Measurement of Resting Cytosolic Ca2+ Concentrations and Ca2+ Store Size in HEK-293 Cells Transfected with Malignant Hyperthermia or Central Core Disease Mutant Ca2+ Release Channels*

Malignant hyperthermia (MH) and central core disease (CCD) mutations were introduced into full-length rabbit Ca2+ release channel (RYR1) cDNA, which was then expressed transiently in HEK-293 cells. Resting Ca2+ concentrations were higher in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1. Cells expressing homotetrameric CCD or MH mutant RyR1 exhibited lower maximal peak amplitudes of caffeine-induced Ca2+ release than cells expressing wild type RyR1, suggesting that MH and CCD mutants might be “leaky.” In cells expressing homotetrameric wild type or mutant RyR1, the amplitude of 10 mm caffeine-induced Ca2+ release was correlated significantly with the amplitude of carbachol- or thapsigargin-induced Ca2+ release, indicating that maximal drug-induced Ca2+ release depends on the size of the endoplasmic reticulum Ca2+ store. The content of endogenous sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b), measured by enzyme-linked immunosorbent assay,45Ca2+ uptake, and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that endoplasmic reticulum Ca2+ storage capacity is increased as a compensatory response to an enhanced Ca2+ leak. When heterotetrameric (1:1) combinations of MH/CCD mutant and wild type RyR1 were expressed together with SERCA1 to enhance Ca2+ reuptake, the amplitude of Ca2+release in response to low concentrations of caffeine and halothane was higher than that observed in cells expressing wild type RyR1 and SERCA1. In Ca2+-free medium, MH/CCD mutants were more sensitive to caffeine than wild type RyR1, indicating that caffeine hypersensitivity observed with a variety of MH/CCD mutant RyR1 proteins is not dependent on extracellular Ca2+ concentration.

Malignant hyperthermia (MH) 1 is an autosomal dominant muscle disorder in which genetically susceptible individuals among populations of humans and domestic animals respond to the administration of potent inhalational anesthetics and depolarizing skeletal muscle relaxants with high fever and skeletal muscle rigidity (1,2). Central core disease (CCD) is a rare, non-progressive myopathy, presenting in infancy and characterized by hypotonia and proximal muscle weakness (1). An important feature of CCD is its close association with MH susceptibility (2).
Although diagnosis of CCD is made on the basis of the lack of oxidative enzymatic activity in central regions of skeletal muscle fibers (3), the diagnostic test for MH susceptibility in humans is the North American caffeine halothane contracture test (4) or its European counterpart, the in vitro contracture test (5). These tests are based on the hypersensitivity of contracture of muscle strips, obtained by biopsy, to caffeine or halothane.
The fact that MH and CCD mutations are found in the same gene suggests that different genotypic variants might result in a spectrum of phenotypic responses of different severity (7,9,10). No significant differences were observed in measurements of caffeine and halothane sensitivity between MH and CCD mutants when they were expressed in homozygous state in HEK-293 cells (22). It is not clear whether there are differences in resting myoplasmic Ca 2ϩ concentrations in muscle fibers of MH and CCD patients (23)(24)(25)(26). Difficulties in obtaining accurate and reproducible measurements of resting Ca 2ϩ concentrations in MH or CCD muscle fibers arise from the possibility that compensation may occur in muscle cells and from the different methods used by different groups who have studied the different mutations.
We have attempted to alleviate these problems by transfecting all MH and CCD mutants into HEK-293 cells, which have a homogenous genetic background, and using a fluorescent (Fura-2) imaging assay to measure resting Ca 2ϩ concentrations in these transfected cells. In an earlier study (22), we showed that 15 MH or CCD (MH/CCD) mutant homotetramers expressed in HEK-293 cells were more sensitive to caffeine and halothane than wild type RyR1. In addition, the response observed for the mutant channels expressed in HEK-293 cells correlated closely with the response observed in contracture tests of muscle samples from humans bearing the same mutations (22). In human MH or CCD individuals, however, heterozygosity is most common. In the present study, we have measured resting Ca 2ϩ concentrations in HEK-293 cells expressing the various mutations, analyzed the relationship between caffeine-induced Ca 2ϩ release and intracellular Ca 2ϩ stores, tested for an increase in sarco(endo)plasmic reticulum Ca 2ϩ -ATPase isoform 2b (SERCA2b) synthesis as a compensatory factor in cells expressing wild type or mutant RyR1, tested the caffeine sensitivity of heterotetrameric MH or CCD mutants expressed in the presence or absence of SERCA1, and tested the influence of extracellular Ca 2ϩ concentration on caffeine responses of MH or CCD mutant channels.

EXPERIMENTAL PROCEDURES
Materials-Enzymes for DNA manipulation were obtained from Boehringer Mannheim, New England Biolabs, Promega, and Amersham Pharmacia Biotech. Tissue culture reagents were purchased from Life Technologies, Inc. Monoclonal antibody 34C (27) was a kind gift from Dr. Judith Airey (University of Nevada, Reno). Fura-2 acetoxymethyl ester (Fura-2/AM) and pluronic F-127 were from Molecular Probes. Caffeine was from Sigma. Halothane was from Fluka. Carbachol and thapsigargin were from Calbiochem. [ 45 Ca 2ϩ ]Cl 2 (10 -40 mCi/mg Ca 2ϩ ) was obtained from Amersham Pharmacia Biotech. All other chemicals were of reagent (or highest available) grade.
Construction of MH/CCD Mutants in RYR1 Cassettes-The cloning, expression, and construction of the full-length rabbit skeletal muscle ryanodine receptor (RYR1) cDNA cassettes were described previously (22,28). The construction and expression of single MH/CCD mutant RYR1 cDNAs were also described previously (22).
Cell Culture and DNA Transfection-Culture and transfection of HEK-293 cells using the calcium phosphate precipitation method of Chen and Okayama (29) were carried out as described earlier (22). Ten g of plasmid DNA were used to transfect 2 ϫ 10 5 cells/60-mm plate. Control cells were treated in the same way, but with no DNA or with expression vector DNA only.
Fluorescence Measurements-Ca 2ϩ photometry and Ca 2ϩ imaging assays were used to measure Ca 2ϩ concentration changes in transfected HEK-293 cells as described previously (22). A Photon Technologies Inc. micro-fluorimetry system was used in photometric assays to measure caffeine-induced changes in Fura-2/AM fluorescence resulting from Ca 2ϩ release through the different ryanodine receptor proteins expressed in HEK-293 cells. Dose-response curves were generated and normalized to the maximal Ca 2ϩ release response observed at 10 mM caffeine for both the caffeine and the halothane responses.
Procedures for Ca 2ϩ imaging were the same as for the Ca 2ϩ photometric assay, except that the cells were loaded with 4 M Fura-2 AM, 0.02% pluronic F-127 for 45 min and the emitted fluorescence was fed into a charge-coupled device (CCD) camera (Photon Technologies Inc. SenSys-KAF 1400) instead of a photomultiplier tube. Acquired digital images (400 ms/frame) were analyzed with Image Master 2.0 software (Photon Technologies Inc.). The 340/380 nm ratios from single cells were converted to Ca 2ϩ concentrations according to Grynkiewicz et al. (30): The R max value of 16 was obtained using 10 M ionomycin. R min was determined to be 0.4, and the proportionality constant (S f2 / S b2 ) was determined to be 15.2. A value of 224 nM was used for the apparent K d of Ca 2ϩ binding to Fura-2 (30).
Microsome Preparation, Ca 2ϩ Uptake, and Enzyme-linked Immunoabsorbent Assay (ELISA)-Microsomes were prepared and assayed for Ca 2ϩ transport activity, and data were analyzed as described previously (31). Aliquots of 50 l of the microsome preparation (1 mg/ml) were analyzed by ELISA, as described previously (32). A primary mouse monoclonal anti-SERCA2 ATPase antibody (IgG2a, Affinity Bioreagents Inc.) was used to detect SERCA2 in microsomes.
Immunofluorescence Labeling and Laser Scanning Confocal Micros-copy-HEK-293 cells transfected with RYR1 cDNA were cultured on coverslips. They were fixed for 20 min in 3.7% formaldehyde at room temperature, washed once with PBS, and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Cells were then incubated with IgG2a (Affinity Bioreagents Inc.) in 1:100 dilution for 1 h, washed 3 times with  PBS, followed by tetramethylrhodamine isothiocyanate-conjugated anti-mouse secondary antibody in 1:100 dilution for 1 h, and washed 3 times with PBS. All antibodies were diluted in PBS containing 0.1% Triton X-100. Coverslips were mounted on microscope slides in Im-munoFloure Mounting Medium obtained from ICN. Cells were observed and analyzed with a Nikon Optiphot II microscope, equipped with epifluorescence illumination, and a Bio-Rad MRC 600 confocal laser scanning microscope system. Immunofluorescent images were recorded on a zip disk and analyzed using NIH Image 5.19 software.
Statistical Methods-All data are expressed as mean Ϯ S.E. Linear regression analysis was performed using Origin software (Microcal Software Ltd., Northampton, MA). An unpaired Student's t test was used for statistical comparisons of mean values between samples. A value of p Ͻ 0.05 was taken to indicate statistical significance.

Comparison of Resting Ca 2ϩ Concentration and Caffeineinduced Ca 2ϩ Release among Wild Type, MH Mutant, and CCD Mutant RyR1
Proteins-In this study, we used Ca 2ϩ photometry and Ca 2ϩ imaging to record the Ca 2ϩ release properties of a number of RyR1 mutants expressed in HEK-293 cells. Ca 2ϩ photometry, which is fast and accurate, provides spatially averaged measurements of the fluorescence used to monitor Ca 2ϩ release. Ca 2ϩ imaging provides spatially resolved measurements and was used to acquire images at 2.5 s/frame, permitting us to analyze events in individual cells in response to caffeine.
We used Ca 2ϩ imaging to detect caffeine-induced Ca 2ϩ release in HEK-293 cells transiently transfected with RYR1 cDNA, thereby distinguishing transfected cells from untransfected cells. Ca 2ϩ photometry indicates that Ca 2ϩ release is a rare event in vector-transfected or nontransfected HEK-293 cells which is lost in the background fluorescence when a cluster of 50 or more cells is analyzed. In single cell imaging of pcDNA vector-transfected cells, 6 out of 200 cells responded to 10 mM caffeine, increasing cytosolic Ca 2ϩ concentrations to an average of 180 Ϯ 54 nM (n ϭ 6) and establishing a background response rate of about 3%. In wild type or MH/CCD mutant RYR1-transfected cells, however, 40 -60% of isolated cells, where higher DNA transfection was observed, responded to 10 mM caffeine, a rate about 13-20-fold higher than background. Accordingly, caffeine-responsive cells in our Ca 2ϩ imaging studies were regarded as RYR1-transfected cells, and an error rate of 3% was considered to be acceptable.
Resting cytoplasmic Ca 2ϩ concentrations and 10 mM caffeine-induced Ca 2ϩ release for wild type and 15 MH/CCD mutant RyR1 proteins were measured in single RYR1-transfected cells. The resting cytoplasmic Ca 2ϩ concentration in untransfected HEK-293 cells was 97 Ϯ 5 nM. Transfection with wild type RyR1 raised the resting cytoplasmic Ca 2ϩ concentration to 112 Ϯ 11 nM (Fig. 1A). The resting cytoplasmic Ca 2ϩ concentration in HEK-293 cells transfected with each of the 10 MH mutants tested (C36R, G249R, G342R, R553W, R615C, R615L, R2163C, G2435R, R2458C, and R2458H) also raised the resting cytoplasmic Ca 2ϩ concentration to values ranging from 103 Ϯ 7 to 119 Ϯ 7 nM with an average value of 110 Ϯ 2 nM (Fig. 1A). There were no significant differences in resting Ca 2ϩ concentrations between wild type and any of the MH mutant forms of RYR1. The resting Ca 2ϩ concentration (Fig. 1A) for each of the 5 CCD mutants tested (R164C, I404M, Y523S, R2163H, and R2436H) was higher than for any of the 10 MH mutants and the average resting Ca 2ϩ concentration for the 5 CCD mutants, 142 Ϯ 9 nM, was significantly higher (p Ͻ 0.001) than the average resting Ca 2ϩ concentration for the 10 MH mutants.
However, when resting Ca 2ϩ concentrations for individual CCD mutants were compared with wild type RYR1, significant differences were found for only 2 of the 5 individual CCD mutants tested, Y523S and R2163H (p Ͻ 0.05 for Y523S and p Ͻ 0.001 for R2163H).
Average caffeine-induced Ca 2ϩ release and maximal 340/380 nm ratio change values for MH mutants were 590 Ϯ 107 nM ( . The caffeine-induced Ca 2ϩ release values for MH mutants was not significantly different from the comparable values for CCD mutants. It should be noted, however, that many individual MH and CCD mutants had a significantly lower response to caffeine, measured by both Ca 2ϩ photometry and Ca 2ϩ imaging, than wild type RYR1 (Fig. 1). Maximal 340/380 nm ratio changes were significantly lower for the Y523S (p Ͻ 0.001) and R2163H (p Ͻ 0.001) mutants (Fig. 1B), which had higher resting Ca 2ϩ concentrations ( Fig. 1A) than wild type RyR1. These results are consistent with the view that CCD mutants might be more leaky than MH mutants, accounting for higher resting Ca 2ϩ concentrations and lower Ca 2ϩ -releasable stores, and that MH mutants might also be more leaky than wild type, accounting for lower concentrations of Ca 2ϩ in caffeine-releasable stores.
To test whether resting Ca 2ϩ concentrations might determine caffeine sensitivity and influence maximal caffeine-induced Ca 2ϩ release, a linear correlation analysis was carried out. Resting Ca 2ϩ concentrations had no linear correlation with caffeine ED 50 values (r ϭ Ϫ0.31, p ϭ 0.24) or with maximal caffeine-induced Ca 2ϩ release (r ϭ Ϫ0.31, p ϭ 0.24). Surpris-ingly, a linear correlation was observed between ED 50 and maximal caffeine-induced Ca 2ϩ release (r ϭ 0.63, p Ͻ 0.05) and between ED 50 and maximal 340/380 nm ratio change (r ϭ 0.67, p Ͻ 0.05, data not shown). A linear correlation analysis was also carried out between maximal caffeine-induced Ca 2ϩ release for 9 MH/CCD mutants obtained in this study and caffeine-induced muscle tension or caffeine threshold obtained through in vitro contracture test in an earlier study (19). A linear correlation was observed between maximal caffeine-induced Ca 2ϩ release and caffeine-induced muscle tension (r ϭ Ϫ0.83, p Ͻ 0.05) and between maximal caffeine-induced Ca 2ϩ release and caffeine threshold (r ϭ 0.83, p Ͻ 0.05).
Relationship among Caffeine-, Carbachol-, and Thapsigargin-induced Ca 2ϩ Release in Transfected Cells-To test whether releasable ER Ca 2ϩ stores were really lower in MH/ CCD mutant-transfected cells, thapsigargin and carbachol were used to gate Ca 2ϩ release, determined by fluorescence measurement of the amount of Ca 2ϩ released. Thapsigargin increases intracellular Ca 2ϩ concentration by irreversible blocking of sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) molecules. Thapsigargin-induced Ca 2ϩ release, therefore, represents the rapid, passive depletion of the internal Ca 2ϩ store (33). Carbachol increases intracellular Ca 2ϩ concentration by activation of the phospholipase C pathway through an endogenous muscarinic receptor found in HEK-293 cells, resulting in elevation of intracellular IP 3 and activation of the IP 3 receptor (34). Thus low ER Ca 2ϩ stores would respond with low Ca 2ϩ release following the application of either of these agents.
Figs. 2, A-C, shows that there was a close linear correlation for Ca 2ϩ release induced by any of these three reagents in 27 RYR1 cDNA-transfected cells. These cells were challenged sequentially by 10 mM caffeine (RyR1), 20 M carbachol (IP 3 receptor), and 1.5 M thapsigargin (SERCA). In cells transfected with different MH/CCD mutant cDNAs to form either homotetrameric or heterotetrameric channels, caffeine-induced Ca 2ϩ release was closely correlated with thapsigargininduced Ca 2ϩ release (Fig. 2D). This close correlation among caffeine-, carbachol-, and thapsigargin-induced Ca 2ϩ release indicates that maximal caffeine-induced Ca 2ϩ release reflects the size of the ER Ca 2ϩ store. The lower maximal caffeineinduced Ca 2ϩ release in MH/CCD mutants is likely to be due to a lower Ca 2ϩ store, which, in turn, is likely to reflect Ca 2ϩ leakage through MH/CCD mutant channels.
Comparison of Ca 2ϩ Stores and SERCA2b Content among Untransfected and RYR1 cDNA-transfected Cells-To compare ER Ca 2ϩ stores among untransfected and RYR1 cDNAtransfected cells, carbachol-induced Ca 2ϩ release was measured by Ca 2ϩ imaging and was regarded as an indicator of the size of the Ca 2ϩ store. Fig. 3 shows that carbachol-induced Ca 2ϩ release in untransfected HEK-293 cells was smaller than Ca 2ϩ release in cells transfected with wild type RYR1 but was higher than Ca 2ϩ release in cells transfected with CCD or MH mutant RYR1. The smaller carbachol-induced Ca 2ϩ release in cells transfected with MH/CCD mutants supports the view that these mutants are more leaky than wild type RyR1.
The enhancement of the Ca 2ϩ store by transfection of wild type RyR1 led us to test the hypothesis that cells transfected with channels that increase release from ER Ca 2ϩ stores may overexpress endogenous SERCA2b in an attempt to reestablish Ca 2ϩ homeostasis by lowering elevated resting Ca 2ϩ concentrations to normal (Fig. 1). To determine whether cells transfected with wild type or mutant RYR1 had the capacity to create a larger Ca 2ϩ store, SERCA2b contents were measured by ELISA (Fig. 4A), and Ca 2ϩ uptake was measured in microsomes extracted from transfected cells (Fig. 4B). When HEK-293 cells were transfected with wild type RYR1, the SERCA2b content was increased to 119% (ELISA) or 120% (Ca 2ϩ uptake). Transfection with MH mutant R615L led to an increase in SERCA2b content to 117% (ELISA) or 120% (Ca 2ϩ uptake), whereas transfection with CCD mutant Y523S led to an increase in SERCA2b content to 127% (ELISA) or 123% (Ca 2ϩ uptake). Although transfection efficiency approaches 50% for non-confluent cells, overall transfection efficiency is only about 25%. Therefore, the real increase in SERCA2b expression in transfected cells would be about 4-fold higher, approaching a doubling of SERCA2b content. Immunofluorescent labeling and confocal microscopy were used in a third measurement of the enhancement of SERCA2b content in single cells transfected with mutant RyR1. Because only mouse anti-RyR1 and mouse anti-SERCA2 monoclonal antibodies are available, green fluorescent protein (GFP) vector (CLONTECH) and rabbit RyR1 mutant Y523S were cotransfected into HEK-293 cells in a 1:9 molar ratio. GFP fluores- The linear analysis method was used to analyze the following: the correlation between Ca 2ϩ release induced by carbachol, an agonist of an endogenous muscarinic receptor which releases IP 3 and acts on an IP 3 receptor, and by caffeine, a drug which acts directly on RyR1 (A); the correlation between Ca 2ϩ release induced by thapsigargin, an irreversible SERCA inhibitor, and by caffeine (B); the correlation between Ca 2ϩ release induced by carbachol and thapsigargin (C); and the correlation between mean thapsigargin-and caffeine-induced Ca 2ϩ release values in MH/CCD mutant homotetramers and heterotetramers (D).
cence in cells was regarded as an indicator of transfected cells. Immunofluorescent staining of rabbit RyR1, detected by mouse monoclonal 34C antibody, showed that about 80% of transfected cells expressed both GFP and RyR1, 17% of transfected cells expressed GFP alone and 3% of transfected cells expressed RyR1 alone (not shown). The SERCA2 fluorescence from Y523S-transfected cells, which were GFP-positive, was increased by 74 Ϯ 4% (n ϭ 45, p Ͻ 0.001) when compared with fluorescence from untransfected cells (not shown), supporting our other measurements showing that the SERCA2 content of Y523S-transfected cells was nearly doubled. These results suggest that compensation in the form of enhanced SERCA2 expression occurs in RYR1 or MH/CCD mutant cDNA-transfected cells.
Caffeine Responses for Homotetrameric and Heterotetrameric MH/CCD Mutant Channels and the Effect of Coexpression with SERCA1-The maximal caffeine-induced Ca 2ϩ release for most MH/CCD mutant homotetrameric channels was smaller than for wild type channels (Fig. 1). This is in contrast to clinical observations which show that muscle fibers from malignant hyperthermia-sensitive individuals contract more strongly in low concentrations of caffeine and halothane than fibers from normal individuals, in line with enhanced Ca 2ϩ release. Although homotetrameric MH/CCD mutant channels expressed in HEK-293 cells have been shown to be more sensitive to caffeine and halothane than wild type RyR1 (22), heterotetrameric MH/CCD mutant channels are most common in humans. In order to determine whether heterotetrameric wild type and mutant RyR1 channels would be less leaky, R615L (MH mutant) and Y523S (CCD mutant) were coexpressed in a 1:1 ratio with wild type RYR1. When HEK-293 cells expressed heterotetrameric channels, their caffeine sensitivities were found to be intermediate between those of cells expressing homotetrameric R615L or Y523S and cells expressing homotetrameric wild type RYR1 (Fig. 5A). Maximal 340/ 380 nm ratio changes (Fig. 5B) and 10 mM caffeine-induced Ca 2ϩ release (Fig. 5D) in heterotetrameric MH/CCD mutants were higher than those in homotetrameric MH/CCD mutants, suggesting that heterozygote channels are less leaky than homozygote channels.
It is assumed that the activity of SERCA pumps is coordinated with the activity of RyR Ca 2ϩ release channels in healthy skeletal muscle to regulate cytosolic Ca 2ϩ concentrations. Thus the 2-fold enhancement of SERCA2 synthesis that we observed in cells expressing MH/CCD mutants could be explained logically as a compensatory event. Nevertheless, enhanced endogenous SERCA2b synthesis was not sufficient to compensate for the higher resting Ca 2ϩ concentrations attributed to leaky mutant channels R615L or Y523S (Fig. 1). Since it is unlikely that compensatory SERCA synthesis had reached equilibrium in these cells over the 48-h period between transfection and analysis, we attempted to increase SERCA synthesis at a more rapid rate by transfection with SERCA1 under conditions where high levels of activity are observed within 48 h (35). Under these conditions, SERCA1 was functional, since it increased the rate of Ca 2ϩ removal from the cytoplasm following caffeine-induced Ca 2ϩ release and lowered the elevated base line of Ca 2ϩ fluorescence resulting from repeated caffeine stimulation of cells cotransfected with wild type or MH/CCD mutant RyR1 (not shown).
Surprisingly, coexpression of mutants R615L or Y523S with SERCA1 raised resting Ca 2ϩ concentrations (Fig. 5C). The coexpression of SERCA1 with Y523S, or with Y523S plus wild type RyR1, or with R615L, or with R615L plus wild type RyR1 decreased caffeine sensitivity (Fig. 5A), increased resting Ca 2ϩ concentration (Fig. 5C), and increased maximal Ca 2ϩ release for both RyR1 mutants (Fig. 5, B and D). These results suggest that SERCA1 can increase Ca 2ϩ removal rates and Ca 2ϩ stores but that increased flux through the mutant channels in the ER, even under conditions where Ca 2ϩ uptake is greatly increased, ultimately results in an increase in resting Ca 2ϩ concentrations.
We used Ca 2ϩ imaging to test caffeine-induced Ca 2ϩ release in MH/CCD mutant heterotetramers. We found that the heterotetrameric R615L mutant plus SERCA1 and the heterotetrameric mutant Y523S plus SERCA1 were significantly more sensitive to low concentrations of caffeine (0.5 and 1 mM) and halothane (0.1 and 0.25 mM) than wild type RyR1 plus SERCA1 (not shown). The MH/CCD mutant heterotetramers plus SERCA1 released more Ca 2ϩ when challenged by low concentrations of caffeine and halothane than wild type RyR1 plus SERCA1 (not shown).
Influence of Extracellular Ca 2ϩ Levels on Caffeine Sensitivity of Wild Type and MH/CCD Mutant RyR1-All of the results described above were obtained from HEK-293 cells incubated in a medium containing 2 mM Ca 2ϩ . To confirm that caffeineinduced Ca 2ϩ release in RYR1-transfected cells was caused by Ca 2ϩ release from ER Ca 2ϩ stores, caffeine-induced Ca 2ϩ release in RYR1-transfected cells was measured in Ca 2ϩ -free medium. Ca 2ϩ release, induced by caffeine or carbachol and measured by Ca 2ϩ imaging in RYR1-transfected cells, was reduced dramatically, and resting Ca 2ϩ concentrations in both transfected and untransfected cells were much lower in a Ca 2ϩfree medium than in Ca 2ϩ -containing medium (not shown). These results indicate that extracellular Ca 2ϩ has a significant influence on intracellular Ca 2ϩ concentration.
In early experiments, we found that caffeine-induced Ca 2ϩ release could be abolished by the addition of thapsigargin in Ca 2ϩ -containing medium (not shown), indicating that caffeineinduced Ca 2ϩ release depends on the ER Ca 2ϩ store. In later studies, RYR1 cDNA-transfected cells were incubated in Ca 2ϩfree medium with Fura-2/AM for 30 min, and caffeine-induced Ca 2ϩ release was measured in Ca 2ϩ -free medium, then in Ca 2ϩ -containing medium, and, finally, in Ca 2ϩ -free medium ( Fig. 6A). In Ca 2ϩ -free medium, caffeine responses were reduced dramatically after the third caffeine stimulation (Fig.  6A). In Ca 2ϩ -containing medium (not shown), caffeine stimulation of Ca 2ϩ release could be induced repeatedly. The addition of Ca 2ϩ -containing medium (2 mM Ca 2ϩ ) resulted in an increase in resting Ca 2ϩ concentration and an increase in the amplitude of caffeine-induced Ca 2ϩ release (Fig. 6A). When the cells were returned to Ca 2ϩ -free medium, the caffeine response was reduced dramatically. Similar results were obtained for carbachol responses in untransfected cells (not shown). These results confirm that extracellular Ca 2ϩ concentrations have a profound influence on cellular Ca 2ϩ homeostasis.
When we measured caffeine dose-response curves for wild type and Y523S and R615L mutants in Ca 2ϩ -free medium, we

FIG. 5. Comparison of resting Ca 2؉ concentrations and caffeine-induced Ca 2؉ release in HEK-293 cells transfected with wild type and MH/CCD mutant RYR1 constructs to form homotetrameric or heterotetrameric RyR1 in the presence or absence of SERCA1.
HEK-293 cells were transfected with wild type or MH/CCD mutant RYR1 cDNA to obtain homotetrameric RyR1 or cotransfected with wild type and CCD or MH mutant RYR1 constructs in a 1:1 molar ratio to obtain heterotetrameric RyR1. Caffeine ED 50 (A) and maximal 340/380 nm fluorescence ratio changes (B) were measured by Ca 2ϩ photometry. Resting Ca 2ϩ concentrations (C) and 10 mM caffeine-induced Ca 2ϩ release (D) in cells transfected with the DNA constructs indicated were measured by Ca 2ϩ imaging. observed that the mutant channels were more sensitive than wild type to caffeine (Fig. 6B), just as they were in Ca 2ϩcontaining medium. The caffeine ED 50 values for RyR1, Y523S, and R615L in Ca 2ϩ -free media were 8.1, 0.99, and 2.64 mM, respectively. These values are 2 to 4 times higher than the ED 50 values measured in Ca 2ϩ -containing medium. The lower caffeine ED 50 values in Ca 2ϩ -free medium may have been due to the lower resting Ca 2ϩ levels in Ca 2ϩ -free media, suggesting that resting Ca 2ϩ concentrations may also influence the caffeine sensitivity of RyR1.

Analysis of RyR1 Mutations in HEK-293
Cells-In a recent paper Querfurth et al. (36) reported that an unspecified isoform of the ryanodine receptor (RyR) is expressed in HEK-293 cells. The level of endogenous RyR expression measured was very low, since RyR could only be detected through highly sensitive immunoprecipitation of radiolabeled cells or by Western blotting of immunoprecipitates. We could not detect RyR proteins in extracts of our HEK-293 cells by Western blotting, although we observed huge amounts of transfected RyR under comparable conditions (22). Although Querfurth et al. (36) reported cellular immuno-staining, a suitable control for background staining was not provided. Querfurth et al. (36) reported increases in intracellular Ca 2ϩ concentration of nearly 500 nM in 23-33% of their untransfected HEK-293 cells by imaging. We cannot confirm this result under the conditions of our experiments. If this high level of endogenous RyR activity were present in our cells, we would, unquestionably, have detected it by Ca 2ϩ photometry when we measured the combined fluorescence emission from at least 50 cells in experiments repeated over many years (22). Instead, we measured a negligible change in the background 340/380 fluorescence ratio from Fura-2 in untransfected HEK-293 cells, but we measured a ratio change in HEK-293 cells transfected with wild type RyR1 that was greater than 0.8.
In this study, in which we present single cell imaging of pcDNA-transfected HEK-293 cells, we also measured the back-ground of endogenous Ca 2ϩ release in untransfected and vector-transfected cells. We found that 6 out of 200 cells responded to 10 mM caffeine, increasing cytosolic Ca 2ϩ concentrations to an average of 180 Ϯ 54 nM and establishing our background response rate at 3%. In wild type or mutant RyR1-transfected cells, 40 -60% of cells responded to 10 M caffeine, a rate 13-20fold higher than background, increasing cytosolic Ca 2ϩ concentrations well above 500 nM (Fig. 1A). The differences in the results of our experiments and those of Querfurth et al. (36) are most likely to be due to the way in which experiments were carried out. Querfurth et al. (36) could not observe caffeineinduced Ca 2ϩ release when they allowed buffer changes to flow over the cells (the conditions of our experiments). In order to see Ca 2ϩ release, they had to interrupt flow and then remove and replace buffer to achieve instant 15 mM caffeine. Thus re-perfusion at high caffeine seemed to sensitize the endogenous RyR activity, even though the resulting spike of Ca 2ϩ release was actually slower than that which we observed. Even if there were re-perfusion artifacts in the Ca 2ϩ -imaging protocol of Querfurth et al. (36) that could account for the exaggerated Ca 2ϩ release attributed to endogenous RyR, they would not be relevant to our studies, since we used a different protocol that, maximally, triggered only rare cases of Ca 2ϩ release in untransfected cells.
Functional Differences in MH and MH/CCD Mutations-In earlier publications (7,9,10), we proposed that there might be spontaneous Ca 2ϩ leakage through MH mutant channels but that compensatory mechanisms would lead to rapid re-establishment of Ca 2ϩ homeostasis. Thus muscle hypertrophy in MH swine might result from spontaneous Ca 2ϩ release from an abnormal Ca 2ϩ release channel leading to spontaneous muscle contracture. We also proposed that CCD mutations might lead to more serious spontaneous Ca 2ϩ release, which would disrupt Ca 2ϩ homeostasis in the core of the cell but not in the periphery where re-establishment of Ca 2ϩ homeostasis would be aided by plasma membrane Ca 2ϩ -ATPases or Na ϩ /Ca 2ϩ exchangers in the plasma membrane (9, 10).

FIG. 6. Ca 2؉ concentration changes induced by caffeine in RYR1-transfected cells in Ca 2؉ -containing and Ca 2؉ -free media (A) and caffeine dose-response curves for wild type and MH/CCD mutant RyR1 in Ca 2؉ -free medium (B).
A, trace of the response of RYR1-transfected cells to caffeine in Ca 2ϩ -free or Ca 2ϩ -containing media recorded by Ca 2ϩ photometry; B, dose-response curves were obtained by Ca 2ϩ photometry of HEK-293 cells transfected with wild type RYR1 or CCD (Y523S) or MH (R615L) mutant RYR1. About 48 h after transfection, the cells were loaded with 1 M Fura-2/AM for 30 min in Ca 2ϩ -free media and stimulated with different concentrations of caffeine. The amplitude of each peak caffeine response was normalized to the amplitude of the peak caffeine response. Changes in the fluorescence ratio are presented as ⌬R ϭ (R Ϫ R min )/(R max Ϫ R min ), where R refers to the 340/380 nm fluorescence ratio at each caffeine concentration, and R min and R max refer to the fluorescence ratio under resting conditions and at the highest response to caffeine. R is plotted as a function of caffeine concentration.
In this study, we observed higher resting Ca 2ϩ concentrations in HEK-293 cells transfected with wild type RYR1, suggesting that even the normal expressed channel might increase the permeability of the ER Ca 2ϩ store. This might explain the difficulty that we have encountered in attempts to obtain a stable HEK-293 cell line expressing RYR1. When the cells were transfected with RYR1 carrying MH mutations, resting cytosolic Ca 2ϩ concentrations were raised over that of wild type RyR1-transfected cells in some cases (Fig. 1), but average values were not significantly different from wild type. Of greater interest was the observation that the average resting cytosolic Ca 2ϩ concentration was elevated significantly over wild type and over MH mutant RyR1 proteins for the five CCD mutant RyR1 proteins expressed in HEK-293 cells. For the individual CCD mutants, Y523S and R2163H, resting cytosolic Ca 2ϩ concentrations were elevated significantly over wild type RyR1.
Maximal caffeine-induced Ca 2ϩ release, measured by both Ca 2ϩ photometry and Ca 2ϩ imaging, was lower in cells transfected with individual MH/CCD mutants than with wild type RYR1. We measured the size of the ER Ca 2ϩ store that was releasable by three different triggers acting through three different mechanisms as follows: caffeine, which releases Ca 2ϩ through RyR; thapsigargin, which inhibits SERCA2, thereby preventing re-uptake of Ca 2ϩ lost through passive leaks; and carbachol, which releases Ca 2ϩ indirectly through the IP 3 receptor. The close correlation of caffeine-, carbachol-, and thapsigargin-induced Ca 2ϩ release indicates that the maximal caffeine-induced Ca 2ϩ release is proportional to the size of the ER Ca 2ϩ store. We noted a large variation in amplitude of Ca 2ϩ release in different cells to the same releasing agents (caffeine, carbachol, and thapsigargin) (Fig. 2). This variation was not caused by DNA transfection, because untransfected cells also showed a large variation in their response to carbachol and thapsigargin. It is more likely that variation was based on factors such as age or cell cycle stage. Despite this variation among individual cells, we were able to obtain clear correlations from measurements of sizes of ER Ca 2ϩ stores in response to different triggering agents. Accordingly, leakage from these stores through an abnormal RyR1 would be predicted to lower the store available for caffeine-induced Ca 2ϩ release and, at the same time, to increase cytosolic Ca 2ϩ concentrations, as observed in Fig. 1. These observations suggest that CCD and MH mutant channels are more leaky than wild type RYR1 channels.
Resting cytosolic Ca 2ϩ concentrations were not correlated with caffeine sensitivity or with maximal caffeine-induced Ca 2ϩ release. This lack of correlation is consistent with clinical observations and with our previous results (22), which showed no differences in caffeine and halothane sensitivity between MH and CCD mutants. The results suggest that resting Ca 2ϩ concentrations do not have a major influence on the caffeine sensitivity of MH/CCD mutants.
Caffeine ED 50 values for Ca 2ϩ release through MH/CCD mutant proteins were linearly correlated with maximal caffeine responses, and the maximal caffeine-induced Ca 2ϩ release was also linearly correlated with clinical caffeine thresholds, indicating that higher ER Ca 2ϩ stores inhibit caffeine responses. In single-channel measurements with rabbit RYR1, an increase in lumenal Ca 2ϩ concentration, from micromolar to millimolar, has been shown to decrease single channel activity (37)(38)(39). Since high lumenal Ca 2ϩ lowers channel open probability, it may raise caffeine ED 50 . The corollary is that low lumenal concentrations may increase caffeine ED 50 . On the other hand, lower Ca 2ϩ concentration in the sarcoplasmic reticulum lumen may provide a compensatory mechanism in MH/CCD skeletal muscle cells. Whether halothane will trigger an MH reaction may depend not only on the sensitivity of MH/CCD mutants to halothane but also on the balance between the decreased inhibitory effect of lumenal Ca 2ϩ concentration and the reduced net Ca 2ϩ efflux from the sarcoplasmic reticulum. This may explain why some individuals who carry MH mutations do not show higher sensitivity to caffeine and halothane.
The observation of lower caffeine-induced Ca 2ϩ release in HEK-293 cells transfected with MH/CCD mutants is not consistent with clinical observations. However, in vivo, MH individuals rarely have homozygous MH/CCD mutations, and an increase in the number of SERCA pumps could compensate for enhanced Ca 2ϩ release. We tested whether MH/CCD mutants had higher caffeine-induced Ca 2ϩ release when they were coexpressed with wild type RyR1 and SERCA1. The caffeine sensitivity of the heterotetrameric MH/CCD mutants was between that of the homotetrameric MH/CCD mutants and wild type RyR1 (Fig. 2), but maximal 340/380 nm ratio changes in MH/CCD heterotetrameric mutants were higher than those in homotetrameric MH/CCD mutants. RyR1 isolated from pigs heterozygous for the R614C MH mutation demonstrated intermediate values for the rate of Ca 2ϩ release and the affinity for [ 3 H]ryanodine (40 -44). Maximal Ca 2ϩ release responses were similar in heterotetrameric MH/CCD mutants and wild type homotetramers, but the heterotetrameric MH/CCD mutants plus SERCA1 were more sensitive to low concentrations of caffeine and halothane than the cells transfected with wild type RyR1 plus SERCA1 (Fig. 5). Overall, these results indicate that coexpression of SERCA1 increases the ER Ca 2ϩ store and that expression of heterotetrameric MH/CCD mutants reduces the abnormal leak of homotetrameric MH/CCD mutant channels.
Compensation in RYR1-transfected Cells-Our overall observations concerning the expression of wild type, MH, and CCD mutant RyR1 in HEK-293 cells can be interpreted as a coherent pattern of events. The expression of wild type RyR1 raises the resting Ca 2ϩ concentration (Fig. 1A), increases the size of the Ca 2ϩ store (Fig. 3), and increases both SERCA2b content and activity (Fig. 4). These observations can be explained on the basis of an increased permeability of the ER Ca 2ϩ store which is compensated for by Ca 2ϩ -induced synthesis of SERCA2b, with a consequent enlargement of the Ca 2ϩ store. Despite this attempt at re-establishment of Ca 2ϩ homeostasis, the enhanced flux of Ca 2ϩ through the ER still results in a higher resting Ca 2ϩ concentration. Expression of MH or CCD mutant RyR1 channels also raises resting Ca 2ϩ concentrations and enhances synthesis of SERCA2b, leading to a higher potential for Ca 2ϩ storage. This potential was not realized, however, since the carbachol-releasable stores for MH and CCD mutants were seen to be depleted (Fig. 3). This suggests that the attempt to re-establish Ca 2ϩ homeostasis for MH and CCD mutants was less successful, possibly because the flux through even more permeable channels would require even higher synthesis of SERCA2b. In attempts to determine whether full compensation could ever be achieved, we coexpressed SERCA1 with homozygous and heterozygous mutant channels (Fig. 5). We found that coexpression of higher levels of SERCA1 did not reduce resting Ca 2ϩ concentrations but did increase caffeinereleasable Ca 2ϩ stores, particularly for RyR1 heterozygotes (Fig. 5). These experiments provide new insights into the way in which diseases arising from defects in Ca 2ϩ regulatory proteins progress and are compensated. Clearly compensation is a much more complex process involving Ca 2ϩ regulatory proteins in the sarcoplasmic reticulum, the plasma membrane, and mitochondria (7,9,10), and the contributions of these systems to disturbances in Ca 2ϩ homeostasis will have to be investigated in future studies.
Our current observations provide support for the hypothesis (7, 10) that MH and CCD mutants have enhanced permeability and that compensatory mechanisms such as enhanced SERCA synthesis are brought into play to restore Ca 2ϩ homeostasis. The synthesis of SERCA1 is enhanced in myoblasts by elevated Ca 2ϩ (45,46). Our results confirm that SERCA2b expression is enhanced in transfected HEK-293 cells and show that the most leaky CCD mutant channel (Y523S) induces the highest overexpression of endogenous SERCA2 (Fig. 4). The correlation between higher permeability and enhancement of Ca 2ϩ stores, however, was not perfect, however. We observed only slightly higher synthesis of SERCA2b for the MH and CCD mutants than for wild type RyR1. This may simply reflect the fact that our observations were made only 48 h after transfection, a period too short for equilibrium to be established. One of the striking morphological features of CCD muscle is a proliferation of the sarcotubular system in the core (47). The development of such cores occurs over a period of months or even years, so that compensatory mechanisms may require a long time to reach equilibrium.
In Fig. 7, we illustrate the Ca 2ϩ concentration changes that we have observed in our studies of the size of the ER Ca 2ϩ stores and resting cytosolic Ca 2ϩ concentrations in cells transfected with wild type, MH, or CCD mutant RyR1. The expression of wild type RyR1 increases resting cytosolic Ca 2ϩ concentrations. In response to increased cytosolic Ca 2ϩ concentration, the transfected cells increase SERCA2b expression, increasing the potential to store more ER Ca 2ϩ . This potential is realized as a higher Ca 2ϩ store for the cells transfected with wild type RyR1. The MH mutant R615L is more leaky than wild type RyR1, resulting in an elevated resting Ca 2ϩ concentration and enhanced SERCA2b synthesis, but the potential for an increased Ca 2ϩ store is not realized because Ca 2ϩ flux out of the store is higher than Ca 2ϩ flux into the store. A lower ER Ca 2ϩ store results. For the same reasons, the CCD mutant, Y523S, being even more leaky, results in higher resting Ca 2ϩ concentration and an even higher SERCA2b synthesis, but because Ca 2ϩ fluxes are not balanced, the Ca 2ϩ store is even lower. The result is that the cells transfected with a CCD mutant have the highest resting Ca 2ϩ concentration and the lowest ER Ca 2ϩ store. These results support the view that MH and CCD mutants can result in a spectrum of phenotypes ranging from muscle hypertrophy, induced by spontaneous Ca 2ϩ leaks, to muscle atrophy, caused by Ca 2ϩ -induced damage to the core of the muscle cell where compensatory function is least effective (7,9,10).
Influence of Extracellular Ca 2ϩ Concentration on Intracellular Ca 2ϩ Homeostasis-Extracellular Ca 2ϩ concentrations were not considered when we compared caffeine or halothane sensitivities among wild type, MH, and CCD mutant RyR1 because we were measuring K activation properties of different mutant proteins. It became a concern, however, when the size of the ER Ca 2ϩ store was measured. Under the conditions of these experiments, we found that extracellular Ca 2ϩ has a profound effect on caffeine-induced Ca 2ϩ release and ER Ca 2ϩ stores (Fig. 6). It is impossible to obtain an accurate RyR1 caffeine ED 50 in a Ca 2ϩ -free medium, because cytosolic Ca 2ϩ is lost to extracellular spaces after it is released from the ER. Accordingly, caffeine ED 50 measured in Ca 2ϩ -free medium reflected not only the sensitivity of the RyR1 channel but also the size of the ER Ca 2ϩ store in the transfected cells. The lower caffeine sensitivity in Ca 2ϩ -free medium than in Ca 2ϩ -containing medium indicates that resting Ca 2ϩ concentrations can influence RyR1 caffeine sensitivity.
In this study, we have identified at least three factors that influence the RyR1 caffeine response. These are the sensitivity of RyR1 proteins, the size of the releasable ER Ca 2ϩ store, and the resting Ca 2ϩ concentration. Several laboratories have shown that MH mutants are more sensitive to a variety of channel activators, including Ca 2ϩ , ATP, caffeine, and halothane, than wild type (9, 22, 38, 40 -44) and are less sensitive to Mg 2ϩ (48). Thus the main cause of MH is the hypersensitivity of RyR1 mutant proteins to very basic stimulus, but the occurrence of MH in humans may also be influenced by the size of the Ca 2ϩ store and resting Ca 2ϩ concentration which may be modulated by a system of regulatory proteins in skeletal muscle.
The present results were obtained with HEK-293 cells transfected with wild type and MH/CCD mutant RYR1 constructs, and the data were obtained 48 h after transfection. HEK-293transfected cells differ from skeletal muscle cells in that they lack many of the proteins that may modulate myoplasmic Ca 2ϩ concentrations. The advantage of the HEK-293 cell expression system is that it permits the expression of homozygous and concentrations, probably by the enhanced permeability of ER Ca 2ϩ stores. In an effort to compensate for increased Ca 2ϩ release to restore Ca 2ϩ homeostasis, the HEK-293 cells express more SERCA2b, increasing the potential for more Ca 2ϩ storage. Transfection with MH mutant RYR1 increases permeability of the ER Ca 2ϩ store even more, so that ER Ca 2ϩ stores begin to be depleted. Resting cytosolic Ca 2ϩ concentrations are higher, and ER Ca 2ϩ stores are lower in CCD mutant RYR1-transfected cells, because of the high permeability of the CCD mutant RyR1 proteins.
heterozygous MH/CCD mutants in a homogeneous genetic background. This has facilitated the detection of functional differences between normal and abnormal channels, which may not be detectable in native skeletal muscle samples. It is difficult or impossible to obtain substantial quantities of human MH mutant muscle, and most human samples are from heterozygotes where it is more difficult to detect small changes in RyR1 function. Moreover, differences between MH/CCD and normal samples might be too small to be detected due to the compensatory effects of other Ca 2ϩ regulatory systems unique to skeletal muscle cells.
The absence of potential Ca 2ϩ regulatory systems in HEK-293 cells is a disadvantage that must be weighed against the advantage of cleaner analysis of the Ca 2ϩ release channel. It would be of interest, if cell lines could be established, to observe the long term compensatory effects that are probably missing in transiently transfected HEK-293 cells. Despite the differences in the assay systems, it is reasonable to believe that MH/CCD mutants, which are more leaky in HEK-293 cells, are also more leaky in skeletal muscle cells.