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Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

2004; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês

10.1074/jbc.m408375200

ISSN

1083-351X

Autores

Shigeki Kobayashi, Mark L. Bannister, Jaya Gangopadhyay, Tomoyo Hamada, Jerome Parness, Noriaki Ikemoto,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

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 (KQ) 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. 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 (KQ) 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. 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). 1The 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′-(β, γ-methylene)triphosphate; MOPS, 3-(N-morpholino)propanesulfonic acid; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid. 1The 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′-(β, γ-methylene)triphosphate; MOPS, 3-(N-morpholino)propanesulfonic acid; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid. MH is a potentially deadly, pharmacogenetically mediated, hypermetabolic response to volatile anesthetics that results from unregulated intramyoplasmic Ca2+ release (1Mickelson J.R. Louis C.F. Physiol. Rev. 1996; 76: 537-592Crossref PubMed Scopus (258) Google Scholar). The drug is known to inhibit excitation-contraction coupling of skeletal muscle (2Ellis K.O. Castellion A.W. Honkomp L.J. Wessels F.L. Carpenter J.E. Halliday R.P. J. Pharmacol. Sci. 1973; 62: 948-995Abstract Full Text PDF PubMed Scopus (56) Google Scholar) by suppressing depolarization-induced sarcoplasmic reticulum (SR) Ca2+ release in normal and MH-susceptible skeletal muscle without affecting voltage sensor activation (3Szentesi P. Collet C. Sarkozi S. Szegedi C. Jona I. Jacquemond V. Kovacs L. Csernoch L. J. Gen. Physiol. 2001; 118: 355-375Crossref PubMed Scopus (76) Google Scholar). In MH, the voltage dependence of contractile activation is shifted to lower voltages (4Gallant E.M. Lentz L.R. Neurosci. Lett. 1992; 28: 181-186Crossref Scopus (31) Google Scholar), whereas in the presence of clinical concentrations of dantrolene, i.e. 10 μm (5Flewellen E.H. Nelson T.E. Jones W.P. Arens J.F. Wagner D.I. Anesthesiology. 1983; 59: 275-280Crossref PubMed Scopus (129) Google Scholar), the voltage dependence of contractile activation is shifted to higher voltages (6Hainaut K. Desmedt J.E. Nature. 1974; 252: 728-730Crossref PubMed Scopus (105) Google Scholar, 7Morgan K.G. Bryant S.H. J. Pharmacol. Exp. Ther. 1977; 201: 138-147PubMed Google Scholar). 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 (8Owen B.J. Taske N.L. Lamb G.D. Am. J. Physiol. 1997; 272: C203-C211Crossref PubMed Google Scholar, 9Lamb G.D. J. Muscle Res. Cell Motil. 1993; 14: 554-556Crossref PubMed Scopus (34) Google Scholar). 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 (3Szentesi P. Collet C. Sarkozi S. Szegedi C. Jona I. Jacquemond V. Kovacs L. Csernoch L. J. Gen. Physiol. 2001; 118: 355-375Crossref PubMed Scopus (76) Google Scholar, 10Van Winkle W.B. Science. 1976; 193: 1130-1131Crossref PubMed Scopus (203) Google Scholar, 11Herbette L. Messineo F.C. Katz A.M. Annu. Rev. Pharmacol. 1982; 22: 413-434Crossref Scopus (35) Google Scholar, 12Otha T. Ito S. Ohga A. Eur. J. Pharmacol. 1990; 178: 11-19Crossref PubMed Scopus (84) Google Scholar). Although dantrolene has been shown to suppress the ryanodine binding activity of the SR (13Fruen B.R. Mickelson J.R. Louis C.F. J. Biol. Chem. 1997; 272: 26965-26971Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 14Zhao F. Li P. Chen S.R. Louis C.F. Fruen B.R. J. Biol. Chem. 2001; 276: 13810-13816Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), this finding is not universal (15Palnitkar S.S. Mickelson J.R. Louis C.F. Parness J. Biochem. J. 1997; 326: 847-852Crossref PubMed Scopus (40) Google Scholar). 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 (16Nelson T.E. Lin M. Zaoata-Sudo G. Sudo R.T. Anesthesiology. 1996; 84: 1368-1379Crossref PubMed Scopus (75) Google Scholar), but others have not been able to see any effect of this drug on single channels (3Szentesi P. Collet C. Sarkozi S. Szegedi C. Jona I. Jacquemond V. Kovacs L. Csernoch L. J. Gen. Physiol. 2001; 118: 355-375Crossref PubMed Scopus (76) Google Scholar). Importantly, dantrolene has been shown to at least partially restore the normal properties of RyR1 Ca2+ channels in SR isolated from MH-susceptible pigs (1Mickelson J.R. Louis C.F. Physiol. Rev. 1996; 76: 537-592Crossref PubMed Scopus (258) Google Scholar, 17Britt B.A. Scott E. Frodis W. Clements M.J. Endrenyi L. Can. Anaesth. Soc. J. 1984; 31: 130-154Crossref PubMed Scopus (23) Google Scholar, 18Ohnishi S.T. Taylor S. Gronert G.A. FEBS Lett. 1983; 161: 103-107Crossref PubMed Scopus (118) Google Scholar). 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 (19Dirksen R.T. Avila G. Trends Cardiovasc. Med. 2002; 12: 189-197Crossref PubMed Scopus (102) Google Scholar, 20Galli L. Orrico A. Cozzolino S. Pietrini V. Tegazzin V. Sorrentino V. Cell Calcium. 2002; 32: 143-151Crossref PubMed Scopus (50) Google Scholar, 21Davis M.R. Haan E. Jungbluth H. Sewry C. North K. Muntoni F. Kuntzer T. Lamont P. Bankier A. Tomlinson P. Sanchez A. Walsh P. Nagarajan L. Oley C. Colley A. Gedeon A. Quinlivan R. Dixon J. James D. Muller C.R. Laing N.G. Neuromuscul. Disord. 2003; 13: 151-157Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 22Monnier N. Ferreiro A. Marty I. Labarre-Vila A. Mezin P. Lunardi J. Hum. Mol. Genet. 2003; 12: 1171-1178Crossref PubMed Scopus (112) Google Scholar, 23Zorzato F. Yamaguchi N. Xu L. Meissner G. Muller C.R. Pouliquin P. Muntoni F. Sewry C. Girard T. Treves S. Hum. Mol. Genet. 2003; 12: 379-388Crossref PubMed Scopus (40) Google Scholar, 24Tammaro A. Bracco A. Cozzolino S. Esposito M. Di Martino A. Savoia G. Zeuli L. Piluso G. Aurino S. Nigro V. Clin. Chem. 2003; 49: 761-768Crossref PubMed Scopus (36) Google Scholar). The pathophysiological consequences of MH mutations are hyperactivation and/or hypersensitization of RyR1 Ca2+ channel activity to stimulating conditions (both pharmacological and voltage-dependent) (1Mickelson J.R. Louis C.F. Physiol. Rev. 1996; 76: 537-592Crossref PubMed Scopus (258) Google Scholar, 18Ohnishi S.T. Taylor S. Gronert G.A. FEBS Lett. 1983; 161: 103-107Crossref PubMed Scopus (118) Google Scholar). In contrast, most mutations conferring susceptibility to central core disease, a rare myopathy showing a different phenotype (19Dirksen R.T. Avila G. Trends Cardiovasc. Med. 2002; 12: 189-197Crossref PubMed Scopus (102) Google Scholar, 25Avila G. O'Connell K.M. Dirksen R.T. J. Gen. Physiol. 2003; 121: 277-286Crossref PubMed Scopus (62) Google Scholar, 26Du G.G. Khanna V.K. Guo X. MacLennan D.H. Biochem. J. 2004; 382: 557-564Crossref PubMed Scopus (30) Google Scholar), are located in the C-terminal putative channel pore region (Ile3916–Ala4942) (19Dirksen R.T. Avila G. Trends Cardiovasc. Med. 2002; 12: 189-197Crossref PubMed Scopus (102) Google Scholar, 26Du G.G. Khanna V.K. Guo X. MacLennan D.H. Biochem. J. 2004; 382: 557-564Crossref PubMed Scopus (30) Google Scholar). 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 (27Yang T. Ta T.A. Pessah I.N. Allen P.D. J. Biol. Chem. 2003; 278: 25722-25730Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). 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 (28Yamamoto T. El-Hayek R. Ikemoto N. J. Biol. Chem. 2000; 275: 11618-11625Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 29Yamamoto T. Ikemoto N. Biochemistry. 2002; 41: 1492-1501Crossref PubMed Scopus (48) Google Scholar, 30Ikemoto N. Yamamoto T. Front. Biosci. 2002; 7: 671-683Crossref PubMed Google Scholar, 31Kobayashi S. Yamamoto T. Parness J. Ikemoto N. Biochem. J. 2004; 380: 561-569Crossref PubMed Scopus (32) Google Scholar). 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 (32Palnitkar S.S. Bin B. Jimenez L.S. Morimoto H. Williams P.G. Paul-Pletzer K. Parness J. J. Med. Chem. 1999; 42: 1872-1880Crossref PubMed Scopus (38) Google Scholar) 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 (33Paul-Pletzer K. Palnitkar S.S. Jiminez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (39) Google Scholar) 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 (34Paul-Pletzer K. Yamamoto T. Bhat M.B. Ma J. Ikemoto N. Jimenez L.S. Morimoto H. Williams P.G. Parness J. J. Biol. Chem. 2002; 277: 34918-34923Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). 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 (29Yamamoto T. Ikemoto N. Biochemistry. 2002; 41: 1492-1501Crossref PubMed Scopus (48) Google Scholar, 31Kobayashi S. Yamamoto T. Parness J. Ikemoto N. Biochem. J. 2004; 380: 561-569Crossref PubMed Scopus (32) Google Scholar). 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 (KQ), 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 KQ 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 PROCEDURESPreparation 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 (35Ikemoto N. Kim D.H. Antoniu B. Methods Enzymol. 1988; 157: 469-480Crossref PubMed Scopus (39) Google Scholar).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 (31Kobayashi S. Yamamoto T. Parness J. Ikemoto N. Biochem. J. 2004; 380: 561-569Crossref PubMed Scopus (32) Google Scholar).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) (36Kang J.J. Tarcsafalvi A. Carlos A.D. Fujimoto E. Shahrokh Z. Thevenin B.J. Shohet S.B. Ikemoto N. Biochemistry. 1992; 31: 3288-3293Crossref PubMed Scopus (32) Google Scholar).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 (37Inui M. Fleischer S. Methods Enzymol. 1988; 157: 490-505Crossref PubMed Scopus (20) Google Scholar). 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 (38El-Hayek R. Lokuta A.J. Arevalo C. Valdivia H.H. J. Biol. Chem. 1995; 270: 28696-28704Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), 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 (31Kobayashi S. Yamamoto T. Parness J. Ikemoto N. Biochem. J. 2004; 380: 561-569Crossref PubMed Scopus (32) Google Scholar). 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™ 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™ 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) (29Yamamoto T. Ikemoto N. Biochemistry. 2002; 41: 1492-1501Crossref PubMed Scopus (48) Google Scholar, 31Kobayashi S. Yamamoto T. Parness J. Ikemoto N. Biochem. J. 2004; 380: 561-569Crossref PubMed Scopus (32) Google Scholar).RESULTSDantrolene 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") (29Yamamoto T. Ikemoto N. Biochemistry. 2002; 41: 1492-1501Crossref PubMed Scopus (48) Google Scholar, 31Kobayashi S. Yamamoto T. Parness J. Ikemoto N. Biochem. J. 2004; 380: 561-569Crossref PubMed Scopus (32) Google Scholar). As shown previously (29Yamamoto T. Ikemoto N. Biochemistry. 2002; 41: 1492-1501Crossref PubMed Scopus (48) Google Scholar), 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 (29Yamamoto T. Ikemoto N. Biochemistry. 2002; 41: 1492-1501Crossref PubMed Scopus (48) Google Scholar). 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) (39Chen S.R. Airey J.A. MacLennan D.H. J. Biol. Chem. 1993; 268: 22642-22649Abstract Full Text PDF PubMed Google Scholar, 40Wu Y. Aghdasi B. Dou S.J. Zhang J.Z. Liu S.Q. Hamilton S.L. J. Biol. Chem. 1997; 272: 25051-25061Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), 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.The domain switch hypothesis predicts that, if the MCA probe attached to the N-terminal domain is buried in the zipped configuration, it will be relatively inaccessible to a macromolecular fluorescence quencher, bovine serum albumin-conjugated QSY. Unzipping should, however, render the MCA probe more accessible to the quencher. DP4 has been previously shown to enhance [3H]ryanodine binding (28Yamamoto T. El-Hayek R. Ikemoto N. J. Biol. Chem. 2000; 275: 11618-11625Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), to induce Ca2+ release from the SR (29Yamamoto T. Ikemoto N. Biochemistry. 2002; 41: 1492-1501Crossref PubMed Scopus (48) Google Scholar), to induce contraction in skinned muscle fiber at an inhibitory Mg2+ concentration (41Lamb G.D. Posterino G.S. Yamamoto T. Ikemoto N. Am. J. Physiol. 2001; 281: C207-C214Crossref PubMed Google Scholar), to increase the frequency of Ca2+ sparks in saponin-permeabilized fibers (42Shtifman A. Ward C.W. Yamamoto T. Wang J. Olbinski B. Valdivia H.H. Ikemoto N. Schneider M.F. J. Gen. Physiol. 2002; 116: 15-31Crossref Scopus (47) Google Scholar), and to increase the open probability of single channels (42Shtifman A. Ward C.W. Yamamoto T. Wang J. Olbinski B. Valdivia H.H. Ikemoto N. Schneider M.F. J. Gen. Physiol. 2002; 116: 15-31Crossref Scopus (47) Google Scholar). Thus, DP4 mimics MH-like hyperactivation and hypersensitization of the RyR1 Ca2+ channel in the otherwise wild-type system. Furthermore, a mutation in DP4 (DP4-mut) that mimics the R2458C MH mutation results in virtually complete loss of the activities of wild-type DP4 (28Yamamoto T. El-Hayek R. Ikemoto N. J. Biol. Chem. 2000; 275: 11618-11625Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 29Yamamoto T. Ikemoto N. Biochemistry. 2002; 41: 1492-1501Crossref PubMed Scopus (48) Google Scholar, 41Lamb G.D. Posterino G.S. Yamamoto T. Ikemoto N. Am. J. Physiol. 2001; 281: C207-C214Crossref PubMed Google Scholar, 42Shtifman A. Ward C.W. Yamamoto T. Wang J. Olbinski B. Valdivia H.H. Ikemoto N. Schneider M.F. J. Gen. Physiol. 2002; 116: 15-31Crossref Scopus (47) Google Scholar). This is consistent with the idea that MH mutations weaken domain-domain interactions within RyR1 that are mimicked by synthetic domain peptide-domain (RyR) interaction. Thus, we used DP4 herein to mimic MH-like channel dysfunction and to investigate the effects of dantrolene on these functions.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 (KQ), is a measure of the degree of domain unzipping. As shown in Fig. 2B, the KQ′/KQ value (where KQ′ is the quenching constant in the presence of DP4 or DP4-mut, and KQ is the quenching constant in their absence) was used to assess the extent of domain unzipping. KQ′/KQ increased with increasing concentrations of DP4 and leveled off at ∼100 μm peptide. Significantly, the DP4 concentration dependence of the increase in KQ′/KQ correlates well with the DP4 concentration dependence of activation of RyR Ca2+ channels (cf.Fig.

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