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.
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ABSTRACT |
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INTRODUCTION |
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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 1s-DHPR
(
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 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+.
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EXPERIMENTAL PROCEDURES |
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HSV-1 Virion ProductionAll 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 Infection1B5 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 6070% 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 ImagingStock 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 1020 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 ImmunoblottingCrude 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 1015 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 AssaysSpecific 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.
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RESULTS |
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Myotubes Expressing MHRyR1s Have Heightened Sensitivity to Depolarization and Direct Channel AgonistsDyspedic 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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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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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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Comparison of the Maximal Cellular Responses to K+, Caffeine, and 4-CmCDose-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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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 AnalysisThe 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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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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DISCUSSION |
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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
1.23.9-fold higher than WTRyR1) and to a greater
extent by Mg2+ (IC50 values 28-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 10100
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.
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FOOTNOTES |
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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.
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