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CARDIOVASCULAR

Effects of Azumolene on Ca²⁺ Sparks in Skeletal Muscle Fibers

Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland

Received January 21, 2005; accepted April 5, 2005.

	Abstract

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Azumolene is an analog of dantrolene, the only approved medicine for treatment of malignant hyperthermia (MH). The pharmacological mechanism of these drugs is to inhibit skeletal muscle sarcoplasmic reticulum (SR) Ca²⁺ release by modulating the activity of the SR ryanodine receptor (RyR) Ca²⁺ release channel. To investigate the effects of azumolene on SR Ca²⁺ channel gating within skeletal muscle fibers, we monitored Ca²⁺ sparks in permeabilized frog skeletal muscle fibers. Application of 0.0001 to 10 µM azumolene suppressed the frequency of spontaneous Ca²⁺ sparks in a dose-dependent manner (EC₅₀ = 0.25 µM; Hill coefficient = 1.44), but it did not cause systematic dose-dependent effects on the properties of the Ca²⁺ sparks. These results suggest that azumolene decreases the likelihood of Ca²⁺ release channel openings that initiate Ca²⁺ sparks, thereby decreasing spark frequency, but it has little effect on aggregate Ca²⁺ channel open times during a spark. To assess azumolene inhibition of RyRs activated in a manner analogous to those activated during an MH episode, we applied DP4, a synthetic peptide corresponding to a central region of RyR1 (Leu2442 to Pro2477), which mimics an MH modification. Azumolene also decreased Ca²⁺ spark frequency in a dose-dependent manner without altering spark properties in the DP4 MH model. We conclude that azumolene suppresses the opening rate but not the open time of RyR Ca²⁺ release channels within skeletal fibers.

In frog skeletal muscle, Ca²⁺ sparks underlie the global Ca²⁺ transient during depolarization (Tsugorka et al., 1995; Klein et al., 1996). Ca²⁺ sparks are brief, highly localized elevations of myoplasmic [Ca²⁺]_free that can be visualized by fluorescent Ca²⁺ indicators (Cheng et al., 1993). These events provide a means to evaluate the opening and closing properties of Ca²⁺ release channels (the RyR) or small groups of channels in functional muscle fibers. In the present study, we sought to identify the effect of azumolene, a more water-soluble analog of dantrolene, on the opening and closing properties of RyR Ca²⁺ release channels in permeabilized frog skeletal muscle fibers. Using this approach it is possible to determine whether the inhibition of Ca²⁺ release by azumolene in muscle fibers is due to changes in Ca²⁺ spark frequency (i.e., channel opening rate), changes in Ca²⁺ spark spatiotemporal properties (i.e., channel open time and/or conductance), or both. Permeabilized muscle fibers maintain most of the macromolecular interactions present in intact muscle fibers and provide a convenient means to apply a range of conditions, reagents, peptides, and proteins to assess their effects on RyR activity (for review, see Schneider and Ward, 2002). The sparks observed in permeabilized fibers are "spontaneous" calcium release events, and their spatiotemporal properties reflect the influence of activating and inhibiting agents in the absence of voltage sensor activity (Klein et al., 1996). Here, we find that azumolene can completely inhibit the frequency of spontaneous Ca²⁺ sparks in a dose-dependent manner, with little alteration in the spatiotemporal properties of the Ca²⁺ sparks. Furthermore, using a synthetic peptide segment of the central domain of RyR1 from Leu2442 to Pro2477 (DP4) to mimic an MH episode (Yamatomo et al., 2000), we show that the increased spontaneous Ca²⁺ spark frequency in the presence of DP4 is also greatly reduced by azumolene in a dose-dependent manner. Our data suggest that azumolene acts to stabilize the closed state of the RyR Ca²⁺ release channel and thereby inhibits the initiation of Ca²⁺ sparks, but it has no effects on the termination of the Ca²⁺ release underlying the Ca²⁺ sparks.

	Materials and Methods

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Frogs (Rana pipiens) were first placed into a crushed ice water slurry for 30 min followed by rapid decapitation and spinal cord destruction according to protocols approved by the University of Maryland Institutional Animal Care and Use Committee. The iliofibularis muscle was removed and pinned in a dissecting chamber containing Ringer's solution: 115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, and 10 mM HEPES, pH 7.0. Small fiber segments (5 mm) were manually dissected in relaxing solution containing: 120 mM K-glutamate, 2 mM MgCl₂, 0.1 mM EGTA, and 5 mM Na-Tris-maleate, pH 7.0, and mounted in an experimental chamber, stretched to 3.6 ± 0.4 µm per sarcomere. The fiber was bathed in a relaxing solution containing 0.01% saponin and 1.0 mM EGTA for 30 to 40 s for chemical permeabilization, allowing solution equilibration into the myoplasm. Immediately after the permeabilization procedure, the fiber was bathed in internal solution containing: 80 mM potassium-glutamate, 5 mM Na₂ATP, 4.79 mM MgCl₂ (0.42 [Mg²⁺]_free), 20 mM Tris-maleate, 0.1 mM EGTA, 20 mM Na₂ creatine phosphate, 5 mM glucose, and 0.05 mM Fluo-3 (pentapotassium salt) (Molecular Probes, Eugene, OR), pH 7.0, supplemented with 8% dextran (41 kDa). The [Ca²⁺]_free (100 nM) and the [Mg²⁺]_free in our internal solution were calculated using WinMaxC 2.5 (Patton et al., 2004). After control data collection, the bathing solution was changed to an experimental internal solution containing 0.2% DMSO plus azumolene sodium, [1-(((5-(4-bromophenyl)-2-oxazolyl)methylene)amino)-2,4-imidazlidinedione; gift from Dr. Jerome Parness, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ] at a final concentration of 0 to 10 µM. The fiber was allowed to equilibrate for 10 min before another period of data collection. To control for effects of solution change, "sham" fibers were exposed to azumolene-free 0.2% DMSO in internal solution. To minimize muscle-to-muscle variation, a group of sham fibers ([azumolene] = 0) was obtained from the same muscles as used for each azumolene concentration.

For experiments mimicking an MH episode, the fiber was first bathed in internal solution with the [Mg²⁺]_free increased to 1.2 mM (6.73 mM total MgCl₂) to decrease the frequency of Ca²⁺ sparks. After data collection in the absence of DP4, the bathing solution was changed to the internal solution containing 150 µM DP4 (a synthetic domain peptide of RyR1 from Leu2442 to Pro2477; a gift from Dr. Noriaki Ikemoto, Harvard Medical School, Boston, MA) plus 0.2% DMSO. The fibers were then allowed to equilibrate for 10 min before the second data collection (control). The bathing solution was then changed to internal solution containing 150 µM DP4, 0.2% DMSO, and 0.04 to 5 µM azumolene (experimental) or 150 µM DP4, 0.2% DMSO, and no azumolene (sham).

Ca²⁺ sparks in fibers were monitored on an inverted microscope (Olympus IX-70 with a 60x, 1.4 numerical aperture oil immersion objective). The line-scan images were recorded with a laser scanning confocal system (Bio-Rad MRC 600, 488-nm excitation) operated in line-scan (x-t) mode, with the scan line parallel to the fiber axis (2 ms per line, 768 pixels per line, 0.18 µm per pixel, 512 lines per image, total line scan image duration 1.024 s). The scan line was 138 µm in length, parallel to the fiber's long axis. To avoid laser damage to the fiber, the line-scan was repeated for five images at one location and then moved 0.9 µm perpendicular to the fiber's long axis between runs. Multiple successive runs of images were recorded in each condition. Line-scan images were computer-processed to identify and record spark locations using a detection algorithm as described previously (Cheng et al., 1999; Shtifman et al., 2000).

Images were corrected for PMT offset and converted to F images by subtraction of resting fluorescence (F) along the scan line averaged in time, excluding the contribution of potential Ca²⁺ spark regions of interest. F images were then normalized pixel by pixel by F and smoothed 3 x 3 to get the F/F images. Spatial and temporal profiles were extracted from each region of interest as described previously (Lacampagne et al., 1999). Events with F/F < 0.4 were excluded from data analysis post hoc.

The frequency of occurrence of Ca²⁺ sparks (number of events per sarcomere per second) was calculated from the number of sparks per image divided by the number of sarcomeres along the line and by the image duration (1.024 s). Spark frequency was determined in each fiber for control and either sham or experimental conditions. Because of the variability in the starting Ca²⁺ spark frequency among fibers, the frequency in a given fiber under experimental or sham conditions was normalized to the mean of the control Ca²⁺ spark frequencies for the same group of experimental or sham fibers at each azumolene concentration. Ca²⁺ spark frequency results are reported as means ± S.E.M. of these normalized frequencies from N fibers divided by the mean normalized frequency for the shams at the same azumolene concentration. Analysis of variance was used as statistical analysis for comparison of means, with a significance level of p < 0.05. The spatiotemporal properties of Ca²⁺ sparks, such as amplitude, rise time, full duration at half-max, full width at half-max (FWHM), and spark mass, were not normally distributed; therefore, a nonparametric analysis of variance was performed (Dunn's) to compare spark properties under different experimental conditions. All statistical analysis was performed with SigmaStat (SPSS Inc., Chicago, IL). Nonlinear curve fitting was performed in SigmaPlot (SPSS Inc.).

	Results

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Azumolene Decreases Spontaneous Ca²⁺ Spark Frequency. To investigate the effects of azumolene on localized Ca²⁺ release permeabilized, cut frog skeletal muscle fibers were first incubated in an internal solution (control), then either in an internal solution either with 0.2% DMSO (sham) or in an internal solution containing the appropriate concentration of azumolene and 0.2% DMSO (experimental). The [Mg²⁺]_free in the internal solution was 0.42 mM. At this low Mg²⁺ concentration, the spontaneous Ca²⁺ spark frequency would be relatively high (Lacampagne et al., 1998), thereby facilitating the acquisition of sufficient numbers of Ca²⁺ sparks for statistical analysis subsequent to an inhibition of spark frequency in the presence of azumolene.

Figure 1 shows representative line-scan fluorescence (F/F) images of permeabilized frog muscle fibers in control (A and C) and after addition of either 0.2% DMSO (B, sham) or 1 µM azumolene with 0.2% DMSO (D) to the bathing solution. Distance along the fiber (x) is represented vertically and time (t) is represented horizontally to give the x versus t image in each panel. Each localized increase in [Ca²⁺] (Ca²⁺ spark) is characterized by a brief and localized increase in fluorescence (Klein et al., 1996; Schneider and Klein, 1996). When added to the permeabilized muscle fibers, 1 µM azumolene seemed to modulate SR Ca²⁺ release by producing a large decrease in the frequency of Ca²⁺ sparks.

View larger version (70K): [in this window] [in a new window]   Fig. 1. Effects of azumolene on spontaneous Ca2+ spark occurrence in permeabilized frog skeletal muscle fibers. Representative line-scan images under control for DMSO (A), DMSO (0.2%) (B), control for azumolene (C), and azumolene (1 µM) + DMSO (0.2%) (D) conditions. Line-scan images are presented for permeabilized frog skeletal muscle fibers bathed in internal solution containing the Ca2+ indicator Fluo-3. Images show the F/F fluorescence resulting from line scan of a 138-µm length of fiber for 1.024 s. Azumolene (1 µM) decreased the Ca2+ spark events dramatically, whereas 0.2% DMSO sham solution had no apparent effect on spark frequency.

(1)

where f is the event frequency normalized to the average control event frequency in the same groups of fibers divided by the mean normalized frequency in the corresponding sham; b is the Hill coefficient, and K is the concentration of azumolene that elicits a 50% decrease in Ca²⁺ spark frequency (EC₅₀). Since spark frequencies at each azumolene concentration were expressed relative to the mean frequency in the corresponding sham fibers, f_max was constrained to 1. Fitting of the data to eq. 1 provided an f_min value of 0.00 ± 0.15, indicating full suppression of all spark activity at saturating azumolene concentrations. Inhibition (50%) occurred at an EC₅₀ of 0.25 ± 0.12 µM azumolene. A Hill coefficient of 1.44 ± 0.99 suggests a single binding site for azumolene on RyR.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Azumolene decreases the calcium spark frequency in a dose-dependent manner. Ca2+ spark frequency in each fiber in the presence of the indicated azumolene was normalized to the average control frequency for that group of fibers before azumolene or sham solution change. Ca2+ spark frequency results are reported as means ± S.E.M. of these normalized frequencies from N fibers divided by the mean normalized frequency for the shams at the same azumolene concentration. The line was obtained by fitting the data to the Hill equation (eq. 1; fmax = 1.00, fmin 0.00), resulting in an EC50 of 0.25 ± 0.12 µM, a Hill coefficient of = 1.44 ± 0.99, and fmin of 0.00 ± 0.15 sarcomere-1 s-1. R2 = 0.93.

Effects of Azumolene on Ca²⁺ Spark Spatiotemporal Properties. To determine whether the decrease in Ca²⁺ spark frequency in the presence of azumolene was associated with changes in the spatiotemporal properties of individual Ca²⁺ release events, we analyzed the spatiotemporal properties of the detected Ca²⁺ sparks. Figure 3 shows box plots of the azumolene concentration dependence of the distribution of values for the spatiotemporal properties of Ca²⁺ release events (F/F 0.4) after addition of 0.2% DMSO (sham; [azumolene] = 0 µM) or after the addition of azumolene (0.0001–1 µM). Although at 1 µM azumolene the frequency of Ca²⁺ spark occurrence was considerably inhibited (Fig. 2), the number of Ca²⁺ spark events was still practical for analysis of their spatiotemporal properties. At 10 µM, azumolene the number of events was too small for analysis of spark properties. Figure 3 shows that there were no systematic concentration-dependent effects of azumolene on the temporal properties (rise time and full duration at half-max), amplitude, or spatial spread (FWHM) of the Ca²⁺ sparks.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Effects of azumolene on the distribution of individual spark spatiotemporal properties. Box plots indicate the 10, 25, 50 (i.e., median), 75, and 90 percentile values of the distribution (*, p < 0.05 compared with its matched sham). The boxes at 0 concentration represent the properties of groups of shams, each matched to the azumolene concentration denoted by the same letter. The solid lines were obtained by fitting the median values of spark properties to the Hill equation (eq. 1; f now representing the indicated spark property). No systematic concentration-dependent effects of azumolene were found on these spatiotemporal properties. The calcium spark numbers are 778 for sham and 1101 for test at 0.0001 µM azumolene, 2349 for sham and 1258 for test at 0.001 µM azumolene, 2536 for sham and 1469 for test at 0.1 µM azumolene, 2488 for sham and 1466 for test at 0.01 µM azumolene, 4056 for sham and 2636 for test at 0.3 µM azumolene, 1622 for sham, and 453 for test at 1 µM azumolene.

To determine whether there was an azumolene-dependent change in the amount of Ca²⁺ released from SR by the individual sparks, spark mass at time of spark peak was calculated for each spark using the equation (Hollingworth et al., 2001):

(2)

Figure 4 shows that azumolene (0.0001–1 µM) caused no systematic concentration-dependent change in spark mass. Overall, the predominant effect of azumolene in permeabilized frog skeletal muscle was to greatly decrease the frequency of spontaneous Ca²⁺ sparks but to cause no systematic changes in spark properties.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effects of azumolene on individual spark mass. Spark mass was calculated by eq. 2. The boxes at 0 concentration represent the masses of shams matched to the azumolene concentration indicated by the same letter. The solid line was obtained by fitting the median values to the Hill equation (eq. 1; f = spark mass). There is no systematic concentration dependent effect of azumolene on spark mass. The numbers of sparks at each azumolene treatments are the same as in the previous figure. Box plots indicate the 10, 25, 50 (i.e., median), 75, and 90 percentile values of the distribution (*, p < 0.05 compared with its matched sham).

View larger version (15K): [in this window] [in a new window]   Fig. 5. A, MH mimic episode generated by DP4. DP4 (150 µM) in an internal solution having a free Mg2+ concentration of 1.2 mM resulted in an 11-fold increase in spark frequency after 10-min exposure to a DP4-containing solution. The increased Ca2+ spark frequency did not change further with time after changing the 150 µM DP4 internal solution to another sample of the internal solution containing the same concentration of DP4 and waiting an additional 10 min. B, azumolene decreases Ca2+ spark frequency in a dose-dependent manner in the 150 µM DP4 MH mimic system. The spark frequency of each fiber in the presence of the indicated DP4 and azumolene was normalized to the average frequency for that group of fibers in DP4 before azumolene or sham solution change. Ca2+ spark frequency results are reported as means ± S.E.M. of these normalized frequencies from N fibers divided by the mean normalized frequency for the shams at the same azumolene concentration. Fitting these data to eq. 1 (fmax = 1.00, fmin 0.00) resulted in an EC50 of 2.35 ± 4.02, Hill coefficient of 1.73 ± 2.72, and fmin of 0.00 ± 1.14. R2 = 0.91.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Effects of azumolene on individual spark spatiotemporal properties in the DP4-MH mimic model. Box plots indicate the 10, 25, 50 (i.e., median), 75, and 90 percentile values of the distribution. The boxes at 0 concentration represent the properties of shams matched to the different azumolene concentration treatments. The solid lines were obtained by fitting the median values to the Hill equation (eq. 1) as in Fig. 3. No systematic concentration dependent effects of azumolene were found on these spatiotemporal properties. The calcium spark numbers are 714 for sham ([DP4] = 150 µM), 746 for DP4 (150 µM) + azumolene (0.04 µM), 341 for sham ([DP4] = 150 µM), 332 for DP4 (150 µM) + azumolene (0.2 µM), 208 for sham ([DP4] = 150 µM), 136 for DP4 (150 µM) + azumolene (2.24 µM), 207 for sham ([DP4] = 150 µM), and 52 for DP4 (150 µM) + azumolene (5 µM) treatment.

	Discussion

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Previous studies have demonstrated that dantrolene decreases the sensitivity of isolated RyR1 and RyR3 to activation by Ca²⁺ in that it shifts the Ca²⁺ dependence of ryanodine binding to higher Ca²⁺ levels (Fruen et al., 1997; Zhao et al., 2001). The results here are also consistent with azumolene shifting Ca²⁺ sensitivity to calcium-induced calcium release (CICR). In our experiments, the global Ca²⁺ concentration is relatively low inside a muscle fiber equilibrated with our internal solution (100 nM), well under the Ca²⁺ concentration for full activation by CICR. Under these conditions, azumolene could decrease the rate of spontaneous openings of RyR channels by CICR, resulting in the observed decrease in the frequency of Ca²⁺ sparks. In contrast, the properties of the Ca²⁺ sparks that do occur may not be changed appreciably because the local Ca²⁺ concentration within the calcium release unit during a spark is high after opening of an RyR Ca²⁺ channel. Ca²⁺ activation of neighboring channels inside this cluster could then be maximal both with and without azumolene at this high local Ca²⁺ concentration, giving a similar local Ca²⁺ release event in the presence or absence of azumolene. This interpretation requires that the elevated local Ca²⁺ within a release unit can open azumolene-bound channels. Alternatively, azumolene would have to fully dissociate from the RyRs in the release unit within a time frame much shorter than the few millisecond rise time of a Ca²⁺ spark. However, either of these requirements would seem to be contrary to the ability of azumolene (and dantrolene) to suppress the Ca²⁺ regenerative aspects of an MH episode.

An alternative interpretation that does not rely on the ability of elevated Ca²⁺ levels to activate azumolene-bound channels during a spark could be based on having only a small number of channels (e.g., 2–4; Shtifman et al., 2000) underlying the generation of a Ca²⁺ spark. In this case, occupancy of a single channel in this small release unit could effectively eliminate that unit from generating a detectable spark. Thus, frequency would be decreased as azumolene concentration was increased, but any events that did occur would be generated by release units without any bound azumolene and would thus exhibit normal spark properties.

There are two RyR isoforms in frog skeletal muscle, RyR and RyR, which are homologues of mammalian RyR1 and RyR3, respectively (Murayama and Ogawa, 2002). These two isoforms of RyR are expressed at about the same level in frog skeletal muscle. Dantrolene was found to decrease Ca²⁺ release from intracellular stores in central neurons (Wei and Perry, 1996; Pelletier et al., 1999; Mattson et al., 2000), which express predominantly RyR3. Heterologously expressed RyR3 was also significantly inhibited by dantrolene and azumolene, and this inhibition was comparable with the dantrolene inhibition of RyR1 (Zhao et al., 2001). In our studies, azumolene (10 µM) completely inhibited Ca²⁺ sparks. Thus, our results are consistent with the notion that azumolene (or dantrolene) may have an inhibitory effect on both RyR1 and RyR3 isoforms.

Nelson et al. (1996) found that dantrolene and azumolene at nanomolar concentrations increased the open-state probability and open dwell time of single RyR1 channels from SR vesicles incorporated into lipid bilayer membranes. In contrast, at higher dantrolene concentration (5 µM), they observed a decrease in the open probability due to a decrease in the channel open time as well as a decrease in single channel conductance. In the present study, low concentrations of azumolene, from 0.1 to 10 nM, did not markedly increase spark frequency, whereas at higher concentrations a decrease in spark frequency, which should correspond to an increase in channel closed time, was observed. There was no systematic alteration in the spatiotemporal properties of Ca²⁺ sparks. These differing results may reflect the different experimental preparations or different RyR isoform composition in frog skeletal muscle. Nelson's experiments used crude RyR1 from mammalian SR. Although dantrolene in micromolar concentrations inhibits the activity of RyR3 (Zhao et al., 2001) and inhibits calcium efflux from the intracellular stores of neurons (Nelson et al., 1999), it has not been determined whether dantrolene or azumolene at nanomolar concentrations would activate RyR3. However, evidence for such activation was not detected in our experiments. In other studies, using purified RyR in lipid bilayers, no effects of dantrolene were observed on purified RyR1 channels, which was interpreted as showing that the dantrolene effect on muscle fibers may be on an accessory protein (Szentesi et al., 2001). Finally, using SR vesicles in patch-clamped bilayers, 50 µM dantrolene was found to decrease the bursts of Ca²⁺ channel activity (Suarez-Isla et al., 1986), consistent with our observation of decreased spark frequency.

DP4 is a synthetic peptide corresponding to a region of the central domain of RyR1 from Leu2442 to Pro2477. Most MH mutations are found in this domain or within an N-terminal domain. According to a "domain switch" model proposed by Ikemoto, these two domains are putative regulatory domains that intricately interact with each other and are involved in the regulation of channel gating (Ikemoto and Yamamoto, 2002). In the resting or nonactivated state, the N-terminal and central domains make contact with each other at several undetermined subdomains, forming the "zipped" configuration that promotes the closed state of RyR. Stimulation via excitation-contraction coupling or application of chemical agents weakens these interdomain contacts, thereby lowering the energy barrier for RyR opening (Ikemoto and Yamamoto, 2002; Kobayashi et al., 2004). For MH mutants in either of these two domains, the domain switch is weakened, making the RyR hypersensitive to RyR agonists. DP4 is thought to weaken this interdomain interaction, producing an MH-like activation/sensitization effect on the channel (El-Hayek et al., 1999; Shtifman et al., 2002; Yamamoto and Ikemoto, 2002; Kobayashi et al., 2004). Here, we applied 150 µM DP4 to permeabilized fibers to mimic an MH episode and have shown that azumolene inhibited the Ca²⁺ spark frequency in a dose-dependent manner in the presence of DP4, whereas the Ca²⁺ spark properties were not changed. Compared with the inhibitory effect in normal fibers, an approximately 10-fold higher concentration of azumolene was needed to get the same inhibitory effect in the presence of DP4.

The cartoon in Fig. 7 integrates our current results with the interdomain interaction model for single RyR channel gating. Azumolene (or dantrolene) might preferentially bind to the closed state of RyR (Fig. 7, top), promoting the domain-domain interaction and thereby stabilizing the closed configuration of RyR (Kobayashi et al., 2005). This stabilization would also be effective in the presence of DP4, which mimics an MH episode, if azumolene preferentially binds to the closed state of the channel (Fig. 7). The basis for the observed lack of effect of azumolene on the properties of the sparks that do occur would then depend on the number of interacting channels within the Ca²⁺ release unit and the nature of their interaction (see above).

View larger version (43K): [in this window] [in a new window]   Fig. 7. Cartoon model for azumolene modulation of the interactions between domains of RyR. Azumolene (AZ) binds to the N-terminal domain of closed RyR1 channels, promoting the stabilization of the closed zipped state of the channel, even in the presence of DP4.

	Acknowledgements

 
We thank Dr. Jerome Parness for the suggestion of this experiment and the azumolene.

	Footnotes

 
This work was supported by National Institutes of Health Grant 5R01NS023346 (to M.S.F.). Y.Z. was partially supported by National Institutes of Health Institutional Training Grant 5T32AR07592 to the Interdisciplinary Program in Muscle Biology.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.084046.

ABBREVIATIONS: MH, malignant hyperthermia; SR, sarcoplasmic reticulum; RyR, ryanodine receptor; CICR, calcium induced calcium release; FWHM, full width at half-max.

Address correspondence to: Dr. M. F. Schneider, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201. E-mail: mschneid{at}umaryland.edu

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