubes was significantly lower than that of wild-type myotubes (p < 0.05; Fig. 4B). However no significant differences in sensitivity to 4-CmC among myotubes expressing the seven MHRyR1s tested were observed.



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FIG. 4.
Ca2+ release stimulated by 4-CmC in dyspedic myotubes transfected with wild-type or mutant RYR1 cDNAs. A, representative traces of Ca2+ responses to incremental doses of 4-CmC of transfected dyspedic myotubes. B, corresponding sigmoidal doseresponse curves for WTRyR1 and MHRyR1s. Calcium imaging, data calculation, and sigmoidal dose-response analysis were performed as shown under "Experimental Procedures." EC50 values for all the constructs are presented in the inset. *, EC50 significantly lower than WTRyR1, p < 0.05 (Tukey's multiple comparison test). Data in B are presented as means ± S.E.

 

Comparison of the Maximal Cellular Responses to K+, Caffeine, and 4-CmC—Dose-response curves for K+, caffeine, and 4-CmC were plotted directly without normalization to maximum response attained in each cell at saturating activator (20 mM caffeine, 500 µM 4-CmC, and 60 mM KCl; see Fig. 5). At low concentrations (0.1, 0.5, 1, and 2 mM) of caffeine, the amplitude of the Ca2+ response was significantly greater for myotubes expressing any of the MHRyR1s tested when compared with those expressing WTRyR-1. However at saturating caffeine (20 mM), the cellular responses for all myotubes expressing any MHRyR1s were significantly smaller than those expressing WTRyR1. The same pattern of responses was consistently observed when myotubes were challenged with low (5, 20, and 100 µM) and high (500 µM) concentrations of 4-CmC. These results could possibly be the result of either (1) partial depletion of the SR Ca2+ stores in myotubes expressing MHRyR1 as previously concluded from previous results from heterologous expression of MHRyR1 in HEK 293 (14), or (2) a diminished efficacy of MHRyR1 channels to fully activate in response to saturating levels of direct channel agonist.



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FIG. 5.
Comparison of the amplitudes of the Ca2+ release responses stimulated by caffeine, 4-CmC, and KCl in dyspedic myotubes transfected with wild-type or mutant RyR1 cDNAs. Dose-response curves for K+, caffeine, and 4-CmC were plotted directly without normalization to the maximum response attained in each cell at the saturating activator concentration (20 mM caffeine, 500 µM 4-CmC, and 60 mM KCl (–, WTRyR1; –, MHRyR1s). Data are presented as means ± S.E.

 

However, the amplitude of K+-induced Ca2+ transients for myotubes expressing any of the seven MHRyR1s tested was significantly higher than that produced in myotubes expressing WTRyR1, at all KCl concentrations including the highest KCl concentration (60 mM). Of note, in myotubes expressing WTRyR1 the amplitude of the response to 20 mM caffeine was similar to that observed with a challenge of 60 mM K+ (52.7 ± 4.6% versus 48.3 ± 4.7%). These data suggest that myotubes carrying MHRyR1s produced larger Ca2+ transients in response to depolarization mimicking EC coupling than responses to the direct channel activators caffeine or 4-CmC. In skeletal myotubes expressing the MHRyR1s studied here, it is unlikely that diminished responses to saturating caffeine or 4-CmC were a result of store depletion since EC coupling in myotubes expressing all the MHRyR1s tested clearly produced significantly larger transients than those expressing WTRyR1.

MHRyR1 Anomalies in Regulation by Ca2+ and Mg2+ Revealed by [3H]Ryanodine Binding Analysis—The binding of nanomolar [3H]ryanodine to conformationally sensitive sites on the RyR1 complex was utilized to directly measure the sensitivity of WTRyR1 and MHRyR1s to activation by Ca2+, and inhibition by Ca2+ or Mg2+, important physiological regulators of EC coupling. [3H]Ryanodine binding analyses were performed using membrane vesicles isolated from transduced myotubes as described under "Experimental Procedures." To quantitatively assess the level of expression of the seven RyR1 constructs studied, radioligand-receptor binding analysis was performed in the presence of high ionic strength (1 M KCl), optimal Ca2+ (50 µM), and near saturating [3H]ryanodine for the high affinity site. Under these condition expression levels in preparations for all seven constructs were similar ranging from 0.5 to 1.2 pmol/mg of protein (Table I). Representative dose-response curves for Ca2+ activation of WTRyR1, R2458H, and T4825I were summarized in Fig. 6A. Maximal activation of [3H]ryanodine binding occurred between 1 and 100 µM Ca2+ for all RyR1 proteins tested, and no significant differences in EC50 were observed among any of the seven MHRyR1s studied and WTRyR1 (Table I). It was interesting to note that, unlike WTRyR1, two of the MHRyR1s (R2458H and T4825I, Fig. 6A) displayed significant levels of specific [3H]ryanodine-binding in the presence of very low (10 and 100 nM) Ca2+ suggesting a failure to completely inactivate at resting [Ca2+].


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TABLE I
EC50 for Ca2+ activation, IC50 for Ca2+/Mg2+ inhibition, and Bmax of [3H]ryanodine binding of WTRyR1 and MHRyR1s

Experiments done twice in triplicate. Data presented as mean ± S.D. S.D. were calculated directly from linear curve fitting of Logit-log analyses.

 


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FIG. 6.
Dependence of [3H]ryanodine binding on Ca2+ and Mg2+ Representative dose-response curves of [3H]ryanodine binding for WTRyR1 and MHRyR1s: activated by Ca2+ (A, left), inhibited by Ca2+ (A, right) and inhibited by Mg2+ (B). [3H]Ryanodine binding was performed in the presence of 1 nM [3H]ryanodine. Data points represent the mean ± S.D. of two separate experiments, each done in triplicate. EC50 and IC50 values are shown in Table I.

 

In marked contrast, when Ca2+ inhibition of the binding of [3H]ryanodine was studied, the dose-response curves of all MHRyR1s were significantly shifted to the right of WTRyR1 (p < 0.05) (Fig. 6A, right and Table I). Of note, T4825I RyR1 was the least sensitive to inhibition by Ca2+ and had a significantly higher IC50 than all other MH mutations tested (p < 0.01). WTRyR1 had its highest binding activity in the presence of 100 µM Ca2+, was inhibited by increasing Ca2+ in a graded dose-dependent manner and was completely inhibited between 4 and5mM Ca2+. In contrast, all MHRyR1s tested exhibited their highest binding activity between 1 and 2 mM Ca2+ and R2458H and T4825I (Fig. 6A, right) could not be completely inhibited at 8 mM Ca2+, the highest concentration tested.

Diminished negative modulation of MHRyR1s by Ca2+ extended to negative modulation by Mg2+. Representative Mg2+ dose-response curves for inhibition of [3H]ryanodine binding to WTRyR1 and MHRyR1s are summarized in Fig. 6B and Table I. All MHRyR1 proteins studied were significantly less sensitive to inhibition by Mg2+ compared with WTRyR1 (IC50 values for all MHRyR1s significantly greater than WTRyR1; p < 0.01). For example, [3H]ryanodine binding to WTRyR1 declined rapidly at Mg2+ concentrations higher than 500 µM, whereas binding activity for R2458H (Fig. 6B) and all other MHRyR1s tested maintained a binding activity of >90% at 1 mM, with the exception of R164C (81% max at 1 mM). In consonance with diminished sensitivity to inhibition by Ca2+, T4825I also had the lowest sensitivity to inhibition by Mg2+, maintaining 90% of maximum activity in the presence of 3 mM Mg2+.

Taken together these results indicated two important anomalies in the regulation of MHRyR1s contributed to the MH phenotype in intact myotubes: 1) a significant diminution in the feedback inhibition of channel activity by Ca2+ and Mg2+, and 2) an incomplete inactivation of many of the MHRyR1 channels in the presence [Ca2+] that mimics the intracellular resting state of intact myotubes. The present results do not indicate an altered sensitivity toward activation of any of the MHRyR1s by Ca2+.

Heightened Efficacy of Caffeine Toward MHRyR1s at Resting Intracellular Ca2+In the present study, the ability of caffeine to activate [3H]ryanodine binding to vesicles isolated from myotubes expressing WTRyR1 and MHRyR1s was performed at a Ca2+ concentration of 100 nM that mimics the conditions present in the typical wild-type myotube. Fig. 7 shows representative data of the amounts of specific [3H]ryanodine binding at 0, 3, and 10 mM caffeine (Fig. 7, A, B, and C, respectively). Interestingly, in the absence of caffeine stimulation (Fig. 7A), R2458H and T4825I both had significantly higher binding activities than WTRyR1. These data suggest that the calcium release channels carrying these MH mutations may not completely inactivate at Ca2+ concentrations seen in normal resting myotubes. In the presence of a fixed 100 nM Ca2+ in the binding medium, [3H]ryanodine binding activity did not change significantly with expressed WTRyR or MHRyR1s in the presence of 0.1, 0.3, 1.0, or 1.5 mM caffeine (data not shown). However, in the presence of 3 mM caffeine (Fig. 7B), all MHRyR1s tested showed significantly enhanced [3H]ryanodine binding activity relative to both WTRyR1 and control (lacking caffeine, Fig. 7A). This difference became even more dramatic at higher concentrations of caffeine: 6 mM (not shown), 10 mM (Fig. 7C), and 30 mM (not shown). The largest increases in binding activity at all caffeine concentrations tested were observed with R2458H and T4825I (7.13- and 14.6-fold increase over control, respectively). In contrast, WTRyR1 failed to be activated by ≤6 mM caffeine. The dose-response relationship increased linearly to 60 mM, the highest concentration tested, for all MHRyR1s studied preventing sigmoidal dose-response analysis of this data.



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FIG. 7.
Caffeine activation of [3H]ryanodine binding. Representative data of caffeine stimulation of [3H]ryanodine binding for WTRyR1 and MHRyR1s at 0 mM (A), 3 mM (B), and 10 mM (C) caffeine. [3H]Ryanodine binding was performed in the presence of 1 nM [3H]ryanodine and 100 nM Ca2+. Data points represent the means ± S.D. of two separate experiments done in triplicate. *, p < 0.05; +, p < 0.01; significantly higher amount of [3H]ryanodine bound compared with WTRyR1. (unpaired Student's t test).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
An important new finding emerging from homologous expression studies was that all seven MHRyR1s significantly enhanced the sensitivity of myotubes to depolarization induced Ca2+ release. The finding indicates that a common phenotype of MHRyR1 mutations in any of the three hot spots is an altered functional interaction between the Ca2+ release channel carrying the MH mutation and DHPR, which functions as the sensor of depolarization of the surface membrane. Potassium-induced muscle contractures (21), an experimental model for the study of depolarization-contraction coupling of skeletal muscle fibers, have been previously used to study the influence of MH mutations on EC coupling. Using bundles of intact muscle fibers dissected from MH susceptible pigs, Gallant and Lentz (22) demonstrated that potassium-induced contractures were significantly larger in R615C pig muscles than in normal muscles, and the threshold for K+ contractures in R615C muscles was significantly lower than in normal muscles (23). In addition, Dietze et al. (24) showed that the Ca2+ released in porcine R615C myotubes was activated at a significantly lower depolarizing potential than control myotubes. These studies indicated an alteration in the depolarization-contraction coupling coincident with and perhaps caused by the R615C mutation. However the possibility that this phenotype was the consequence of coexisting differences in affected myotubes unrelated to EC coupling could not be excluded. The present study utilized genetically homogeneous dyspedic myotubes whose sole difference is the genotype of the RyR1 that is expressed. We demonstrated here for the first time that expression of any one of seven MHRyR1 alone was both necessary and sufficient to increase the sensitivity of skeletal EC coupling to K+-induced depolarization compared with myotubes expressing WTRyR1. Based on these results we propose that enhanced sensitivity to depolarization may be a common anomaly for all MH mutations, regardless of their location within the linear sequence of the RyR1 protomer. The thresholds for K+-induced Ca2+ release for WTRyR1 (20 mM) and MHRyR1 (≤10 mM) seen in the present study are both lower than the threshold values for K+-induced contracture in Gallant's study (40 and 15 mM for WTRyR1 and R615C, respectively) (23). An increased sensitivity of the EC coupling machinery to depolarization as a direct result of MHRyR1 may therefore play an dominant role in the occurrence of MH episodes, but the exact mechanisms related to the triggering events remains to be explored (24).

Caffeine sensitivity is routinely used for the diagnosis of MH susceptibility. A muscle specimen is defined as MH susceptible if it exhibits a specified amount of contracture force (e.g. ≥2mN) after separate application of both ≤2.0 mM caffeine and ≤0.44 mM halothane (25). Several different preparations have been utilized for functional analysis of caffeine sensitivity of MHRyR1. These have included measurements of caffeine-induced Ca2+ release from heavy SR membranes prepared from MH pig muscles (26), as well as cellular responses to caffeine utilizing either cultured primary myotubes prepared from human or pig muscle biopsies (22, 27), or non-muscle cells expressing MHRyR1s (12). Collectively these diverse preparations have shown that an increased caffeine sensitivity was a common characteristic for each MH mutation. Tong et al. (11) investigated the caffeine sensitivity of calcium release in HEK-293 cells for 15 MH mutants including five of those investigated in this study, R164C, G342R, R615C, R2163C, and R2458H. Consistent with our results, cells expressing all the 15 MH mutations had significantly increased caffeine sensitivity of calcium release compared with cells expressing WTRyR1. Unexpectedly no statistically significant difference in the EC50 values was detected among the MH mutations tested. Moreover the mean EC50 of the fifteen MHRyR1 expressed in HEK-293 was similar to the EC50 obtained for the seven MHRyR1 expressed in myotubes here (0.69 ± 0.21 mM versus 0.81 ± 0.15 mM). R2163C and R2458H showed relatively higher sensitivity than G342R and R615C in both studies. Interestingly in Tong's study, R164C was relatively more sensitive than G342R and R615C, which was opposite to our result in myotubes. The EC50 for caffeine of WTRyR1 expressed in dyspedic myotubes is almost 2-fold higher than that seen in HEK cells (2.7 mM versus 1.4 mM). These differences may be due to differences between the homologous and heterologous expression systems and/or the fact that Ca2+ measurements were obtained from cell populations (11) versus individual myotubes (present study).

4-CmC appears to have a similar effect to caffeine in muscle and myotubes but causes skeletal muscle contractures at considerably lower concentrations than caffeine (28, 29). A recent multi-center study to evaluate the usefulness of an IVCT with 4-CmC found that the predictive value of 4-CmC for the diagnosis of MH susceptibility is as high as that of caffeine, and its use in IVCT has been proposed to refine diagnosis of MH susceptibility (30). Transient expression of R615C and WTRyR1 in COS-7 cells first revealed a significantly lower threshold for 4-CmC-induced Ca2+ mobilization for MHRyR1 (56 ± 3 versus 166 ± 12 µM respectively, Ref. 10). Additional evidence of heightened sensitivity toward 4-CmC in MH came from Epstein-Barr virus-immortalized B-cells isolated from MH-susceptible individuals carrying the V2168M mutation, and primary cultured myotubes from a patient with the T2206M mutation (27, 31). The present study extends the comparison of 4-CmC sensitivity to seven MHRyR1s expressed in dyspedic myotubes. Myotubes expressing all seven MH mutations were significantly more sensitive to 4-CmC than those expressing WTRyR1 strongly suggesting that heightened sensitivity of myotubes carrying MH mutations to 4-CmC was a universal characteristic, and supports the idea that 4-CmC could be used to supplement caffeine in the IVCT for the diagnosis of MH susceptibility.

Estimates of the calcium content of the sarcoplasmic reticulum have been made in HEK-293 cells expressing MH mutants (13, 14), in primary myotubes derived from biopsies of MHS patients (27) and in normal amphibian skeletal muscle fibers (32) using the calcium-sensitive dye fura2-AM to measure the maximal amplitude of caffeine or 4-CmC-induced Ca2+ release. In these studies, seventeen MH mutations were consistently associated with a significant reduction of the maximum amplitude of caffeine or 4-CmC-induced Ca2+ release, suggesting that the SR calcium stores were reduced in MH muscle. In the present study, the maximum responses to caffeine (20 mM) and 4-CmC (0.5 mM) were also diminished in all myotubes expressing MHRyR1 compared with those expressing WTRyR1 as shown in Fig. 5. However, a significant observation that argues against depletion of SR stores in myotubes expressing MHRyR1 was the consistently greater maximal responses achieved with 60 mM K+ depolarization compared with myotubes expressing WTRyR1. This unexpected observation appears to be coupled with the observation that the slope of the KCl dose-response curve is much less steep for all 7 MHRyRs than for WTRyR (Fig. 2B) and begs for an explanation. Gallant and Lentz (22) has previously shown that the slope of the K+ response was similar to the one found in the present study and we (33) have previously shown that MHS pig muscle always releases more Ca2+ in response to partial depolarization by K+ than MHN muscle. As an explanation of this phenomenon we offer the following two possibilities. 1. It has been previously shown that not all RyRs are in an "active" state being able to either respond to a direct agonist (caffeine) or bind ryanodine, but these "inactive" channels can be converted to "active" channels with compounds like Bastadin 10 (34). We hypothesize that one possible mechanism for this difference is that the stores are not depleted but that a higher percentage of MH channels are in the leak conformation and are unresponsive to direct agonists. However, "physiologic" activation of the ryanodine receptors is able to convert passive leak channels into active release channels in a graded manner and thus the graded increased response. A second possible mechanism for this difference could come from our observation that RyR1 is coupled to SOC entry (35). In this hypothesis the cause for the ascending Ca2+ release response to depolarization is that although the SR Ca2+ stores are in fact depleted in MH myotubes, upon progressive partial depolarization the MHRyR1s assume a conformation that facilitates SOC entry in a graded manner. Thus while the direct agonist caffeine is able to demonstrate the reduction in SR Ca2+ stores this reduced store is masked by external Ca2+ entry during depolarization induced Ca2+ responses. Thus it is uncertain at this point that the diminished responsiveness of MH myotubes to direct channel agonists is not the result of SR store depletion. However it is certain that the differences in maximal responses observed with myotubes expressing MHRyR1s were related to the nature of the stimulus.

The molecular mechanisms underlying the MH phenotype was further examined using [3H]ryanodine binding analysis with membranes isolated from myotubes expressing WTRyR-1 and MHRyR1. Quantitative differences in the response of wild-type and MH susceptible channels to Ca2+ and Mg+, two physiologically important regulators of EC coupling, were found to be significant. Consistent with previous results using SR membranes from MH susceptible porcine muscle (R615C; Refs. 36 and 37), human muscle biopsies (G2434R; Ref. 38), myotubes and HEK cells expressing A2350T (39), and R615C pigs using single-channel recordings (2, 40, 41), we demonstrated that MH RyRs from all three MH/CCD hot spots are universally associated with decreased sensitivity for inhibition by both calcium and magnesium (inhibition curves shifted strongly to the right). For the six MH mutations tested in the present study inhibition by Ca2+ (IC50 values 1.2–3.9-fold higher than WTRyR1) and to a greater extent by Mg2+ (IC50 values 2–8-fold higher than WTRyR1) were significantly impaired. In this regard, previous studies of [3H]ryanodine receptor-binding analysis and analysis of the gating activity of single channels reconstituted in BLM have consistently shown significant reduction in the sensitivity of MHRyR1 to inhibition by Ca2+ and Mg2+ over a broad range of experimental conditions. Given the important role of Ca2+ and Mg2+ in negative regulation of SR Ca2+ release, it is reasonable to conclude that a reduction in negative feedback control of RyR1 is a principle contributor to the MH phenotype. By comparison, experiments aimed at assessing quantitative differences between MHRyR1s and WTRyR1 toward activation by Ca2+ have been somewhat more variable and dependent on the experimental conditions used. In the present study, no significant differences in EC50 values for Ca2+ activation were measured for any MH mutation tested compared with WTRyR1. Mickelson et al. (42) reported that the EC50 for Ca2+ activation of [3H]ryanodine binding was not statistically different between porcine WTRyR1 and R615C when measured with 10, 100, 300 nM radioligand, although apparent differences in EC50 was more evident when measurements were performed with >10 nM [3H]ryanodine. Interestingly they found a nearly 2-fold lower EC50 (p < 0.05) in R615C in the presence of 30 nM [3H]ryanodine (Table I in Ref. 42). Subsequent studies with porcine SR preparations have reported from no difference to a 2.7-fold lower EC50 for Ca2+ activation of the porcine MH mutation R615C compared with wild type when measured with 10–100 nM radioligand (3638, 43). The differences in EC50 values may have been, at least in part, due to differences in ionic strength in the assay buffer that ranged from 0.1 M (38) to 1 M (43). Interestingly measurable differences in Ca2+ activation were essentially negated by the presence of high ionic strength in the assay buffer (43). Another important experimental factor likely to influence the dose-response relationship toward Ca2+ is the final concentration of ryanodine in the binding assay. Ryanodine has been proposed to allosterically influence RyR1 in a dose-dependent manner (4447). Recent studies have provided experimental evidence that one consequence of ryanodine allosterism on RyR1 (48) and RyR2 is the enhanced sensitivity toward activation by Ca2+ (49, 50). Therefore we cannot discount the possibility that the free concentration of [3H]ryanodine used in previous binding studies differentially altered the sensitivity of MHRyR1 and WTRyR-1 toward Ca2+ activation, especially at concentrations >10 nM. Two observations support this possibility. First, the KD for high affinity [3H]ryanodine binding sites is ~4-fold lower (higher affinity) for R615C compared with WTRyR1 when measured in low ionic strength (38). Second, the EC50 for activation of single channel gating activity by Ca2+ does not appear to be significantly different between R615C and WTRyR1 (2, 43). Specifically functional analysis of single R615C and WTRyR1 channels incorporated in bilayer lipid membranes showed that they were progressively activated in a similar fashion by cytoplasmic (cis) Ca2+ from pCa 7 to pCa 4. However at Ca2+ above 100 µM cis, A615C channels remained open for significantly longer times than WTRyR-1 channels, indicating that the small increase in sensitivity toward Ca2+ activation observed with R615C was very likely to have been mediated by altered negative regulation contributed by low affinity Ca2+ (inhibitory) binding sites (2). Results from the present study reaffirms that, regardless of the location of the mutation, a common characteristic of MHRyR1 was a significant and selective diminution in negative feedback regulation by Ca2+ and Mg2+ and that this difference is possibly mediated by changes at low affinity cation binding sites.

Another functional characteristic of some, though not all, MHRyR1s examined in the present study, was the inability to completely inactivate specific [3H]ryanodine binding at below [Ca2+]i typically found in resting skeletal myotubes (Figs. 6A and 7A). Interestingly two MHRyR1s, R2458H and T4825I, maintained significantly higher binding activity than WTRyR1 at 100 nM Ca2+ in the absence of caffeine (p < 0.05 for R2458H, p < 0.001 for T4825I), indicating that these channels were resistant to complete inactivation under physiologic conditions of the sarcoplasm at rest. Although, at these low Ca2+ conditions all MHRyR1 proteins studied were much more sensitive to caffeine (≤3 mM) compared with WTRyR-1, R2458H, and T4825I responded to caffeine to a greater extent than the other MHRyR1s. The greater response of these mutants may be attributed to their inability to completely inactivate at low Ca2+. Recently Jiang et al. (51) expressed the R4496C mutation of mouse RyR2, the equivalent to human RyR2 mutation R4497C linked to arrythmogenic right ventricular cardiomyopathy (ARVC), in HEK-293 cells. [3H]Ryanodine binding studies revealed that R4496C exhibited abnormally high RyR2 channel activity particularly at low Ca2+ concentrations. The same results obtained here with the T4825I RyR1 mutation revealed that a common molecular dysfunction, incomplete inactivation of RyR channels at a Ca2+ concentration present in resting muscle cells, may significantly contribute to the etiology of tissue specific diseases; MH susceptibility in skeletal muscle and ARVC in cardiomyocytes.

Taken together these results obtained from skeletal myotubes indicated that the heightened sensitivity of myotubes expressing MHRyR1s to caffeine (seen as a left shift in EC50; Fig. 3B) is the result of: 1) significantly heightened sensitivity to caffeine at resting Ca2+ concentrations found in the resting myotubes, and 2) diminished feedback inhibition of Ca2+ release by Ca2+ and Mg2+ once it has been initiated as a consequence of disabled low affinity cation binding sites. These results were consistent with, and provide a mechanistic framework for, previous results which also identified that caffeine (>2 mM) enhanced the binding of [3H]ryanodine to a significantly greater extent with SR vesicles from porcine muscles (R615C; Ref. 29), SR vesicles from muscle biopsies of MH patients for G2434R (38), and SR vesicles from transfected HEK-293 cells for A2350T (39) compared with WTRyR-1.

Moreover these data demonstrated, for the first time, that there were quantitative functional differences among individual MH mutations. We cannot explain why the C-terminal mutation T4825I produced such a striking functional change. In one recent study on CCD mutations (52), 12 different C-terminal mutations of RyR1 in 16 unrelated families were identified while none of the previously defined N-terminal and neutral region MH/CCD mutations were found. Unlike previous studies in which CCD mutations were found largely in patients who had a MH phenotype during anesthesia, only patients with both clinical symptoms of congenital myopathy and histological abnormalities in skeletal muscle were recruited in this study. Their findings indicated that the C-terminal mutations tend to be related to more severe CCD phenotype. Interestingly, among the 12 C-terminal CCD mutations found, only one mutation, R4825C that is immediately adjacent to the T4826I mutation, was identified to be associated with MHS phenotype by IVCT. Our data strongly suggest that the C-terminal mutation T4825I is associated with a more "leaky" calcium release channel but this awaits direct proof in the future.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Brigham and Women's Hospital, 20 Shattuck St. SR 158, Boston, MA 02115. Tel.: 617-732-6881; Fax: 617-732-6927; E-mail: tyang{at}zeus.bwh.harvard.edu.

1 The abbreviations used are: MH, malignant hyperthermia; EC, excitation-contraction; EC50, effective concentration needed to produce half-maximal binding; IC50, inhibiting concentration needed to produce half-maximal binding; RyR1, ryanodine receptor type 1; SR, sarcoplasmic reticulum; WT, wild type; 4-CmC, 4-chloro-m-cresol; CCD, central core disease; MOI, multiplicity of infection. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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