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Reduced Threshold for Luminal Ca2+ Activation of RyR1 Underlies a Causal Mechanism of Porcine Malignant Hyperthermia*

Naturally occurring mutations in the skeletal muscle Ca2+ release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca2+ release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca2+ release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process we refer to as store overload-induced Ca2+ release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca2+ activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca2+ and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca2+ activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca2+ regulation of RyR1 represents a primary causal mechanism of MH.

by volatile anesthetics (e.g. halothane) and depolarizing muscle relaxants and is characterized by muscle rigidity and a hypermetabolic state (1)(2)(3)(4)(5). MH also occurs in pigs, in which it is caused by stress and known as porcine stress syndrome (6,7). A single point mutation, R615C, in the pig skeletal muscle ryanodine receptor RyR1 is responsible for all cases of porcine MH (6,7). On the other hand, human MH has been linked to a large number of mutations in RyR1 (4,5,8). Some human RyR1 mutations have also been linked to central core disease, which is often associated with MH. Although the genetic basis of MH has been well defined, the molecular mechanisms by which RyR1 mutations confer MH susceptibility and volatile anesthetics and stress trigger MH are not completely understood.
The pig model has proven invaluable in investigating the molecular basis of MH, and these studies have consistently demonstrated that Ca 2ϩ release from MH-susceptible (MHS) pig skeletal muscle or sarcoplasmic reticulum (SR) membrane vesicles is enhanced upon exposure to various stimuli (1, 4, 9 -14). However, to date no clear mechanistic basis of this enhanced responsiveness of MHS RyR1 channels to stimuli has emerged. For example, some studies reported that this enhanced activity of MHS RyR1 channels was associated with changes in the apparent sensitivity of the channel to cytosolic Ca 2ϩ or Mg 2ϩ , whereas others found no marked difference in the apparent sensitivity to cytosolic Ca 2ϩ activation between MHS and normal RyR1 channels (1,(13)(14)(15)(16)(17)(18)(19)(20). Hence, the intrinsic properties of the RyR1 channel that are altered by the MH R615C mutation have remained undefined.
Increasing evidence has highlighted the importance not only of cytosolic Ca 2ϩ , but also of luminal Ca 2ϩ , in controlling the activity of the RyR channel (21)(22)(23). However, in comparison with the extensive investigations of the sensitivity of MHS RyR1 channels to cytosolic Ca 2ϩ and Mg 2ϩ , the effects of the MH R615C mutation on the luminal Ca 2ϩ sensitivity of the channel remain largely unexplored. A notable exception is found among the earliest work investigating the effect of the MH R615C mutation on Ca 2ϩ handling by isolated SR membranes (13,24,25). Nelson and colleagues (13,24,25) originally reported that the luminal Ca 2ϩ load required to trigger spontaneous SR Ca 2ϩ release was markedly reduced in SR membranes isolated from MHS animals, suggesting that a defect in intraluminal Ca 2ϩ regulation may underlie MH.
We have recently shown that disease-causing mutations in the cardiac ryanodine receptor RyR2 increase the sensitivity of the channel to activation by luminal Ca 2ϩ and enhance the propensity for spontaneous Ca 2ϩ release during store Ca 2ϩ overload, a process we have termed store overload-induced Ca 2ϩ release (SOICR) (26,27). Interestingly, disease-linked RyR2 mutations are located in regions corresponding to the MH/central core disease mutation regions in RyR1 (28). This similar distribution suggests that disease-linked RyR2 and RyR1 mutations may exert similar effects on the intrinsic properties of the channel. To test this hypothesis, in the present study, we assessed the impact of the MH R615C mutation and the MHtriggering agent, halothane, on the response of RyR1 to luminal Ca 2ϩ and the propensity for SOICR. We found that the R615C mutation and halothane potentiated luminal Ca 2ϩ response and SOICR. On the other hand, dantrolene, the only treatment for MH, suppressed SOICR. We propose that a reduced threshold for SOICR as a result of augmented luminal Ca 2ϩ activation of RyR1 represents a primary defect underlying the pathogenesis of MH.

EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis-The R615C RyR1 MH mutation in the rabbit RyR1 cDNA was made by the PCR-based overlap extension method (29). The sequence of the PCR-amplified region was confirmed by DNA sequencing. The full-length RyR1 wt and R615C mutant cDNAs were subcloned into the mammalian expression vector pcDNA3.
Single Channel Recordings-Pig RyR1 wt and R615C mutant proteins were partially purified from normal and MHS pig SR microsomes by sucrose density gradient centrifugation. Heart phosphatidylethanolamine and brain phosphatidylserine (Avanti Polar Lipids), dissolved in chloroform, were combined in a 1:1 ratio (w/w), dried under nitrogen gas, and suspended in 30 l of n-decane at a concentration of 12 mg lipid/ml. Bilayers were formed across a 250-m hole in a Delrin partition separating two chambers. The trans chamber (800 l) was connected to the head stage input of an Axopatch 200A amplifier (Axon Instruments, Austin, TX). The cis chamber (1.2 ml) was held at virtual ground. A symmetrical solution containing 250 mM KCl and 25 mM Hepes (pH 7.4), was used for all recordings, unless indicated otherwise. A 4-l aliquot (Ϸ1 g of protein) of the sucrose density gradient-purified wt or mutant RyR1 proteins was added to the cis chamber. Spontaneous channel activity was always tested for sensitivity to EGTA and Ca 2ϩ . The chamber to which the addition of EGTA inhibited the activity of the incorporated channel presumably corresponds to the cytoplasmic side of the Ca 2ϩ release channel. The direction of single channel currents was always measured from the luminal to the cytoplasmic side of the channel, unless mentioned otherwise. Recordings were filtered at 5,000 Hz. Data analyses were carried out using the pclamp 8.1 software package (Axon Instruments). Free Ca 2ϩ concentrations were calculated using the computer program of Fabiato and Fabiato (30).
Generation of Stable, Inducible HEK293 Cell Lines-Stable, inducible HEK293 cell lines expressing RyR1 wt and the R615C mutant were generated using the Flp-In T-REx core kit from Invitrogen. Briefly, the full-length cDNA encoding the RyR1 wt or mutant channel was subcloned into the inducible expression vector, pcDNA5/FRT/TO (Invitrogen). Flp-In T-REx-293 cells were then co-transfected with the inducible expression vector containing the RyR1 wt or mutant cDNA and the pOG44 vector encoding the Flp recombinase in 1:5 ratios using the Ca 2ϩ phosphate precipitation method. Transfected cells were washed with phosphate-buffered saline (137 mM NaCl, 8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 2.7 mM KCl) 1 day after transfection and allowed to grow for 1 more day in fresh medium. The cells were then washed again with phosphate-buffered saline, harvested, and plated onto new dishes. After the cells had attached (ϳ 4 h), the growth medium was replaced with a selective medium containing 200 g/ml hygromycin (Invitrogen). The selective medium was changed every 3-4 days until the desired number of cells was grown. The hygromycin-resistant cells were pooled, aliquoted, and stored at Ϫ80°C. These positive cells are believed to be isogenic, because the integration of the RyR1 cDNA is mediated by the Flp recombinase at a single target site. Each HEK293 cell line was tested for RyR1 expression using Western blotting analysis and immunocytofluorescence staining.
Single Cell Ca 2ϩ Imaging (Cytosolic Ca 2ϩ )-Intracellular Ca 2ϩ transients in stable inducible HEK293 cells expressing the RyR1 wt or the R615C mutant channels were measured using single-cell Ca 2ϩ imaging and the fluorescent Ca 2ϩ indicator dye fura-2 acetoxymethyl ester (AM) as described previously (26). Cells grown on glass coverslips for 24 h after induction by 1 mg/ml tetracycline (Sigma) were loaded with 5 M fura-2 AM in Krebs-Ringer-Hepes (KRH) buffer (125 mM NaCl, 5 mM KCl, 1.2 mM KH 2 PO 4 , 6 mM glucose, 1.2 mM MgCl 2 , 25 mM Hepes, pH 7.4) plus 0.02% pluronic F-127 (Molecular Probes) and 0.1 mg/ml bovine serum albumin for 20 min at room temperature. The coverslips were then mounted in a perfusion chamber (Warner Instruments, Hamden, CT) on an inverted microscope (Nikon TE2000-S) equipped with an S-Fluor 20ϫ/0.75 objective. The cells were continuously perfused with KRH buffer containing various concentrations of CaCl 2 (0.2-10 mM) at room temperature. 10 mM caffeine was applied at the end of each experiment to confirm the expression of active RyR1 channels. Time lapse images (0.33 frames s Ϫ1 ) were captured and analyzed with the Compix Inc. Simple PCI6 software.
Single Cell Ca 2ϩ Imaging (Luminal Ca 2ϩ )-To monitor PCI6 luminal Ca 2ϩ transients in HEK293 cells, we used the Ca 2ϩsensitive fluorescence resonance energy transfer (FRET)-based cameleon protein D1ER (31). Stable, inducible HEK293 cells expressing RyR1 wt or mutant channels were transfected using the Ca 2ϩ phosphate precipitation method, with D1ER cDNA 24 h before the induction of RyR1 expression. The cells were perfused continuously with KRH buffer (125 mM NaCl, 5 mM KCl, 1.2 mM KH 2 PO 4 , 6 mM glucose, 1.2 mM MgCl 2 , 25 mM Hepes, pH 7.4) containing various concentrations of CaCl 2 (0 or 5 mM) and tetracaine (1 mM) or caffeine (20 mM) at room temperature. The images were captured with Compix Inc. Simple PCI 6 software at 470-and 535-nm emission, with excitation at 430 nm, every 2 s using an inverted microscope (Nikon TE2000-S) equipped with a S-Fluor 20ϫ/0.75 objective. The amount of FRET was determined from the ratio of the emissions at 535 and 470 nm.

RESULTS
The Porcine MH R615C Mutation Enhances the Luminal Ca 2ϩ Activation of Single RyR1 Channels-To directly assess the impact of the R615C mutation on luminal Ca 2ϩ activation, we incorporated single normal (RyR1 wt) and MHS (RyR1-R615C) RyR1 channels into planar lipid bilayers and examined their responses to increasing concentrations of luminal Ca 2ϩ . As shown in Fig. 1A, elevating the luminal Ca 2ϩ concentration from ϳ45 nM to 50 mM had little effect on single wt channels but markedly activated single R615C mutant channels with an activation threshold of about 0.5 mM luminal Ca 2ϩ (Fig. 1A, panel e).
For instance, at 1.2 mM luminal Ca 2ϩ , the average open probability (P o ) for single R615C mutant channels was 0.070 Ϯ 0.024 (n ϭ 14) (Fig.  1A, panel d), which was significantly greater than that of single wt channels (0.001 Ϯ 0.0004, n ϭ 8) (p Ͻ 0.05) (Fig. 1A, panel b). These observations directly demonstrate that the R615C mutation enhances the response of the RyR1 channel to luminal Ca 2ϩ activation.

The R615C Mutation Has Little Effect on the Cytosolic Ca 2ϩ
Dependence of Single RyR1 Channels-We next determined the response of wt and R615C to cytosolic Ca 2ϩ using single channel recordings. As shown in Fig. 1B, both single wt and R615C channels were maximally activated by ϳ10 M cytosolic Ca 2ϩ with an activation threshold about ϳ100 nM and were completely inhibited by ϳ5 mM Ca 2ϩ . Although the extent of maximum activation of the R615C channels by cytosolic Ca 2ϩ was greater than that of the wt channels (Fig. 1B, panel a), the cytosolic Ca 2ϩ dependence of activation or inactivation of the wt and R615C channels was similar, as seen from the normalized Ca 2ϩ responses (Fig. 1B, panel b). These observations indicate that the R615C mutation does not markedly alter the sensitivity of single RyR1 channels to cytosolic Ca 2ϩ activation or inactivation.
Halothane Potentiates the Luminal Ca 2ϩ Response of Single RyR1 Channels-The effect of halothane on the luminal Ca 2ϩ activation of single RyR1 channels was also investigated (Fig. 2). A single R615C mutant channel exhibited little activity in the presence of 45 nM cytosolic and luminal Ca 2ϩ ( Fig. 2A). The addition of 600 M luminal Ca 2ϩ slightly activated the channel (Fig. 2B). A subsequent addition of 20 mM halothane to the cytosolic side of the channel markedly increased the channel activity (Fig. 2C). The average P o after the addition of halothane was 0.176 Ϯ 0.049 (n ϭ 6) in the presence of 600 M luminal Ca 2ϩ , which was significantly greater than that before the addition of halothane (0.030 Ϯ 0.004) (n ϭ 6) (p Ͻ 0.03). It should be noted that halothane has been shown to have little effect on the apparent affinity of EGTA for Ca 2ϩ (32). Importantly, this halothane-induced enhancement was dependent on the presence of luminal Ca 2ϩ . Reducing the luminal Ca 2ϩ from 600 M to ϳ35 nM decreased the channel activity to the basal level (Fig. 2D). Similarly, halothane also activated single RyR1 wt channels in a luminal Ca 2ϩdependent manner (not shown). It should be noted that because of its highly volatile nature, the actual concentration of halothane in the bilayer recording solution would be much lower than that added to the chamber. Nevertheless, these data indicate that at low cytosolic Ca 2ϩ , halothane potentiates the response of RyR1 to luminal Ca 2ϩ .
Halothane and the R615C Mutation Enhance the Propensity for SOICR in HEK293 Cells-It has long been demonstrated that halothane induces spontaneous contracture in MHS muscle, but not in normal muscle in an external Ca 2ϩ -dependent manner (33), and that the halothane-induced spontaneous SR Ca 2ϩ release is dependent on the SR Ca 2ϩ load (34). To determine whether halothane and the R615C mutation also augment RyR1-mediated spontaneous Ca 2ϩ release or SOICR in a nonmuscle environment, we generated stable, inducible HEK293 cell lines expressing wt or R615C. We have previously shown that elevated [Ca 2ϩ ] o induces SOICR in HEK293 cells expressing RyR2 (26,27). Unlike RyR2-expressing cells, cells expressing RyR1 wt or the R615C mutant did not show SOICR in response to elevated [Ca 2ϩ ] o in the absence of stimuli (Fig. 3A,  panel b). However, in the presence of low concentrations of halothane, elevated [Ca 2ϩ ] o triggered SOICR in both RyR1 wtand R615C mutant-expressing HEK293 cells (Fig. 3A, panels a   and b). Analyzing a number of oscillating cells revealed that HEK293 cells expressing R615C displayed a greater propensity for SOICR than cells expressing wt (Fig. 3A, panel b). The frequency of Ca 2ϩ oscillations in cells expressing the R615C mutant was also much higher than that in cells expressing wt (Fig. 3A, panel c). Similar results were obtained when halothane was replaced with caffeine ( Fig. 3A, panels d and e). The parental HEK293 cells do not express a detectable level of RyRs (26,35). These observations indicate that the MH R615C mutation, halothane, and caffeine can enhance the propensity for SOICR in a nonmuscle environment, suggesting that the response of RyR1 to Ca 2ϩ overload is a major determinant of halothane-or caffeine-induced spontaneous Ca 2ϩ release.
The R615C Mutation Reduces the Luminal Ca 2ϩ Threshold at Which SOICR Occurs-To directly measure the luminal Ca 2ϩ threshold at which SOICR occurs, we used a FRET-based endoplasmic reticulum (ER) Ca 2ϩ sensor protein, D1ER (31), to monitor the ER luminal Ca 2ϩ dynamics during store Ca 2ϩ overload in HEK293 cells expressing RyR1 wt or R615C. As shown in Fig. 3B (panels a and b), elevated [Ca 2ϩ ] o increased the level of ER luminal Ca 2ϩ . When the luminal Ca 2ϩ reached a threshold level, SOICR occurred, displaying as downward deflections in the FRET signal. SOICR was then suppressed by 1.0 mM tetracaine, an inhibitor of RyR1, to estimate the maximum luminal Ca 2ϩ level. Caffeine (20 mM), an activator of RyR1, was then used to estimate the minimum luminal Ca 2ϩ level by emptying the store. Fig. 3B (panel c) shows that the luminal Ca 2ϩ threshold (percentage of maximum luminal Ca 2ϩ store capacity) at which SOICR occurs is significantly lower in cells expressing R615C (76.1 Ϯ 0.8%, n ϭ 243) than in cells expressing wt (89.5 Ϯ 1.0%, n ϭ 106) (p Ͻ 0.00001) in the presence of 2 mM caffeine. There was no significant difference in the maximum luminal Ca 2ϩ store capacity between the R615C-expressing cells (105.5 Ϯ 6.2%) and the wt-expressing cells (100%) (p ϭ 0.25), which was calculated by subtracting the minimum FRET signal (in the presence of 20 mM caffeine) from the maximum FRET signal (in the presence of 1 mM tetracaine). Similarly, the luminal Ca 2ϩ threshold at which SOICR occurs is significantly lower in cells expressing R615C (80.0 Ϯ 3.1%, n ϭ 107) than in cells expressing wt (93.2 Ϯ 0.8%, n ϭ 108) (p Ͻ 0.00001) in the presence of 2 mM halothane. The resting luminal Ca 2ϩ level in the presence of near 0 mM external Ca 2ϩ and 2 mM caffeine is also lower in cells expressing R615C (34.0 Ϯ 2.9%) than in cells expressing wt (54.5 Ϯ 4.5%) (p Ͻ 0.00001). Similarly, the resting luminal Ca 2ϩ level in the presence of near 0 mM external Ca 2ϩ and 2 mM halothane is lower in cells expressing R615C (62.8 Ϯ 3.3%) than in cells expressing wt (81.5 Ϯ 2.4%) (p Ͻ 0.00001). Taken together, these data are consistent with those of single channel studies showing that the R615C mutation and halothane enhance the response of RyR1 to luminal Ca 2ϩ , leading to a reduced SOICR threshold and resting ER Ca 2ϩ level.
Dantrolene Abolishes SOICR in HEK293 Cells Expressing RyR1-R615C but Not in Cells Expressing RyR2-N4104K-It has also long been shown that dantrolene suppresses caffeine-or halothane-induced spontaneous contracture in MHS muscle (36,37) and that dantrolene inhibits spontaneous Ca 2ϩ release from skeletal muscle SR but not from cardiac muscle SR (38). Single channel activities of the R615C mutant were recorded as described in the legend to Fig. 1. The Ca 2ϩ concentration on both the cytosolic and luminal sides of the channel was adjusted to ϳ45 nM (A). The luminal Ca 2ϩ concentration was then increased to 600 M (B). C, single channel current traces after the addition of halothane to the cytosolic side of the channel. The luminal Ca 2ϩ concentration was then reduced to ϳ35 nM in the continued presence of halothane (D). The holding potential was Ϫ20 mV, and the openings are downward.
To determine whether dantrolene can also suppress caffeineinduced spontaneous Ca 2ϩ release in a nonmuscle environment, we assessed the impact of dantrolene on SOICR in HEK293 cells. As shown in Fig. 4, HEK293 cells expressing the RyR1-R615C mutant exhibited Ca 2ϩ oscillations in the presence of 5 mM [Ca 2ϩ ] o plus 2.0 mM caffeine. The addition of 100 nM dantrolene diminished these oscillations, reducing the number of oscillating cells by ϳ80% (Fig. 4, A and B). Fig. 4C shows that dantrolene suppressed SOICR in HEK293 cells expressing either the R615C mutant or RyR1 wt with an IC 50 of ϳ10 -20 nM. Interestingly and in contrast, dantrolene did not abolish Ca 2ϩ oscillations in HEK293 cells expressing the disease-causing RyR2 mutation, N4104K, even at high concentrations (10 M) (Fig. 4D). These data demonstrate that dantrolene potently suppresses RyR1-mediated, but not RyR2-mediated, SOICR in HEK293 cells and therefore suggest that the inhibition of RyR1-mediated SOICR may represent a primary therapeutic action of dantrolene.

DISCUSSION
Investigations over the past decades have greatly advanced our understanding of the molecular and cellular mechanisms of MH. However, some fundamental questions still remain, including: 1) what intrinsic properties of the RyR1 channel altered by mutations are principally responsible for MH pathogenesis? 2) how do volatile anesthetics trigger MH? and 3) how does dantrolene suppress MH? In the present study, we demonstrated that both the MH R615C mutation and MH-triggering agent, halothane, sensitize the RyR1 channel to activation by luminal Ca 2ϩ and reduce the threshold for SOICR. In contrast, dantrolene, an effective treatment for MH, suppresses SOICR. Based on these observations, we propose that volatile anesthetics, by further reducing the already reduced threshold for SOICR in the MHS muscle, trigger spontaneous Ca 2ϩ release and thus MH, whereas dantrolene suppresses spontaneous Ca 2ϩ release and MH by increasing the SOICR threshold.
The MH R615C Mutation Sensitizes the RyR1 Channel to Luminal Ca 2ϩ Activation-Despite the consistent observation that the MHS pig RyR1 channel is more active than the normal RyR1 channel upon stimulation, many studies have failed to detect major changes in the intrinsic sensitivity of the MHS RyR1 channel to cytosolic Ca 2ϩ activation (1,13,15,16,19,20). Consistent with these previous observations, we observed little effect of the MH R615C mutation on the cytosolic Ca 2ϩ dependence of single RyR1 channels (Fig. 1B). How then do the MHS and normal RyR1 channels with similar intrinsic sensitivities to cytosolic Ca 2ϩ activation respond so differently to activation by various stimuli? Given the importance of luminal Ca 2ϩ in regulating SR Ca 2ϩ release, we reasoned that MH mutations may alter the response of the RyR1 channel to luminal Ca 2ϩ . To address this possibility, in the present study, we directly assessed the impact of the MH R615C mutation on the sensitivity of single RyR1 channels to luminal Ca 2ϩ activation. We found that the MH R615C mutation markedly enhanced the luminal Ca 2ϩ activation of RyR1 (Fig. 1A) and that halothane potentiated this response (Fig. 2). These results therefore suggest that an enhanced sensitivity of the RyR1 channel to luminal Ca 2ϩ may be a primary defect resulting from the R615C mutation and contribute to the observed enhanced activity.
It remains, however, to be determined how the R615C mutation, which is located in the cytoplasmic domain of the channel, affects luminal Ca 2ϩ activation. It has been proposed that the MH R615C mutation weakens the interactions between the N-terminal and the central domains and consequently destabilizes the closed state of the channel (20). It is possible that these domain-domain interactions are involved in the activation of the channel by luminal Ca 2ϩ and that weakening these interactions facilitates luminal Ca 2ϩ activation.
The R615C Mutation and Halothane Reduce the Threshold for SOICR-Because SOICR is triggered by the luminal Ca 2ϩ activation of RyR, an enhanced luminal Ca 2ϩ response would lead to an increased propensity for SOICR. In line with this, we found that halothane and caffeine enhanced SOICR in HEK293 cells expressing the RyR1 wt or the R615C mutant (Fig. 3A) and that HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing RyR1 wt (Fig. 3B). Consistent with these observations, a number of human MH RyR1 mutations have also been shown to increase the incidence of spontaneous Ca 2ϩ release when expressed in dyspedic myotubes (39). Hence, an enhanced propensity for SOICR may be a common defect of MH RyR1 mutations.
Our observations are also consistent with the results of early studies of the MHS and normal pig skeletal muscles. Nelson et al. (33) demonstrated that halothane induced spontaneous contracture in MHS muscle, but not in normal muscle, and that this halothane-induced contracture is dependent on the external Ca 2ϩ concentration. Nelson et al. (13,24,25) also showed that the threshold amount of SR Ca 2ϩ load required for Ca 2ϩinduced Ca 2ϩ release to occur in MHS pig SR vesicles was lower than that in normal pig SR and that halothane lowered this threshold. These observations have led to the proposal that a defect in some intraluminal regulatory site for Ca 2ϩ release may underlie MH. Notably, these observations preceded the cloning of the RyR1 cDNA and the identification of the porcine MH mutation R615C. Similarly, Ohnishi et al. (34) demonstrated that the SR Ca 2ϩ content required for halothane-induced Ca 2ϩ release in MHS pig SR was also lower than that in normal SR. Interestingly, when the SR Ca 2ϩ load was below the threshold level, halothane was unable to induce Ca 2ϩ release from either normal or MHS SR vesicles (34). Consistent with these observations, we found that the activation of single RyR1 channels by halothane was also dependent on luminal Ca 2ϩ (Fig. 2) and that halothane and caffeine increased the propensity for SOICR. Taken together, the results of our single channel and SOICR studies and those of previous SR Ca 2ϩ release studies suggest that a reduced threshold for SOICR as a result of enhanced luminal Ca 2ϩ activation of RyR1 underlies a causal mechanism of MH.
Suppression of SOICR Is a Primary Action of Dantrolene-Dantrolene, an effective treatment for MH, markedly suppresses spontaneous muscle contracture and SR Ca 2ϩ release (36,37). Despite its remarkable impact on MH, dantrolene up to ϳ50 M only partially inhibits voltage-or Ca 2ϩ -induced SR Ca 2ϩ release, suggesting that the primary action of dantrolene is unlikely to lie in the suppression of voltage-or Ca 2ϩ -induced SR Ca 2ϩ release (40 -42). On the other hand, if enhanced SOICR activity caused by a reduced SOICR threshold is the cause of MH, one would expect that dantrolene at clinically relevant concentrations (1-10 M) would be able to diminish RyR1-mediated SOICR activity even in a nonmuscle environment. Indeed, we found that dantrolene inhibited SOICR in HEK293 nonmuscle cells expressing the R615C mutant with an IC 50 of ϳ20 nM (Fig. 4). Similarly, Zhang et al. (43) have recently shown that azumolene, a dantrolene analog, decreased the frequency of spontaneous Ca 2ϩ sparks in skeletal muscle fibers with an IC 50 of ϳ250 nM. The fact that much lower concentrations of dantrolene are required for suppressing RyR1-mediated SOICR compared with those required for inhibiting voltage-triggered SR Ca 2ϩ release suggests that the suppression of RyR1-mediated SOICR may be a primary action of dantrolene.
Whether direct and specific binding of dantrolene to the RyR1 channel fully accounts for its therapeutic actions as a skeletal muscle relaxant and antidote for MH remains an open question (44 -46). In this regard, we were unable to detect significant effects of dantrolene on either the cytosolic or luminal Ca 2ϩ activation of single RyR1 channels incorporated into lipid bilayers (not shown), despite the marked effects of dantrolene on RyR1-mediated SOICR that we observed. The reasons for this apparent discrepancy are unknown. It has been shown that dantrolene binds to the N-terminal region of RyR1 and stabilizes domain-domain interactions, indicating that RyR1 or its complex is a direct target of dantrolene (47,48). However, the functional manifestation of dantrolene binding to RyR1 may require the presence of other factors such as calmodulin, ATP, or other RyR1-associated proteins (41,42). Importantly, we found that dantrolene did not affect SOICR in cells expressing a mutant RyR2 channel (Fig. 4). Thus our results convincingly demonstrate that dantrolene suppression of SOICR is strictly dependent on the expression of the RyR1 isoform. It has recently been shown that RyR1 is associated with components of the store-operated Ca 2ϩ entry pathway and that azumolene suppresses the store-operated Ca 2ϩ entry pathway that is coupled to RyR1 (49,50). It is therefore possible that the interaction between RyR1 and components of store-operated Ca 2ϩ entry pathway or other unknown RyR1-associated proteins is required for the action of dantrolene. Clearly, further studies are needed to understand the molecular mechanism by which dantrolene suppresses SOICR.
A New View of MH-Our results support a model in which the MH R615C mutation and the MH-triggering agent halothane reduce the threshold for luminal Ca 2ϩ activation of RyR1 and thus the threshold for SOICR, whereas the MH-suppressing agent, dantrolene, inhibits SOICR (Fig. 5). In this view, the threshold for SOICR from normal SR is higher than the SR-free Ca 2ϩ level under both resting and stimulated conditions (Fig.  5A). Therefore, there is little or no Ca 2ϩ spillover from normal SR. On the other hand, in the MHS SR, the threshold for SOICR is reduced because of mutations in RyR1 (Fig. 5B). Under resting conditions, the reduced threshold for mutant channels remains higher than the resting SR-free Ca 2ϩ level, so that there is again little or no Ca 2ϩ spillover. However, in the presence of MH-triggering agents (e.g. halothane), the threshold is further reduced, and SOICR is more likely to occur. The resulting SR Ca 2ϩ spillover leads to spontaneous muscle contraction and MH. Dantrolene reverses the effect of the MH mutations and of volatile anesthetics by increasing the SOICR threshold.
In this working model, SR Ca 2ϩ handling in MHS muscles at rest is expected to be normal, if the reduced SOICR threshold would still be significantly higher than the resting SR-free Ca 2ϩ level. Consistent with this prediction, the majority of MHS individuals are asymptomatic in the absence of triggering agents.
However, if the reduced SOICR threshold were too close to the resting SR Ca 2ϩ level because of more severe RyR1 mutations, Ca 2ϩ spillover could occur even under the resting conditions. Such uncontrolled SR Ca 2ϩ release at rest may account for the muscle defects seen in those MHS individuals with central core disease (1)(2)(3)(4)(5).
Implication for Stress-induced MH-In addition to volatile anesthetics, physical and emotional stress can also trigger MH in pigs and humans, but the mechanism underlying stress-induced MH is unclear. It is well known that in the heart, physical and emotional stress activates the ␤-adrenergic receptor/cAMP/cAMP-dependent protein kinase signaling pathway, leading to an increase in the SR Ca 2ϩ load and SR Ca 2ϩ release (51). Rudolf et al. (52) have recently demonstrated directly that, as in cardiac muscle, ␤-adrenergic receptor stimulation also increases the SR Ca 2ϩ load and Ca 2ϩ release in skeletal muscle. Because the occurrence of SOICR is determined not only by its threshold but also by the SR Ca 2ϩ load (Fig. 5), physical and emotional stress may induce SOICR by increasing the SR Ca 2ϩ load. Because the threshold for SOICR is reduced in the MHS SR, SOICR and thus spontaneous muscle FIGURE 5. A proposed model for the action of the porcine MH mutation, the MH-triggering agent, halothane, and the MH-suppressing agent, dantrolene. The threshold for SOICR and the SR-free Ca 2ϩ level in normal (A) and the MHS SR (B) in the resting and stimulated states are schematically shown. The SOICR threshold, which is reduced in the MHS SR as a consequence of the R615C RyR1 mutation (short arrow), is depicted by a red bar. The SR-free Ca 2ϩ level is represented by the blue area. The anesthetic-reduced SOICR threshold is depicted by a yellow bar. When the SOICR threshold decreases to the SR-free Ca 2ϩ level, SOICR occurs, leading to a large SR Ca 2ϩ spillover, which can trigger spontaneous Ca 2ϩ release, muscle contraction, and MH. Dantrolene restores the reduced SOICR threshold.
contraction would be more likely to occur in MHS pigs or individuals during stress-induced SR Ca 2ϩ overload. Therefore, conditions that affect either the SOICR threshold or SR Ca 2ϩ loading will have a profound impact on the occurrence of SOICR and thus the susceptibility to MH. This multi-factorial dependence of the occurrence of SOICR may provide an explanation for the unsolved heterogeneity of MH expression in MHS individuals carrying even the same RyR1 mutation.
Conclusions-In summary, the present study sheds novel insight into the causal mechanism of MH and proposes a unifying model for MH susceptibility, MH triggering, and MH suppression. We have demonstrated that both the MH R615C mutation and the MH trigger halothane act to reduce the threshold for luminal Ca 2ϩ activation and SOICR, whereas the MH antidote dantrolene suppresses SOICR. Together these data suggest that the sensitivity of the RyR1 channel to luminal Ca 2ϩ activation plays a central role in the pathogenesis of MH.