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Store-operated Ca2+ Entry in Malignant Hyperthermia-susceptible Human Skeletal Muscle*

      In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca2+ triggers sarcolemmal Ca2+ influx via store-operated Ca2+ entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca2+] either within the resealed t-system ([Ca2+]t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca2+ release or Ca2+t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca2+ release and Ca2+t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca2+ release took the form of a propagated wave, which was temporally coupled to a wave of Ca2+t-sys depletion. SOCE was potently inhibited by “extracellular” application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca2+ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca2+ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca2+ pool may contribute to the maintained rise in cytosolic [Ca2+] that underlies MH.

      Introduction

      Although recent studies have demonstrated the presence of store-operated Ca2+ entry (SOCE)
      The abbreviations used are: SOCE
      store-operated Ca2+ entry
      MH
      malignant hyperthermia
      MHN
      MH normal
      MHS
      MH-susceptible
      SR
      sarcoplasmic reticulum
      ECCE
      excitation-coupled Ca2+ entry
      APACC
      action potential-activated Ca2+ current
      IVCT
      in vitro contracture test
      DMSO
      dimethyl sulfoxide
      HDTA
      hexamethylenediamine-tetraacetate
      STIM1
      stromal interaction molecule 1.
      in skeletal muscle (
      • Launikonis B.S.
      • Barnes M.
      • Stephenson D.G.
      ,
      • Kurebayashi N.
      • Ogawa Y.
      ), the role of this Ca2+ influx mechanism remains uncertain. SOCE has typically been studied using protocols that involve caffeine-induced activation of RyR1 and/or inhibition of sarcoplasmic reticulum Ca2+-ATPase to induce a profound depletion of sarcoplasmic reticulum (SR) Ca2+ (
      • Launikonis B.S.
      • Barnes M.
      • Stephenson D.G.
      ,
      • Kurebayashi N.
      • Ogawa Y.
      ,
      • Launikonis B.S.
      • Ríos E.
      ). However, under physiological conditions, the amount of Ca2+ released during a tetanic contraction is typically <10% of the total SR Ca2+ content (
      • Posterino G.S.
      • Lamb G.D.
      ,
      • Launikonis B.S.
      • Zhou J.
      • Royer L.
      • Shannon T.R.
      • Brum G.
      • Ríos E.
      ) and apparently insufficient to activate SOCE (
      • Kurebayashi N.
      • Ogawa Y.
      ,
      • Launikonis B.S.
      • Stephenson D.G.
      • Friedrich O.
      ). This suggests that SOCE is unlikely to have a significant role in Ca2+ homeostasis during exercise of moderate intensity. SOCE might, however, be activated during intensive exercise leading to fatigue (
      • Zhao X.
      • Yoshida M.
      • Brotto L.
      • Takeshima H.
      • Weisleder N.
      • Hirata Y.
      • Nosek T.M.
      • Ma J.
      • Brotto M.
      ), where SR luminal Ca2+ declines (
      • Kabbara A.A.
      • Allen D.G.
      ,
      • Steele D.S.
      • Duke A.M.
      ). Consistent with this possibility, a reduced capacity for SOCE has been linked to impaired fatigue resistance in aged murine skeletal muscle (
      • Zhao X.
      • Weisleder N.
      • Thornton A.
      • Oppong Y.
      • Campbell R.
      • Ma J.
      • Brotto M.
      ).
      Another possibility not yet considered is that SOCE might contribute to the pathological rise in cytosolic [Ca2+] that underlies human malignant hyperthermia (MH). In most cases of MH, susceptibility is conferred by mutations in the gene encoding RyR1 (RYR1, chromosome 19q13.1) (
      • MacLennan D.H.
      • Duff C.
      • Zorzato F.
      • Fujii J.
      • Phillips M.
      • Korneluk R.G.
      • Frodis W.
      • Britt B.A.
      • Worton R.G.
      ,
      • McCarthy T.V.
      • Healy J.M.
      • Heffron J.J.
      • Lehane M.
      • Deufel T.
      • Lehmann-Horn F.
      • Farrall M.
      • Johnson K.
      ). During an MH episode, activation of RyR1 by a volatile anesthetic induces SR Ca2+ release, muscle contracture, and a potentially fatal rise in core body temperature. Given this established sequence of events, it is generally assumed that the rise in cytoplasmic [Ca2+] during MH solely reflects Ca2+ efflux from the SR. However, activation of RyR1 and consequent depletion of SR Ca2+ might also induce SOCE, thereby triggering a sustained Ca2+ influx from an effectively infinite extracellular pool.
      Interestingly, the involvement of extracellular Ca2+ in MH is suggested by findings that the anesthetic-induced contracture during the in vitro contracture test (IVCT) is markedly reduced in solutions lacking extracellular Ca2+ (
      • Adnet P.J.
      • Krivosic-Horber R.M.
      • Adamantidis M.M.
      • Reyford H.
      • Cordonnier C.
      • Haudecoeur G.
      ,
      • Fletcher J.E.
      • Huggins F.J.
      • Rosenberg H.
      ). However, this observation is inconclusive because reduced binding of extracellular Ca2+ to the dihydropyridine receptor has been shown to inhibit pharmacological activation of RyR1 independently from Ca2+ entry (
      • Brum G.
      • Ríos E.
      • Stéfani E.
      ), and thus the role of SOCE in human MH remains uncertain.
      The aim of the present study was to establish whether SOCE is activated by volatile anesthetic exposure in human MH-susceptible (MHS) skeletal muscle. Experiments were done on normal (MHN) or MHS vastus medialis muscle obtained from patients undergoing MH diagnosis. Fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca2+] within the sealed t-system or within the cytosol, using fluo-5N or fluo-3, respectively. The data provide the first evidence of SOCE in adult human skeletal muscle and show that clinically relevant levels of volatile anesthetic can induce Ca2+ influx, secondary to a submaximal SR Ca2+ depletion in MHS but not in MHN fibers. The characteristics of SOCE in human skeletal muscle and the possible involvement of sarcolemmal STIM1 in the Ca2+ influx mechanism are addressed.

      EXPERIMENTAL PROCEDURES

       Skinned Fibers

      Samples of vastus medialis muscle were obtained by open biopsy from patients attending for MH diagnosis at St. James's University Hospital, Leeds, UK. Approximately 1 g of muscle was removed for use in the IVCT. With institutional Research Ethics Committee approval and informed patient consent, an additional bundle (0.2 g) was taken to provide material for mechanically skinned muscle experiments. All procedures were done according to the Declaration of Helsinki. The IVCT provided the primary method of categorizing tissue as MHN or MHS, according to the criteria for MH research of the European MH Group (
      The European Malignant Hyperpyrexia Group
      ). This ensures a high sensitivity and specificity of the MHS diagnosis (98 and 94% respectively)(
      • Ording H.
      • Brancadoro V.
      • Cozzolino S.
      • Ellis F.R.
      • Glauber V.
      • Gonano E.F.
      • Halsall P.J.
      • Hartung E.
      • Heffron J.J.
      • Heytens L.
      • Kozak-Ribbens G.
      • Kress H.
      • Krivosic-Horber R.
      • Lehmann-Horn F.
      • Mortier W.
      • Nivoche Y.
      • Ranklev-Twetman E.
      • Sigurdsson S.
      • Snoeck M.
      • Stieglitz P.
      • Tegazzin V.
      • Urwyler A.
      • Wappler F.
      ).
      With institutional review board approval, all patients consented to provide a blood sample for DNA analysis. DNA was extracted from whole blood, and RYR1 mutation analysis was conducted (
      • Robinson R.
      • Carpenter D.
      • Shaw M.A.
      • Halsall J.
      • Hopkins P.
      ). The MHS samples used in this study originated from 13 patients (supplemental Table 1). 11 of the samples were from patients with one of 34 recurrent RYR1 mutations (supplemental Table 2). The mutations were distributed among three hot spot regions (see the legend of supplemental Table 1 for details).
      Muscle samples were placed in paraffin oil to displace the extracellular fluid before isolation and mechanical skinning of individual fibers with fine forceps. Skinned fibers were suspended in a bath with an “internal” solution designed to mimic the intracellular environment (see below). Vastus medialis is of mixed fiber type, and Sr2+ sensitivity was used to classify the fibers as type 1 or type 2 (
      • Lynch G.S.
      • Stephenson D.G.
      • Williams D.A.
      ). Most preparations did not generate tension at negative logarithm (base 10) of the molar concentration of Sr2+ 5.2, confirming that most fibers selected for skinning were type-2. Preparations generating tension at negative logarithm (base 10) of the molar concentration of Sr2+ 5.2 were excluded from the study.

       Solutions

      Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (Poole, UK). A basic internal solution was prepared comprising (in mm): 50 HDTA; 8 ATP; 37 Na+; 126 K+; 10 phosphocreatine; 0.1 EGTA; 90 HEPES. The free [Mg2+] was adjusted to 0.8 mm by the addition of MgO. The free [Ca2+] was 60 nm. In most experiments, 50 μm n-benzyl-p-toluene sulfonamide (Calbiochem, Nottingham, UK) was added to inhibit Ca2+ activation of the myofilaments (
      • Cheung A.
      • Dantzig J.A.
      • Hollingworth S.
      • Baylor S.M.
      • Goldman Y.E.
      • Mitchison T.J.
      • Straight A.F.
      ) and associated movement. All experiments were done at room temperature (20–22 °C), pH 7.1.
      A modified Tyrode's “external” solution was used, which comprised (in mm): 140.3 NaCl; 2.48 KCl; 1.5 CaCl2; 1 MgCl2; 5 HEPES; 5 glucose; and 5 sodium pyruvate. A halothane stock solution was prepared in a dimethyl sulfoxide (DMSO). The final concentration of DMSO did not exceed 0.1%. Once prepared, solutions with halothane were contained within airtight syringes with zero dead space to prevent vaporization (
      • Duke A.M.
      • Hopkins P.M.
      • Halsal J.P.
      • Steele D.S.
      ). Stock fluo-5N and fluo-3 (Biotium, Hayward, CA) solutions (1 m) were prepared in deionized water. In some experiments, a STIM1 antibody (20 μg/ml; BD Biosciences, Dorset, UK) was introduced into the t-system before skinning. This antibody targets the extracellular N terminus of STIM1 within the plasma membrane (
      • Spassova M.A.
      • Soboloff J.
      • He L.P.
      • Xu W.
      • Dziadek M.A.
      • Gill D.L.
      ).

       Western Blot

      MHN muscle samples were fractionated using detergent-free methods (
      • Calaghan S.
      • Kozera L.
      • White E.
      ). In brief, peripheral membrane proteins were extracted in 500 mm Na2CO3 containing 0.5 mm EGTA and 1% protease inhibitor mixture (Sigma), homogenized, sonicated (three times each for 20 s at full power), and layered on a discontinuous sucrose gradient (45–35-5%). 12 fractions were collected following centrifugation for 17 h (280,000 × g) at 4 °C. STIM1 (610954; BD Biosciences), caveolin-3 (610420; BD Biosciences), and β-adaptin (610382; BD Biosciences) were measured by Western blotting following SDS-PAGE. As described recently (
      • Edwards J.N.
      • Murphy R.M.
      • Cully T.R.
      • von Wegner F.
      • Friedrich O.
      • Launikonis B.S.
      ), the STIM1 antibody identified additional bands at two additional bands at ∼50 and ∼42 kDa in human muscle (not shown). Consequently, although extracellular application of the STIM1 antibody produced a striated pattern consistent with binding to the t-system (supplemental Fig. 1), this finding should be interpreted with caution.

       Apparatus

      Mechanically skinned muscle fibers were mounted in a shallow bath with a glass coverslip base as described previously (
      • Duke A.M.
      • Steele D.S.
      ). The experimental bath was placed on the stage of an S200 Nikon Diaphot inverted microscope (Nikon, Surrey, UK). Muscle fibers were viewed via ×40 Fluor objective (Nikon CF Fluor, NA 0.75). A confocal laser-scanning unit (Cellmap, Bio-Rad, Herts, UK) was attached to the side port of the microscope. Fluorophores were excited with the 488-nm line of a diode laser and emitted fluorescence was measured at >515 nm. In most experiments, the system was configured with a “signal-enhancing lens system” in the light path, which improves detection efficiency while slightly reducing confocality (
      • Reichelt S.
      • Amos W.B.
      ). This configuration minimizes bleaching and allows more prolonged scanning of live cells.

       Mechanical Skinning and Compartmentalization of fluo-5N within the Resealed T-system

      When fibers are mechanically skinned under oil, the necks of the t-tubules close off at the periphery, thereby trapping the extracellular fluid within the resealed t-system (
      • Launikonis B.S.
      • Barnes M.
      • Stephenson D.G.
      ). Thereafter, the cytosolic space becomes an extension of the bathing solution. There was no apparent difference in susceptibility to damage between MHS and MHN fibers.
      Changes in [Ca2+] within the t-system were detected using fluo-5N, which was trapped within the t-system using one of two methods. In the first method, a thin film of external solution containing fluo-5N was introduced at the interface between the fiber and the surrounding oil (
      • Launikonis B.S.
      • Barnes M.
      • Stephenson D.G.
      ), allowing the dye to enter the t-system before mechanical skinning and resealing. Supplemental Fig. 2A shows a confocal x-y image (average of four frames) obtained from a human MHN fiber, which was skinned using this method. As indicated by the line profile, the characteristic “M” pattern associated with the t-tubule arrangement in mammalian muscle is clearly visible (
      • Launikonis B.S.
      • Stephenson D.G.
      ). In other figures shown in this study, the t-tubule pattern is typically less distinct. This is because the frames were not averaged and because of slight movement of the fiber despite the presence of n-benzyl-p-toluene sulfonamide and the use of the signal-enhancing lens system.
      In some experiments, an alternative method was used, which exploits the tendency for fluorophores to be transported from the cytosol to the extracellular space (
      • Di Virgilio F.
      • Fasolato C.
      • Steinberg T.H.
      ). Briefly, skinned fibers were placed in an internal solution containing 100 μm fluo-5N, which was progressively transported from the cytosol into the t-system. Adequate dye loading was obtained after ∼20 min, and uptake into the t-system was potently inhibited by the nonspecific anion transport inhibitor probenecid (see supplemental Fig. 3), suggesting the involvement of an anion transporter (
      • Di Virgilio F.
      • Fasolato C.
      • Steinberg T.H.
      ). In all experiments, fluo-5N was removed from the bathing solution for at least 5 min before commencing the experiment, leaving the dye in the t-system only. There was no apparent difference between the properties of SOCE measured using the two methods.
      In control experiments with the signal-enhancing lens system lens in place, a slow decrease in t-system fluorescence occurred with time (supplemental Fig. 2B). This presumably reflects the combined effects of dye bleaching and loss. All cumulative data graphs were corrected for this loss of t-system fluorescence.

       Rationale for SR Ca2+ Release Protocols

      In previous studies, we have characterized the effects of both halothane and sevoflurane on RyR1 activation in human MHS and MHN skinned fibers (
      • Duke A.M.
      • Hopkins P.M.
      • Halsal J.P.
      • Steele D.S.
      ,
      • Duke A.M.
      • Hopkins P.M.
      • Halsall P.J.
      • Steele D.S.
      ). Once triggered, the properties of SR Ca2+ release are similar with both anesthetics, although the probability of triggering release is lower with sevoflurane (
      • Duke A.M.
      • Hopkins P.M.
      • Halsall P.J.
      • Steele D.S.
      ). In the clinical setting, sevoflurane is the more commonly used volatile anesthetic. However, halothane was used in the present study (i) because it induces SR Ca2+ release more consistently than sevoflurane in MHS muscle and (ii) because single fiber data can be compared with the IVCT from the same patient, where the dose-response relationship to halothane is the basis of the standardized diagnostic test.
      Halothane was applied at 0.5 mm, which is within the range that occurs clinically during anesthetic induction (
      • Davies D.N.
      • Steward A.
      • Allott P.R.
      • Mapleson W.W.
      ) (and therefore known to induce MH) but does not normally trigger SR Ca2+ release in MHN fibers. In experiments involving cytosolic Ca2+ measurement (see Fig. 1), a maximal SR Ca2+ depletion was induced by rapid exposure to 20 mm caffeine, whereas simultaneously decreasing the free [Mg2+] to 20 μm. However, in experiments involving SOCE where x-y images were taken (see FIGURE 2, FIGURE 3, FIGURE 4), maximal SR Ca2+ depletion was induced by decreasing the free [Mg2+] to 20 μm alone (
      • Lamb G.D.
      • Stephenson D.G.
      ). Although less rapid, this method of SR Ca2+ depletion was preferred because it minimized movement associated with myofilament activation.
      Figure thumbnail gr1
      FIGURE 1Differential sensitivity of cytosolic SR Ca2+ release to halothane in MHS and MHN fibers. A, typical example showing an endogenously Ca2+-loaded MHN fiber, which was initially exposed to a solution containing 20 mm caffeine and 20 μm Mg2+ to induce a maximal release (MR) of Ca2+ from the SR, resulting in a transient increase in fluo-3 fluorescence within the cytosol (upper panel). The SR was then reloaded to the same by exposure to a solution with a free [Ca2+] of ∼200 nm, and another maximal Ca2+ release was induced. After reloading the SR again, the MHN fiber was subjected to stepwise increases in [halothane] until SR Ca2+ release occurred at 2 mm in this example. When the same protocol was applied to MHS fibers, the threshold for halothane-induced Ca2+ release was consistently ≤0.5 mm (lower panel). B, sequential x-y confocal images (0.83 Hz) from an MHS fiber in which halothane exposure induced a slow propagated Ca2+ wave. Scale bar indicates 80 μm. C, cumulative data showing the proportion of MHS and MHN fibers responding to 0.5 mm halothane in the presence of 0.8 or 0.2 mm Mg2+. The total number of halothane-responsive fibers (one per patient) is indicated above each bar.
      Figure thumbnail gr4
      FIGURE 4Effect of reduced [ Mg2+] on the response of MHN fibers to halothane. A, typical x-y confocal images of fluo-5N fluorescence within the resealed t-tubules of a skinned MHN fiber in the presence of 0.8 mm (upper panel) or 0.2 mm Mg2+ (lower panel) free [Mg2+]. At 0.8 mm Mg2+, halothane had no apparent effect on fluo-5N fluorescence, whereas a subsequent maximal SR Ca2+ release (MR) induced a sustained decrease in [Ca2+]t-sys. In the presence of 0.2 mm Mg2+, the introduction of halothane resulted in a transient increase in [Ca2+]t-sys followed by a sustained decrease. B, cumulative data showing the mean fluorescence obtained from sequential x-y images before and during exposure to 0.5 mm halothane in the presence of 0.8 mm (n = 8, red circles) or 0.2 mm (n = 8, black circles) free Mg2+. Each data point represents mean ± S.E. * indicates significantly different from control, p < 0.05.
      Figure thumbnail gr3
      FIGURE 3Halothane-induced wave of SOCE in human MHS fibers. Sequential surface plots of x-y confocal images taken from an MHS fiber (0.83 Hz), which exhibited a propagated wave of t-tubule [Ca2+] change following exposure to 0.5 mm halothane, are shown. An initial wave front of raised [Ca2+]t-sys was followed by sustained [Ca2+]t-sys depletion. Similar results were obtained in two other preparations.
      Figure thumbnail gr2
      FIGURE 2Properties of halothane-induced SOCE. A, typical x-y confocal images of fluo-5N fluorescence obtained with the dye trapped within the resealed t-tubules of a mechanically skinned MHN or MHS fiber. In MHN fibers (upper panel), the introduction of 0.5 mm halothane had no apparent effect on fluo-5N fluorescence within 60 s. However, after initiation of a maximal SR Ca2+ release (MR) by reducing in the free [Mg2+] to 20 μm, t-tubule fluorescence decreased markedly. In MHS fibers (lower panel), the introduction of 0.5 mm halothane resulted in an initial increase in fluo-5N fluorescence followed by a decrease, which approached a new steady state after ∼60 s. Thereafter, initiation of a maximal SR Ca2+ release induced a further decrease in fluo-5N fluorescence. B, cumulative data showing the mean fluorescence obtained from sequential x-y images before and during exposure to halothane or maximal SR Ca2+ release (MR), in MHS (n = 8, red circles) or MHN (n = 8, black circles) human skeletal muscle fibers. Each data point represents mean ± S.E. * indicates significantly different from control, p < 0.05.

       Data Analysis

      Analysis was done using Image Pro Plus (Media Cybernetics) and Image J software (National Institutes of Health). The change in t-tubule fluorescence was expressed as a percentage of the maximum steady-state fluorescence immediately prior to induction of SOCE. The zero point was taken to be that remaining after the fiber had been exposed to 50 μg/ml saponin for 10 min to permeabilize the t-system. In the cumulative data, each point represents the average pixel value of the corresponding x-y frame. Data are presented as the mean ± S.E., with the number of observations indicated in parentheses (n). Statistical significance (p < 0.05) was determined using a one-way analysis of variance. All statistical analysis was performed using Origin software (MicroCal).

      RESULTS

       Halothane Sensitivity of MHS and MHN Fibers

      Initial experiments were done to establish whether the differential halothane sensitivity of MHN and MHS muscle apparent in the diagnostic IVCT could be demonstrated in skinned fibers (Fig. 1A). After skinning, both MHN and MHS fibers were perfused for 5 min with a weakly Ca2+-buffered intracellular solution. A solution containing 20 mm caffeine and 20 μm [Mg2+] (low Mg2+) was then applied until the response reached a maximal level after ∼10 s. The amplitude of the resulting fluo-3 fluorescence transient was used as an index of the SR Ca2+ content. A further ∼5-min perfusion was typically sufficient to allow the SR to re-reload to a similar level, as indicated by the amplitude of the second response to 20 mm caffeine/low Mg2+. Using this SR Ca2+ load and release protocol, several reproducible Ca2+ transients could be obtained with little change in the amplitude or time course of the response.
      After reloading the SR for a third time, fibers were subjected to stepwise increases in [halothane] over the range 0.2–5 mm or until SR Ca2+ release occurred. In MHN fibers (upper panel), SR Ca2+ release consistently occurred at ≥2 mm halothane under these conditions. In contrast, MHS fibers typically exhibited a robust SR Ca2+ release at ≤0.5 mm halothane (lower panel). Following exposure to halothane, the SR was reloaded, and a final maximal SR Ca2+ release was induced to confirm the viability of the preparation.
      In ∼10% of MHS fibers, the SR Ca2+ release induced by halothane took the form of a propagated cytosolic Ca2+ wave. Fig. 1B shows sequential x-y confocal images obtained from an MHS fiber under control conditions and following the introduction of 0.5 mm halothane. Application of halothane was associated with SR Ca2+ release, which originated outside the field of view and propagated from right to left in sequential images, consistent with Ca2+-induced Ca2+ release. As in previous studies (
      • Duke A.M.
      • Hopkins P.M.
      • Halsal J.P.
      • Steele D.S.
      ), propagated SR Ca2+ release was not observed in MHN fibers under these conditions.
      Cumulative data showing the number of MHS and MHN fibers that exhibited SR Ca2+ release in response to 0.5 mm halothane under control conditions and in the presence of 0.8 mm Mg2+ or 0.2 mm Mg2+ is shown in Fig. 1C. At 0.8 mm [Mg2+]i, a level within the normal physiological range (
      • Westerblad H.
      • Allen D.G.
      ), 0.5 mm halothane discriminated effectively between MHS and MHN fibers with 8/8 and 1/8 fibers responding to 0.5 mm halothane, respectively. Consistent with previous studies on human fibers (
      • Steele D.S.
      • Duke A.M.
      ), a decrease in the cytosolic [Mg2+] to 0.2 mm increased the responsiveness of MHN fibers to the anesthetic such that 0.5 mm halothane induced SR Ca2+ release in all MHS and MHN fibers.
      Although one MHN fiber responded unexpectedly to 0.5 mm halothane, these data on skinned fibers demonstrate a high degree of consistency with the IVCT diagnosis. Therefore, as 0.5 mm halothane is also within the range expected to occur during anesthetic induction (
      • Davies D.N.
      • Steward A.
      • Allott P.R.
      • Mapleson W.W.
      ), this concentration was used in all subsequent experiments.

       Evidence of SOCE in Human MHS and MHN Fibers

      Experiments were carried out to establish whether the SR Ca2+ release induced by volatile anesthetic exposure (Fig. 1) triggers Ca2+ influx via SOCE. MHN or MHS fibers were mechanically skinned, and fluo-5N was trapped within the resealed t-system (see “Experimental Procedures”). In this and all subsequent protocols addressing SOCE, dyes were absent from the cytosol. Fig. 2A shows representative confocal x-y images of fluo-5N trapped with the resealed t-system of an MHN (upper panel) and an MHS (lower panel) human skeletal muscle fiber. In the MHN fiber, rapidly increasing [halothane] to 0.5 mm for 60 s had no apparent effect on fluo-5N fluorescence. However, a subsequent maximal SR Ca2+ release induced by a decrease in the [Mg2+] from 0.8 mm to 20 μm (
      • Lamb G.D.
      • Stephenson D.G.
      ) was associated with a pronounced reduction in fluo-5N fluorescence, consistent with SOCE. In contrast, exposure of the MHS fiber to 0.5 mm halothane caused a transient increase in [Ca2+] within the resealed t-system ([Ca2+]t-sys) followed by a gradual decrease due to SOCE. A subsequent maximal SR Ca2+ release induced a further depletion of Ca2+t-sys, indicating that the effect of 0.5 mm halothane on SOCE was submaximal.
      Cumulative data showing the time-dependent effects of halothane on fluo-5N fluorescence are given in Fig. 2B. MHN fibers consistently failed to respond to 0.5 mm halothane. However, in MHS fibers, fluorescence transiently increased on halothane exposure to 107 ± 1.2% (n = 8) before decreasing toward a new steady-state level (69 ± 3.6%, n = 8) after ∼60 s. In both MHN and MHS fibers, maximal depletion of SR Ca2+ ([Mg2+] reduced to 20 μm) decreased the fluo-5N fluorescence to 20 ± 1.5 and 21 ± 2% of the control value, respectively. These values were not significantly different (p > 0.05).

       Halothane Induced Waves of SOCE in Human MHS Fibers

      As shown in Fig. 1B, in some MHS fibers, SR Ca2+ release took the form of a propagated cytosolic Ca2+ wave. Fig. 3 shows a series of x-y images rendered as surface plots, obtained from an MHS fiber following the introduction of 0.5 mm halothane. This example clearly shows a wave crest of raised [Ca2+]t-sys, which propagated from right to left. As in Fig. 2, the initial increase in Ca2+t-sys was followed by a sustained decrease, consistent with SOCE. Although cytosolic Ca2+ could not be imaged simultaneously in this study, it seems likely that the wave of [Ca2+]t-sys change was secondary to a wave of SR Ca2+ release (Fig. 1B). Similar results were obtained in two other preparations.

       Effects of Mg2+ on Halothane-induced SOCE in MHN Fibers

      Previous work has shown that in fibers from MHN patients, decreasing the [Mg2+]i facilitates Ca2+-induced Ca2+ release and increases the sensitivity of RyR1 to halothane (
      • Duke A.M.
      • Hopkins P.M.
      • Halsall P.J.
      • Steele D.S.
      ). As shown in Fig. 1C, decreasing [Mg2+] from 0.8 to 0.2 mm normalized the responses such that all MHN and MHS fibers exhibited SR Ca2+ release on the introduction of 0.5 mm halothane (Fig. 1C). Fig. 4A shows representative x-y images from an MHN fiber perfused continuously with a solution containing 0.2 mm [Mg2+]. The introduction of 0.5 mm halothane induced a transient increase in t-tubule fluorescence followed by a sustained decrease, consistent with SOCE. This response to halothane in MHN fibers at 0.2 mm Mg2+ was qualitatively similar to that obtained in MHS fibers at 0.8 mm Mg2+ (Fig. 2A). The cumulative data show that in the presence of 0.2 mm Mg2+, MHN fibers exhibit a transient increase in fluorescence to 107 ± 2.7% (n = 8) after ∼5 s of exposure to halothane, before decreasing to 73 ± 9% after 60 s (Fig. 4B). Again, there was no significant effect of 0.5 mm halothane in the presence of 0.8 mm Mg2+.

       Effects of STIM1 Antibody on SOCE in MHS Fibers

      STIM1 is located within SR/endoplasmic reticulum membrane, where it acts as a Ca2+ sensor, communicating store depletion to sarcolemmal store-operated Ca2+ channels (
      • Westerblad H.
      • Allen D.G.
      ,
      • Zhang S.L.
      • Yu Y.
      • Roos J.
      • Kozak J.A.
      • Deerinck T.J.
      • Ellisman M.H.
      • Stauderman K.A.
      • Cahalan M.D.
      ). However, recent work suggests that STIM1 is also present within the sarcolemma in some cell types and that inhibition of the protein with a blocking antibody can inhibit SOCE (
      • Spassova M.A.
      • Soboloff J.
      • He L.P.
      • Xu W.
      • Dziadek M.A.
      • Gill D.L.
      ,
      • Dziadek M.A.
      • Johnstone L.S.
      ). As STIM1 is present in skeletal muscle sarcolemma (
      • Stiber J.
      • Hawkins A.
      • Zhang Z.S.
      • Wang S.
      • Burch J.
      • Graham V.
      • Ward C.C.
      • Seth M.
      • Finch E.
      • Malouf N.
      • Williams R.S.
      • Eu J.P.
      • Rosenberg P.
      ), experiments were carried out to establish whether extracellular application of a STIM1-blocking antibody influences SOCE in human muscle.
      When the STIM1-blocking antibody was trapped within the resealed t-system before exposure of an MHS fiber to 0.5 mm halothane, the initial increase in t-system fluorescence was unaffected (Fig. 5A, upper panel). Thereafter, however, although the fluorescence gradually decayed toward the control level, there was no significant depletion of Ca2+t-sys within 60 s. In contrast, when the antibody was denatured prior to application (see “Experimental Procedures”), 0.5 mm halothane induced a typical SOCE response, i.e. t-system fluorescence initially increased and peaked after ∼5 s before decreasing toward a new steady-state after 60 s (lower panel).
      Figure thumbnail gr5
      FIGURE 5Effects of STIM1 antibody on the response of MHS fibers to halothane. A, x-y confocal images of fluo-5N fluorescence within the resealed t-tubules of a skinned MHS fiber in which a STIM1 antibody had been introduced into the t-system prior to skinning. In this case, exposure to halothane was followed by a characteristic increase in [Ca2+]t-sys (upper panel). However, fluo-5N fluorescence then returned slowly toward the control level, and there was little evidence of a sustained decrease within 60 s or subsequently after a maximal SR Ca2+ release (MR). This procedure was repeated in an MHS fiber using a sample of the antibody, which had been denatured by heating (lower panel). After heat treatment, application of halothane resulted in a typical SOCE response. B, cumulative data showing the mean fluorescence obtained from sequential x-y images in MHS fibers with either the active STIM1 antibody (n = 8, red triangles) or the denatured antibody (n = 8, open squares) trapped within the t-system. * indicates statistically different from control level, p < 0.05. C, representative Western blot from human vastus medialis muscle. STIM1 is indentified in whole muscle human homogenate and is also enriched in the buoyant fraction 6, which contains sarcolemmal lipid rafts and caveolae. β-Adaptin (a marker of non-raft membranes) is present at low levels in fractions 5 and 6, whereas caveolin-3 (a marker of caveolae) is enriched. Fractions 7–12 are mostly non-raft membranes and cytosolic proteins. Unlike some other caveolin isoforms, caveolin-3 is found outside buoyant membrane fractions as observed here in fractions 9–12 (
      • Head B.P.
      • Patel H.H.
      • Roth D.M.
      • Lai N.C.
      • Niesman I.R.
      • Farquhar M.G.
      • Insel P.A.
      ).
      In Fig. 5B, the cumulative data showing the characteristic response to 0.5 mm halothane in MHS fibers are shown superimposed upon the data obtained with the STIM1 antibody present within the sealed t-system. The persistence of the initial rising phase following antibody exposure suggests that SR Ca2+ release and subsequent efflux into the t-system are unaffected by the STIM1 antibody, whereas Ca2+ efflux due to store depletion is markedly inhibited. Indeed, the fluorescence after 60 s was not significantly different from the control value prior to halothane exposure (p > 0.05, n = 8). However, SOCE was not inhibited when the antibody was denatured by heating. In further control experiments involving measurement of cytosolic [Ca2+], the STIM1 antibody did not affect SR Ca2+ release induced by either caffeine or halothane (not shown).
      A typical Western blot obtained from human vastus medialis (Fig. 5C) confirms the presence of STIM1 in whole muscle homogenate. STIM1 is also enriched in fraction 6, which is a buoyant fraction containing cholesterol-enriched sarcolemma (lipid rafts and caveolae). Note the relative absence of β-adaptin (a marker of non-raft membranes) in fractions 5 and 6, whereas caveolin-3 (a marker of caveolae) is enriched.

      DISCUSSION

      This is the first study to demonstrate 1) SOCE in adult human skeletal muscle and 2) sustained sarcolemmal Ca2+ influx in MHS muscle, under conditions that precipitate pathological SR Ca2+ release. In both MHN and MHS fibers, changes in t-system [Ca2+] only occurred under conditions that precipitated SR Ca2+ depletion, e.g. in MHS muscle fibers, where 0.5 mm halothane consistently induced SR Ca2+ release (Fig. 1), the introduction of the anesthetic caused an initial rise in [Ca2+]t-sys followed by a sustained decrease (Fig. 2, A and B). In MHN fibers, halothane consistently failed to initiate SR Ca2+ release (Fig. 1A), or to induce changes in t-system [Ca2+] (Fig. 2, A and B).
      When MHN fibers were perfused with a reduced level of cytosolic [Mg2+] (0.2 mm), halothane induced both SR Ca2+ release (Fig. 1C) and changes in [Ca2+]t-sys, similar to those obtained in MHS fibers (Fig. 4). This effect of low [Mg2+] on MHN fibers likely reflects the fact that RyR1 gating is potently inhibited by Mg2+ in resting skeletal muscle (
      • Lamb G.D.
      • Stephenson D.G.
      ). Decreasing the [Mg2+] to 0.2 mm is sufficient to facilitate agonist-induced activation of RyR1 but does not itself cause loss of Ca2+ from the SR (
      • Duke A.M.
      • Hopkins P.M.
      • Halsal J.P.
      • Steele D.S.
      ). The fact that SOCE can be induced in MHN fibers by facilitating halothane-induced SR Ca2+ release suggests that the phenomenon occurs due to store depletion and is not a unique characteristic of MH or particular RYR1 mutations. This conclusion is supported by the fact that the RYR1 mutations identified in the present study are spread throughout all three hot spot regions of RYR1 (see supplemental Table 1).
      The characteristic biphasic change in [Ca2+]t-sys that occurred in MHS fibers in response to halothane is qualitatively similar to that reported in mechanically skinned rat skeletal muscle fibers following the addition of caffeine and/or inhibition of sarcoplasmic reticulum Ca2+-ATPase (
      • Launikonis B.S.
      • Ríos E.
      ). The initial increase in [Ca2+]t-sys can be explained if sarcolemmal Ca2+ extrusion mechanisms facilitate a net efflux of Ca2+ from the cytosol into the t-system when cytosolic [Ca2+] is raised. Consistent with this, previous studies have shown that the initial transient rise in [Ca2+]t-sys can be abolished by raising the Ca2+ buffer capacity of the cytosolic medium (
      • Launikonis B.S.
      • Ríos E.
      ). The later sustained decrease in [Ca2+]t-sys can be explained by a Ca2+ flux from the sealed t-system into the cytosolic space, triggered by depletion of SR Ca2+, i.e. SOCE. However, subsequent maximal depletion of SR Ca2+ induced a further decrease in [Ca2+]t-sys, suggesting that SOCE was not fully activated by halothane (Fig. 2, A and B).

       Waves of T-tubule Ca2+ Depletion

      Previous studies have reported markedly different rates of SOCE in skinned skeletal muscle fibers, e.g. Brotto and colleagues (
      • Zhao X.
      • Yoshida M.
      • Brotto L.
      • Takeshima H.
      • Weisleder N.
      • Hirata Y.
      • Nosek T.M.
      • Ma J.
      • Brotto M.
      ,
      • Zhao X.
      • Weisleder N.
      • Thornton A.
      • Oppong Y.
      • Campbell R.
      • Ma J.
      • Brotto M.
      ,
      • Hirata Y.
      • Brotto M.
      • Weisleder N.
      • Chu Y.
      • Lin P.
      • Zhao X.
      • Thornton A.
      • Komazaki S.
      • Takeshima H.
      • Ma J.
      • Pan Z.
      ,
      • Zhao X.
      • Weisleder N.
      • Han X.
      • Pan Z.
      • Parness J.
      • Brotto M.
      • Ma J.
      ) reported that [Ca2+]t-sys declined slowly in murine skeletal muscle, only approaching a new steady-state 8–10 min after initiation of SR Ca2+ release. In contrast, Launikonis and Ríos (
      • Launikonis B.S.
      • Ríos E.
      ) reported a much more rapid depletion of [Ca2+]t-sys in rat fibers, which was detectable within 1 s of SR Ca2+ release and declined toward a new steady state over ∼30–50 s. These apparent differences in the temporal properties of SOCE might reflect species variation or aspects of the methodology, e.g. the rate of solution exchange or inadequate compensation for dye loss/bleaching. However, the present study on human skeletal muscle clearly demonstrates a rapid Ca2+ flux associated with SOCE, which occurred on a similar time scale to that described by Launikonis and Ríos in rat muscle (Fig. 2B) (
      • Launikonis B.S.
      • Ríos E.
      ). Furthermore, we show for the first time that in MHS fibers, waves of [Ca2+]t-sys depletion (Fig. 3) can occur on a similar tine course to halothane-induced SR Ca2+ waves (Fig. 1B), suggesting a causal relationship. This further emphasizes the close temporal coupling between SR Ca2+ release and activation of SOCE.

       STIM1 and SOCE in Skeletal Muscle

      Recent findings implicate STIM1 and Orai1 as key molecular components underlying SOCE in skeletal muscle, e.g. (i) SOCE is abolished following STIM1 knockdown or by expression of a dominant negative/permeation-defective Orai1 (
      • Lyfenko A.D.
      • Dirksen R.T.
      ) and (ii) myotubes from mice lacking functional STIM1 exhibit defects in force generation and impaired fatigue resistance (
      • Stiber J.
      • Hawkins A.
      • Zhang Z.S.
      • Wang S.
      • Burch J.
      • Graham V.
      • Ward C.C.
      • Seth M.
      • Finch E.
      • Malouf N.
      • Williams R.S.
      • Eu J.P.
      • Rosenberg P.
      ). These and related findings have led to the suggestion that in skeletal muscle, STIM functions as the SR Ca2+ sensor, whereas Orai1 mediates sarcolemmal Ca2+ influx during SOCE, either alone acting as the Ca2+ release-activated Ca2+ channel or in combination with other channels (
      • Stiber J.
      • Hawkins A.
      • Zhang Z.S.
      • Wang S.
      • Burch J.
      • Graham V.
      • Ward C.C.
      • Seth M.
      • Finch E.
      • Malouf N.
      • Williams R.S.
      • Eu J.P.
      • Rosenberg P.
      ). Interestingly, STIM1 has been identified within the plasma membrane in a number of cell types including human epidermal cancer cells, HEK293 cells, and vascular smooth muscle cells (
      • Spassova M.A.
      • Soboloff J.
      • He L.P.
      • Xu W.
      • Dziadek M.A.
      • Gill D.L.
      ,
      • Dziadek M.A.
      • Johnstone L.S.
      ,
      • Zimina O.A.
      • Glushankova A.
      • Skopin A.Y.
      • Alexeenko V.A.
      • Vigont V.A.
      • Mozhayeva G.N.
      • Kaznacheyeva E.V.
      ,
      • Li J.
      • Sukumar P.
      • Milligan C.J.
      • Kumar B.
      • Ma Z.Y.
      • Munsch C.M.
      • Jiang L.H.
      • Porter K.E.
      • Beech D.J.
      ) These studies also showed that extracellular application of the BD Biosciences STIM1 antibody can inhibit SOCE.
      A previous study on rabbit skeletal muscle identified STIM1 in t-tubule membranes (
      • Stiber J.
      • Hawkins A.
      • Zhang Z.S.
      • Wang S.
      • Burch J.
      • Graham V.
      • Ward C.C.
      • Seth M.
      • Finch E.
      • Malouf N.
      • Williams R.S.
      • Eu J.P.
      • Rosenberg P.
      ), and the present findings on human muscle confirm its presence in a buoyant sarcolemmal fraction enriched with lipid rafts and caveolae (Fig. 5C). Furthermore, trapping the STIM1 antibody within the sealed t-system markedly inhibited the SOCE response to halothane in human MHS fibers (Fig. 5A). The initial transient increase in [Ca2+]t-sys was, however, unaffected by the antibody, suggesting that SR Ca2+ release had occurred. SR Ca2+ release was not affected by the antibody, and denaturing the protein abolished its inhibitory effect (Fig. 5B). On balance, previous work suggests that sarcolemmal STIM1 modulates but is not essential for SOCE (
      • Hewavitharana T.
      • Deng X.
      • Wang Y.
      • Ritchie M.F.
      • Girish G.V.
      • Soboloff J.
      • Gill D.L.
      ). Nevertheless, inhibition of SOCE by application of an extracellular antibody provides a novel approach to studying the role of SOCE in adult skeletal muscle, where other commonly used inhibitors (e.g. 2-APB) are ineffective.
      A. M. Duke, P. M. Hopkins, S. C. Calaghan, J. P. Halsall, and D. S. Steele, unpublished observations.

       Relationship to Previous Studies

      In addition to SOCE, two other forms of Ca2+ influx have been identified in skeletal muscle. “Excitation-coupled Ca2+ entry” (ECCE) has mostly been characterized in myotubes and is triggered by repeated or sustained membrane depolarization (
      • Cherednichenko G.
      • Hurne A.M.
      • Fessenden J.D.
      • Lee E.H.
      • Allen P.D.
      • Beam K.G.
      • Pessah I.N.
      ). ECCE does not require store depletion and probably occurs via the L-type Ca2+ channel (
      • Cherednichenko G.
      • Hurne A.M.
      • Fessenden J.D.
      • Lee E.H.
      • Allen P.D.
      • Beam K.G.
      • Pessah I.N.
      ,
      • Hurne A.M.
      • O'Brien J.J.
      • Wingrove D.
      • Cherednichenko G.
      • Allen P.D.
      • Beam K.G.
      • Pessah I.N.
      ,
      • Bannister R.A.
      • Pessah I.N.
      • Beam K.G.
      ). More recently, a “t-system action potential-activated Ca2+ current” (APACC) has been described in adult rat skeletal muscle (
      • Launikonis B.S.
      • Stephenson D.G.
      • Friedrich O.
      ). APACC differs from ECCE in that it is activated by a single action potential but inactivated by repeated depolarizations. These forms of Ca2+ entry are distinct from SOCE, which does not require membrane depolarization, is initiated by SR Ca2+ depletion to a threshold level, and is only inactivated by store refilling (
      • Kurebayashi N.
      • Ogawa Y.
      ,
      • Launikonis B.S.
      • Ríos E.
      ,
      • Launikonis B.S.
      • Stephenson D.G.
      • Friedrich O.
      ).
      As the Ca2+ fluxes associated with both APACC and ECCE require depolarization, these mechanisms may play a physiological role during normal patterns of activity by correcting any efflux of Ca2+ from the cell that occurs via sarcolemmal Ca2+ extrusion mechanisms (
      • Launikonis B.S.
      • Stephenson D.G.
      • Friedrich O.
      ,
      • Hurne A.M.
      • O'Brien J.J.
      • Wingrove D.
      • Cherednichenko G.
      • Allen P.D.
      • Beam K.G.
      • Pessah I.N.
      ). Interestingly, it has been reported that ECCE is enhanced in myotubes from mice with the R163C RyR1 MH mutation (
      • Cherednichenko G.
      • Ward C.W.
      • Feng W.
      • Cabrales E.
      • Michaelson L.
      • Samso M.
      • López J.R.
      • Allen P.D.
      • Pessah I.N.
      ). As sarcolemmal depolarization and consequent fasciculations are known to occur in the presence of the muscle relaxant succinylcholine (
      • Hopkins P.M.
      ), enhanced ECCE current might contribute to the rise in cytosolic [Ca2+] that underlies MH.
      SOCE does not appear to be activated during individual twitch or tetanic responses (
      • Launikonis B.S.
      • Ríos E.
      ,
      • Allard B.
      • Couchoux H.
      • Pouvreau S.
      • Jacquemond V.
      ) but might play a role in fatigue (
      • Pan Z.
      • Yang D.
      • Nagaraj R.Y.
      • Nosek T.A.
      • Nishi M.
      • Takeshima H.
      • Cheng H.
      • Ma J.
      ), where substantial SR Ca2+ depletion can occur (
      • Kabbara A.A.
      • Allen D.G.
      ). Importantly, the present data also suggest that in human MHS muscle, a level of halothane (0.5 mm) within the concentration range that occurs during anesthetic induction (
      • Davies D.N.
      • Steward A.
      • Allott P.R.
      • Mapleson W.W.
      ) can induce sufficient SR Ca2+ depletion to activate SOCE. Such an effect could be large enough to adversely affect cell function. In normal mammalian skeletal muscle, the Ca2+ flux associated with SOCE has been calculated to be ∼19 μm s−1, which corresponds to 1.1–2.2 mm Ca2+ within 1–2 min (
      • Launikonis B.S.
      • Ríos E.
      ), sufficient to completely refill the SR within this period (
      • Kurebayashi N.
      • Ogawa Y.
      ). Furthermore, given the properties of SOCE, Ca2+ influx would be expected to continue until the SR Ca2+ content is restored despite persistent RyR1 activation. On this basis, we propose that sarcolemmal Ca2+ influx via SOCE likely contributes to the sustained increase in cytosolic [Ca2+] that underlies MH.

       Summary

      We show for the first time in human MHS skeletal muscle that (i) a level of halothane within the range that occurs during anesthesia induces sufficient SR Ca2+ release to activate SOCE; (ii) waves of SR Ca2+ release can induce corresponding waves of Ca2+t-sys depletion, suggesting a close temporal relationship between Ca2+ release and SOCE; and (iii) extracellular application of a STIM1-blocking antibody potently inhibits the decrease in [Ca2+]t-sys, suggesting that sarcolemmal STIM1 has an important modulatory role in SOCE. Finally, we suggest that during an MH episode, SOCE may contribute to and sustain the pathological increase in cytosolic [Ca2+].

      Acknowledgments

      We thank Professor David Beech for advice during this study.

      Supplementary Material

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