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J. Biol. Chem., Vol. 280, Issue 8, 6580-6587, February 25, 2005

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Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor^*

Shigeki Kobayashi, Mark L. Bannister, Jaya P. Gangopadhyay, Tomoyo Hamada, Jerome Parness, and Noriaki Ikemoto¶||

From the Boston Biomedical Research Institute, Watertown, Massachusetts 02472, the Departments of Anesthesia, Pharmacology, Pediatrics, and Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, and the ^¶Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, July 23, 2004 , and in revised form, December 13, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca²⁺ channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K_Q) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 °C and was unaffected by calmodulin. [³H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca²⁺ release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 °C, yet required the presence of calmodulin at 22 °C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca²⁺ channel opening rather than the domain switch itself.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Dantrolene (hydrated 1-(((5-(4-nitrophenyl)-2-furanyl)methylene)amino)-2,4-imidazolidinedione sodium salt) is an intracellularly acting skeletal muscle relaxant used for the treatment of malignant hyperthermia (MH).¹ MH is a potentially deadly, pharmacogenetically mediated, hypermetabolic response to volatile anesthetics that results from unregulated intramyoplasmic Ca²⁺ release (1). The drug is known to inhibit excitation-contraction coupling of skeletal muscle (2) by suppressing depolarization-induced sarcoplasmic reticulum (SR) Ca²⁺ release in normal and MH-susceptible skeletal muscle without affecting voltage sensor activation (3). In MH, the voltage dependence of contractile activation is shifted to lower voltages (4), whereas in the presence of clinical concentrations of dantrolene, i.e. 10 µM (5), the voltage dependence of contractile activation is shifted to higher voltages (6, 7). Normalization of the voltage dependence of contractile activation may therefore be one of the important components of the therapeutic action of dantrolene. Dantrolene also confers a normal Mg²⁺ sensitivity to MH-susceptible muscle fibers, which would otherwise show a considerably reduced sensitivity to the normal inhibitory action of myoplasmic Mg²⁺ on the SR Ca²⁺ release mechanism (8, 9). Conferring normal Mg²⁺ sensitivity to mutated ryanodine receptor (RyR1) may be another key component of dantrolene therapeusis in MH.

Extensive studies have been carried out to examine the effect of dantrolene on the function of isolated skeletal muscle SR. It has been shown that both dantrolene and its equipotent, water-soluble analog azumolene suppress SR Ca²⁺ release induced by Ca²⁺ and various pharmacological agents (3, 10–12). Although dantrolene has been shown to suppress the ryanodine binding activity of the SR (13, 14), this finding is not universal (15). Dantrolene (1–5 µM) has been reported to have a biphasic effect on the open probability of RyR1 channels in lipid bilayers, first activating and then blocking at nanomolar concentrations (16), but others have not been able to see any effect of this drug on single channels (3). Importantly, dantrolene has been shown to at least partially restore the normal properties of RyR1 Ca²⁺ channels in SR isolated from MH-susceptible pigs (1, 17, 18). Thus, these studies together suggest that dantrolene interacts with the RyR to suppress the channel dysfunction that occurs with MH mutations.

As widely recognized, MH mutations are not randomly distributed along the RyR1 sequence. The vast majority of them are localized to two restricted regions, the N-terminal (Cys³⁵–Arg⁶¹⁴) and the central (Asp²¹²⁹–Arg²⁴⁵⁸) domains, whereas a third, C-terminal region (Ile³⁹¹⁶–Gly⁴⁹⁴²) contains fewer MH mutations (19–24). The pathophysiological consequences of MH mutations are hyperactivation and/or hypersensitization of RyR1 Ca²⁺ channel activity to stimulating conditions (both pharmacological and voltage-dependent) (1, 18). In contrast, most mutations conferring susceptibility to central core disease, a rare myopathy showing a different phenotype (19, 25, 26), are located in the C-terminal putative channel pore region (Ile³⁹¹⁶–Ala⁴⁹⁴²) (19, 26). The Ca²⁺ release properties of expressed RyR1 channels containing randomly selected MH mutations from the N-terminal and central domains have been shown to display similar properties of hyperactivation and hypersensitization (27). To explain these results, we have proposed a model of channel regulation that involves interdomain interactions between the N-terminal and central domains of RyR1 serving as a "domain switch" for Ca²⁺ channel regulation (28–31). In the resting state, the N-terminal and central domains make close contact at several as yet undetermined subdomains. The conformational constraints imparted by the "zipped" configuration of these two domains stabilize and maintain the closed state of the Ca²⁺ channel. Stimulation of the RyR via excitation-contraction coupling weakens these critical interdomain contacts (unzipping of the domain switch), thereby lowering the energy barrier for Ca²⁺ channel opening. Partial unzipping or weakening of the domain switch may also occur secondary to MH mutations in either the N-terminal or central domain. As a result of this, MH-susceptible RyR1 channels are hypersensitive to agonist stimuli.

Recently, Parness and co-workers (32) localized a dantrolene-binding site within the primary structure of RyR1 using [³H]azidodantrolene, a pharmacologically active photoaffinity analog of dantrolene. [³H]Azidodantrolene specifically photolabels the N-terminal 1400-amino acid fragment of RyR1 cleaved by an endogenous, SR membrane-bound calpain (33) and is localized within sequence Leu⁵⁹⁰–Cys⁶⁰⁹ based on the following evidence. 1) Of several synthetic RyR1 domain peptides examined, [³H]azidodantrolene specifically photolabels only peptides containing the Leu⁵⁹⁰–Cys⁶⁰⁹ sequence (named DP1 for domain peptide-1), and 2) an anti-RyR1 monoclonal antibody recognizing DP1 inhibits [³H]azidodantrolene photolabeling of RyR1 (34). The dantrolene-binding site is therefore located within the domain comprising the N-terminal portion of our proposed domain switch. Since dantrolene seems to inhibit Ca²⁺ release, these findings have led us to suggest that dantrolene may act by reinforcing interactions between the N-terminal and central portions of the RyR1 domain switch that favor a zipped conformation. Here, we present new evidence suggesting that this is indeed at least a portion of the mechanism of action of dantrolene.

In this study, we produced MH-like conditions of RyR1 (i.e. hyperactivation and hypersensitization of Ca²⁺ channels) in SR isolated from normal skeletal muscle by adding DP4, a synthetic domain peptide corresponding to amino acids 2442–2477 of RyR1, or anti-DP4 antibody, which, as we have recently shown, induces spectroscopic changes consistent with domain unzipping (29, 31). The magnitude of domain unzipping was determined from the accessibility of a fluorescence probe, methylcoumarin acetate (MCA), attached to the N-terminal domain, to a macromolecular fluorescence quencher (bovine serum albumin-conjugated QSY). The Stern-Volmer quenching constant (K_Q), a measure of domain unzipping, increased with the addition of either DP4 or anti-DP4 antibody. Here, we show that the addition of dantrolene reversed the increase in K_Q that was produced by DP4 or anti-DP4 antibody with an IC₅₀ of 0.3 µM. This blocking effect of domain unzipping by dantrolene was present at both 37 and 22 °C and was independent of calmodulin (CaM). Although, at 37 °C, dantrolene alone inhibited DP4 enhancement of [³H]ryanodine binding and activation of SR Ca²⁺ release, both measures of Ca²⁺ channel activation, it required calmodulin to do so at 22 °C. These results suggest that inhibition of RyR1 domain unzipping plays a role in the therapeutic action of dantrolene. The present data also suggest that the temperature and CaM dependence of dantrolene activity on RyR1 is due to their effects on the mechanism by which the domain switch is coupled to Ca²⁺ channel function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Preparation of SR Vesicles—The SR microsomes were prepared from rabbit back paraspinous and hind leg skeletal muscles (Pel-Freez Biologicals, Rogers, AR) using the method of differential centrifugation described previously (35).

Domain Peptide and Site-specific Antibody—We used a domain peptide (DP4) corresponding to Leu²⁴⁴²–Cys²⁴⁷⁷ of RyR1 both as a channel-activating reagent and as a site-directed carrier to incorporate the fluorescence probe MCA into RyR1. The peptide was synthesized on an Applied Biosystems Model 431A synthesizer, purified by reversed-phase high-pressure liquid chromatography, and evaluated by mass spectroscopy. To localize the DP4-binding site within the primary structure of RyR1, the site-specific anti-DP1 (Leu⁵⁹⁰–Cys⁶⁰⁹), anti-DP4 (Leu²⁴⁴²–Cys²⁴⁷⁷), and anti-DP3 (Asp³²⁴–Val³⁵¹) polyclonal antibodies were used (31).

Site-specific MCA Labeling at the DP4-binding Site of RyR1—Site-specific fluorescence labeling of the DP4-binding sites on RyR1 in skeletal muscle SR was performed using the cleavable heterobifunctional cross-linking reagent sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1, 3'-dithiopropionate (SAED) (36).

Localization of the Site of MCA Attachment within the RyR—MCA-labeled RyR1 was purified from fluorescently labeled SR using heparin and hydroxylapatite affinity columns as described by Inui and Fleischer (37). MCA-labeled RyR1 was digested with trypsin (1:100, 1:10 trypsin/protein ratio) at 22 °C for 45 min, and the resultant tryptic fragments were analyzed for fluorescence after SDS-PAGE on 8% polyacrylamide gels. For Western blot analysis, tryptic fragments were transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) at 90 V in 10% methanol and 10 mM CAPS (pH 11.0) at 4 °C. The blots were developed with anti-DP1, anti-DP3, and anti-DP4 primary antibodies and peroxidase-conjugated secondary antibodies.

[³H]Ryanodine Binding Assay—Isolated skeletal muscle SR (0.5 mg/ml) that had been labeled with MCA was incubated with 10 nM [³H]ryanodine (68.4 Ci/ml; PerkinElmer Life Sciences) and the desired concentration of DP4 at 22 °C for 12 h or at 37 °C for 1.5 h. Solutions contained 0.15 M KCl, 1 mM AMP-PCP, and either 10 µM dantrolene or an equivalent volume of methanol vehicle (final concentration of 0.7%) and 20 mM MOPS (pH 7.2). The [Ca²⁺] was kept at 0.1 µM using BAPTA/calcium buffer (0.449 mM CaCl₂, 1 mM BAPTA, and 20 mM MOPS (pH 7.2)). The effect of CaM (1 µM) on radioligand binding was determined under identical assay conditions. Assays were carried out in duplicate according to established protocols (38), and each datum point was obtained by averaging the duplicates.

Ca²⁺ Release Assay—Isolated skeletal muscle SR (0.2 mg/ml) was incubated at 22 or 37 °C in a solution containing 0.15 M KCl, 0.1 µM Ca²⁺ (BAPTA/calcium buffer), and either 10 µM dantrolene or an equivalent volume of methanol vehicle (final concentration of 0.7%), 2.0 µM fluo-3, and 20 mM MOPS (pH 7.2) in the presence or absence of CaM. Ca²⁺ uptake was initiated by the addition of 1 mM MgATP to the cuvette, and the time course of Ca²⁺ uptake was monitored in a Cary Eclipse spectrophotometer (Varian Inc.) using fluo-3 (excitation at 488 nm and emission at 525 nm) as a Ca²⁺ indicator (31). After Ca²⁺ uptake had reached a plateau, various concentrations of DP4 were added, and the resultant Ca²⁺ release was monitored. During the Ca²⁺ uptake/release reaction, the sample was continuously stirred, and the temperature was kept constant at either 22 or 37 °C.

Spectroscopic Monitoring of Domain Unzipping—To make a macromolecular collisional quencher, QSY^TM 7 carboxylic acid (2.5 mM) was conjugated with 0.5 mM bovine serum albumin by incubation in 20 mM HEPES (pH 7.5) at 22 °C for 60 min in the dark. Unreacted QSY^TM 7 carboxylic acid was removed by Sephadex G-50 gel filtration. Fluorescence quenching by the conjugate was performed by measuring the steady-state fluorescence of SR labeled with MCA (excitation at 348 nm and emission at 445 nm; Cary Eclipse spectrophotometer). Fluorescently labeled SR (0.2 mg/ml) was incubated at either 22 or 37 °C in a solution containing 0.15 M KCl, 1 mM AMP-PCP, 0.1 µM Ca²⁺ (BAPTA/calcium buffer), and either various concentrations of dantrolene or an equivalent volume of methanol vehicle (final concentration of 0.7%) and 20 mM MOPS (pH 7.2). Various concentrations of DP4 or 20 µg/ml anti-DP4 antibody was used to induce domain unzipping, and DP4-mut, in which one mutation was made to mimic R2458C MH mutation, was used as a negative control. The effect of dantrolene on domain unzipping was investigated by determining the effect of this drug on RyR1 MCA fluorescence in the presence or absence of 1 µM CaM. The data were analyzed using the Stern-Volmer equation: F₀/F = 1 + K[Q], where F and F₀ are fluorescence intensities in the presence and absence of added quencher, respectively; K is the quenching constant, a measure of the accessibility of the protein-bound probe to the quencher; and [Q] is the concentration of the quencher (QSY) (29, 31).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Dantrolene Inhibits an Abnormal Unzipping of the Domain Switch—To investigate the effect of dantrolene on the mode of interdomain interaction, we utilized the MCA fluorescence quenching technique we developed to determine the extent of unzipping of the RyR1 domain switch (see "Experimental Procedures") (29, 31). As shown previously (29), DP4 mediates the specific MCA labeling of the N-terminal 1400-amino acid segment of RyR1. The precise location of DP4-mediated MCA labeling of RyR1 within the N-terminal 1400-amino acid region has not been determined, and we do not yet know whether it occurs in the N-terminal MH domain (Cys³⁵–Arg⁶¹⁴). To better define where MCA is incorporated, we used DP4 conjugated with the heterobifunctional cross-linker SAED, as a site-directing carrier, as described previously (29). The SAED-DP4 conjugate was photo-cross-linked to its binding site, and the DP4 moiety was removed under reducing conditions. MCA-labeled RyR1 was purified and subjected to tryptic digestion. The process of degradation of the fluorescently labeled polypeptide chain was then followed by SDS-PAGE. As shown in Fig. 1, partial digestion of fluorescently labeled RyR1 with a low concentration of trypsin (100:1 (w/w) RyR protein/trypsin ratio) resulted in the appearance of two major fluorescently labeled fragments with approximate molecular masses of 155 and 140 kDa. Digestion with a higher concentration of trypsin (10:1 RyR protein/trypsin ratio) resulted in the appearance of several fluorescently labeled fragments. As shown in the Western blot of Fig. 1, the 155- and 140-kDa MCA-labeled bands stained with both anti-DP1 (Leu⁵⁹⁰–Cys⁶⁰⁹) and anti-DP3 (Asp³²⁴–Val³⁵¹) antibodies, whereas the 51-kDa MCA-labeled band stained with anti-DP3 antibody, but not with anti-DP1 antibody. Given the established trypsin cleavage sites of RyR1 (Fig. 1, scheme, arrows) (39, 40), these results suggest that the MCA-labeled RyR1 polypeptide chain degrades in the following order: 550 kDa > N-terminal 155 kDa > N-terminal 140 kDa > N-terminal 51 kDa, as illustrated in the scheme shown in Fig. 1. Since the 51-kDa MCA-labeled region is included in the N-terminal MH domain encompassed by Cys³⁵–Arg⁶¹⁴, these results indicate that DP4-mediated MCA labeling has taken place within the presumed N-terminal domain portion of the domain switch. These results also suggest that DP4, hence Leu²⁴⁴²–Pro²⁴⁷⁷ of the central domain of RyR1, binds to the N-terminal domain, as predicted from our domain switch hypothesis.

View larger version (43K): [in this window] [in a new window]   FIG. 1. Site-directed MCA labeling of RyR1 by DP4 results in the labeling of a 51-kDa N-terminal tryptic fragment. Purified MCA-labeled RyR1 from SR was digested with varying protein/trypsin ratios. Potential trypsin cleavage sites are denoted by the short arrows in the scheme. The epitopes for anti-DP1 and anti-DP3 antibodies are labeled with a Y. Note that the 51-kDa MCA-labeled fragment stained with anti-DP3 antibody, but not with anti-DP1 antibody.

We first determined the fluorescence intensity of bound MCA as a function of increasing concentrations of the quencher in the absence or presence of DP4 at 22 °C. Fig. 2A shows the Stern-Volmer plot of fluorescence quenching of MCA attached to the N-terminal domain in the presence of various concentrations of DP4. The slope of the plot, which is equal to the Stern-Volmer quenching constant (K_Q), is a measure of the degree of domain unzipping. As shown in Fig. 2B, the K_Q'/K_Q value (where K_Q' is the quenching constant in the presence of DP4 or DP4-mut, and K_Q is the quenching constant in their absence) was used to assess the extent of domain unzipping. K_Q'/K_Q increased with increasing concentrations of DP4 and leveled off at 100 µM peptide. Significantly, the DP4 concentration dependence of the increase in K_Q'/K_Q correlates well with the DP4 concentration dependence of activation of RyR Ca²⁺ channels (cf. Fig. 5). As shown in Fig. 2B, the single MH mutation in DP4-mut has made the peptide incapable of enhancing fluorescence quenching, presumably due to mutation-induced loss of affinity for the N-terminal domain. DP4-induced domain unzipping provides a simple and versatile model for the study of pathogenic and therapeutic mechanisms of MH.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Fluorescence quenching analysis of domain unzipping by a fluorescence MCA probe attached to the RyR1 N-terminal domain. A, a family of Stern-Volmer plots of fluorescence quenching as a function of increasing concentrations of a macromolecular fluorescence quencher, bovine serum albumin (BSA)-conjugated QSY, in the presence of increasing concentrations of DP4. The slope of each plot, which is equivalent to the Stern-Volmer quenching constant (KQ), was used to assess the degree of fluorescence quenching. B, KQ'/KQ versus synthetic domain peptide concentrations. KQ' and KQ are the Stern-Volmer constants in the presence and quenching absence of various concentrations of DP4 (•) or DP4-mut (), respectively. DP4 increased the extent of domain unzipping in a concentration-dependent manner, leveling off at 100 µM. DP4-mut had no effect. Each datum point represents the mean ± S.D. obtained from at least four different experiments.

View larger version (15K): [in this window] [in a new window]   FIG. 5. DP4-induced activation of [3H]ryanodine binding versus DP4 or DP4-mut concentration at 22 °C. The data were fitted to the equation y = aK2x2/(1 + K2x2), where y is the amount of ryanodine bound at x concentration of DP4, a is the maximal binding, and K is the association constant. Each datum point represents the mean ± S.D. of at least four experiments carried out in duplicate.

View larger version (11K): [in this window] [in a new window]   FIG. 3. Dantrolene inhibits macromolecular fluorescence quenching induced by DP4. A, Stern-Volmer plots of fluorescence quenching in the absence and presence of dantrolene. Dantrolene (1 µM) reduced the DP4-maximized KQ' toward control values. BSA, bovine serum albumin. B, the concentration dependence of the dantrolene-induced decrease in the degree of domain unzipping (KQ'/KQ) (•). The effect of dantrolene was seen between 0.1 and 1.0 µM. Half-maximal inhibition occurred at 0.3 µM, and the maximal effect occurred at 1 µM. Dantrolene produced no effect on KQ'/KQ in the absence of DP4 (). C, effect of increasing concentrations of DP4 on KQ'/KQ in the presence of 10 µM dantrolene. The drug suppressed fluorescence quenching at all concentrations of DP4. Each datum point represents the mean ± S.D. obtained from at least four different experiments.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Dantrolene inhibits macromolecular fluorescence quenching induced by anti-DP4 antibody. Shown is the concentration dependence of the dantrolene-induced decrease in the degree of domain unzipping (KQ'/KQ) (). Dantrolene produced no effect on KQ'/KQ in the absence of antibody (). Each datum point represents the mean ± S.D. of at least four experiments.

We next investigated the effect of temperature and CaM on the ability of dantrolene to inhibit DP4-induced SR Ca²⁺ release, as measured by the time- and Ca²⁺-dependent changes in fluo-3 fluorescence spectrophotometry (Fig. 6). These Ca²⁺ release experiments were carried out with an MCA-unlabeled SR preparation because MCA labeling did not produce appreciable changes in the RyR properties in preliminary experiments (data not shown). Approximately 20 s after Ca²⁺ uptake was initiated by the addition of MgATP, 100 µM DP4 was added to induce Ca²⁺ release, and the effect of 10 µM dantrolene on the Ca²⁺ release time course in the absence and presence of 1 µM CaM was analyzed. The Ca²⁺ release time courses (shown in Fig. 6) were fitted to a double exponential equation: y = y₀ + a(1 – e^–k₁t) + b(1 – e^–k₂t), where a and b are the magnitude of the fast and slow phases of release, respectively; and k₁ and k₂ are the rate constants of the fast and slow phases, respectively. The calculated values of the initial rate of Ca²⁺ release ((dy/dt)_t>0 = ak₁ + bk₂) and the amount of Ca²⁺ release (a + b) are listed in Table III. At 22 °C and in the absence of CaM, dantrolene produced no appreciable effect on the initial rate of Ca²⁺ release, although there was a small but appreciable inhibition of the slow phase of Ca²⁺ release (Fig. 6A and Table III). The addition of 1 µM CaM resulted in a significant increase in the initial rate of Ca²⁺ release. Under these conditions, 10 µM dantrolene produced a significant reduction in the initial rate ( 41%) and an appreciable reduction in the total amount of Ca²⁺ released, as well. Interestingly, raising the experimental temperature to 37 °C produced an effect similar in magnitude to that produced by CaM at 22 °C. Thus, at 37 °C, dantrolene produced a significant reduction in both the initial rate and the amount of Ca²⁺ release even in the absence of CaM. These results are again consistent with the view that CaM and temperature seem to affect the dantrolene-blocking signal from the domain switch to the Ca²⁺ channel pore domain.

View larger version (14K): [in this window] [in a new window]   FIG. 6. Dantrolene inhibits DP4-induced Ca2+ release with a temperature and CaM dependence. A, time course of DP4-induced Ca2+ release in the absence of dantrolene (black trace) and in the presence of 10 µM dantrolene (gray trace) at 22 °C. After the Ca2+ uptake initiated by the addition of 1 mM MgATP had reached a plateau, various concentrations of DP4 were added, and the resultant Ca2+ release was monitored by fluo-3 fluorescence spectrophotometry. B, the same experiment as described for A, except that 1 µM CaM was present during the course of the Ca2+ release experiment. C, the same experiment as described for A, except that it was carried out at 37 °C. Each Ca2+ release trace presented here was obtained by signal averaging a total of 35–75 traces originating from four to seven experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We proposed the domain switch model to explain both the mechanism of channel activation under normal conditions and the mechanism of pathogenesis of channel dysfunction. The model assumes that the mode of interaction between the N-terminal and central domains of the RyR (the two major regions harboring many of the reported MH mutations) is involved in Ca²⁺ channel regulation and also in the pathogenesis of MH. In the resting state, the two domains make close contact at several subdomains forming the zipped configuration (i.e. the "off" configuration of the on/off switch), and this configuration stabilizes the closed state of the Ca²⁺ channel. Stimulation of the RyR by T tubule depolarization or chemical agonists causes unzipping of the domain switch (the "on" configuration of the switch), leading to Ca²⁺ channel opening. Mutations in conformationally active portions of either region of the domain switch weaken the interdomain interactions, causing partial unzipping of the switch. As a result, MH-susceptible RyR1 channels are more readily opened as manifested in hyperactivation/hypersensitization effects. In our recent studies, the domain switch hypothesis was tested by examining the effects of synthetic peptides corresponding to the putative critical domains of the RyR (domain peptides) and antibodies raised against these domain peptides. DP4 enhances ryanodine binding (28), induces Ca²⁺ release from the SR (29), induces contraction in skinned muscle fibers at an inhibitory Mg²⁺ concentration (41), increases the sensitivity to caffeine (41), increases the frequency of Ca²⁺ sparks in saponin-permeabilized fibers (42), and increases the open probability of single channels (42). Similarly, anti-DP4 polyclonal antibody, directed against the Leu²⁴⁴²–Pro²⁴⁷⁷ region (a portion of the central domain) of RyR1, produces hyperactivation of Ca²⁺ channels and hypersensitization of Ca²⁺ channels to agonists (31).

The most important aspect of this study is the finding that dantrolene inhibits the DP4-induced increase in K_Q, i.e. dantrolene stabilizes a zipped configuration of the domain switch even in the presence of domain-unzipping agents such as DP4 and anti-DP4 antibody. Since domain unzipping produced by DP4 or anti-DP4 antibody seems to simulate the channel dysfunction caused by MH mutations, the present findings suggest that the molecular mechanism of therapeutic action of dantrolene in MH is to stabilize a zipped configuration of the domain switch, despite the tendency of the mutation to destabilize the domain switch. As a result, the channel hyperactivity and hypersensitivity caused by MH mutations are suppressed by dantrolene. Since the dantrolene-binding site is located in the Leu⁵⁹⁰–Cys⁶⁰⁹ region of the proposed RyR1 domain switch (37), interdomain stabilization by dantrolene must be produced by direct binding of the drug to a region of the domain switch.

In agreement with previous reports (7, 12, 13, 45, 46), we found that the extent of dantrolene inhibition of DP4-induced RyR1 channel activation, as determined by both [³H]ryanodine binding and SR Ca²⁺ release, varies with the temperature, i.e. significant inhibition was observed at 37 °C, with only negligible inhibition at 22 °C. An interesting new finding in this study is that dantrolene inhibition of DP4-induced domain unzipping is independent of temperature. Thus, it seems that the requirement for physiological temperatures for dantrolene activity is not due to a temperature dependence for the stabilization of a zipped configuration of the domain switch. Rather, the temperature requirement is for the downstream mechanism(s) of actual channel opening. According to our working hypothesis, the on/off signal elicited in the domain switch is transmitted to the putative channel gating/pore domain by mediation of some intraprotein coupling mechanism that requires higher temperatures for activation or inhibition.

According to an earlier report (13), the addition of CaM is required to observe dantrolene inhibition of RyR1 channel function. We addressed the question of the possible role of CaM in modulating dantrolene activity in this study. We detected no further effect of CaM on dantrolene inhibition of ryanodine binding or Ca²⁺ release at 37 °C, where there was already significant inhibition by dantrolene. However, at 22 °C, where there was very little dantrolene intrinsic inhibition, the extent of dantrolene inhibition of both ryanodine binding and Ca²⁺ release was considerably increased in the presence of CaM. At 22 °C, however, CaM had no effect on the extent of dantrolene inhibition of domain unzipping. These results suggest that, like raising the temperature from 22 to 37 °C, CaM may lower the conformational energy barrier for the proposed coupling between the domain switch and the channel gating/pore domain and enhance blocking signals elicited in the domain switch to more effectively transmit them to the functional Ca²⁺ channel. These results further suggest that the CaM-binding site on RyR1 is an intervening domain that mediates inhibitory signals from the domain switch to the channel gating/pore domain. Clearly, more work is required to elucidate the detailed molecular mechanism of pharmacological and therapeutic actions of dantrolene.

In summary, we speculate that dantrolene binding to the Leu⁵⁹⁰–Cys⁶⁰⁹ region of the N-terminal portion of the domain switch produces a local conformational change that results in reinforcement of the interdomain interactions between itself and its mating subdomain, likely located within the central domain portion of the domain switch. This stabilizes a zipped (i.e. closed) configuration. Thus, further characterization of the domain-domain interactions involving the dantrolene-binding region will be significant for further resolution of the molecular mechanism of dantrolene action on RyR1 in health and disease, as well as the mechanism of intraprotein control of channel opening.

	FOOTNOTES

 
^* This work was supported by NIAMS Grants AR16922 (to N. I.) and AR045593 (to J. P.) and NHLBI Grant HL072841 (to N. I.) from the National Institutes of Health and by the Banyu Fellowship Award in Cardiovascular Medicine (to S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Boston Biomedical Research Inst., 64 Grove St., Watertown, MA 02472. Tel.: 617-658-7774; Fax: 617-972-1761; E-mail: ikemoto{at}bbri.org.

¹ The abbreviations used are: MH, malignant hyperthermia; SR, sarcoplasmic reticulum; RyR, ryanodine receptor; MCA, methylcoumarin acetate; CaM, calmodulin; SAED, sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1, 3'-dithiopropionate; CAPS, 3-(cyclohexylamino)propanesulfonic acid; AMP-PCP, adenosine 5'-(, -methylene)triphosphate; MOPS, 3-(N-morpholino)propanesulfonic acid; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid.

	ACKNOWLEDGMENTS

 
We thank Dr. Renne C. Lu, Dr. Paul Leavis, David Schrier, and Elizabeth Gowell for help in the synthesis and purification of the peptides.

	REFERENCES

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