scholarly journals Calmodulin sensitivity of the sarcoplasmic reticulum ryanodine receptor from normal and malignant-hyperthermia-susceptible muscle

1996 ◽  
Vol 319 (2) ◽  
pp. 421-426 ◽  
Author(s):  
Sean O'DRISCOLL ◽  
Tommie V. McCARTHY ◽  
Hans M. EICHINGER ◽  
Wolf ERHARDT ◽  
Frank LEHMANN-HORN ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Central Region ◽  
Dependent Manner ◽  
Release Channel ◽  
Malignant Hyperthermia Susceptible ◽  
Potential Binding ◽  
Cam Regulation

Ca2+ release from sarcoplasmic reticulum (SR) of malignant-hyperthermia-susceptible (MHS) muscle is hypersensitive to Ca2+ and caffeine. To determine if an abnormal calmodulin (CaM) regulation of the SR Ca2+-release-channel-ryanodine-receptor complex (RYR1) contributes to this hypersensitivity, we investigated the effect of CaM on high-affinity [3H]ryanodine binding to isolated SR vesicles from normal and MHS pig skeletal muscle. CaM modulated [3H]ryanodine binding in a Ca2+-dependent manner. In the presence of maximally activating Ca2+ concentrations, CaM inhibited [3H]ryanodine binding with no differences between normal and MHS vesicles. In the absence of Ca2+, however, CaM activated [3H]ryanodine binding with a 2-fold-higher potency in MHS vesicles. Significant differences between normal and MHS tissue were observed for CaM concentrations between 50 nM and 10 µM. A polyclonal antibody raised against the central region of RYR1 specifically inhibited this activating effect of CaM without affecting the inhibition by CaM. This indicates that the central region of RYR1 is a potential binding domain for CaM in the absence of Ca2+. It is suggested that in vivo an enhanced CaM sensitivity of RYR1 might contribute to the abnormal high release of Ca2+ from the SR of MHS muscle.

Caffeine stimulation of malignant hyperthermia-susceptible sarcoplasmic reticulum Ca2+ release channel

1994 ◽  
Vol 267 (5) ◽  
pp. C1253-C1261 ◽  
Author(s):  
N. H. Shomer ◽  
J. R. Mickelson ◽  
C. F. Louis
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Malignant Hyperthermia ◽  
Single Channel ◽  
Release Channel ◽  
Muscle Contracture ◽  
Malignant Hyperthermia Susceptible ◽  
Open Time ◽  
Pig Muscle ◽  
Channel Mutation ◽  
The Difference

The altered caffeine sensitivity of malignant hyperthermia-susceptible (MHS) muscle contracture is one basis of the diagnostic test for this syndrome. To determine whether the Arg615-to-Cys615 mutation of the porcine sarcoplasmic reticulum (SR) Ca2+ release channel is directly responsible for this altered caffeine sensitivity, the single-channel kinetics of purified MHS and normal pig Ca2+ release channels were examined. Initial studies demonstrated that decreasing the pH of the medium in either the cis- or trans-chamber decreased the Ca2+ release channel percent open time (Po). The half-inhibitory pH of MHS channels (6.86 +/- 0.04, n = 17) was significantly different from that of normal channels (7.08 +/- 0.07, n = 14). At pH 7.4, in either 7 or 0.12 microM Ca2+, MHS channel Po was not significantly different from that of normal channels over the range 0-10 mM caffeine. Although at pH 6.8 in 7 microM Ca2+ MHS channel Po was greater than that of normal channels over the range 0-20 mM caffeine, the difference could be eliminated by dividing each mean MHS Po by a scaling factor of 3.2. Thus the MHS Ca2+ release channel mutation does not appear to be directly responsible for the altered caffeine sensitivity of MHS pig muscle contracture. Rather, this altered caffeine sensitivity may result from an altered resting myoplasmic Ca2+ concentration or the altered pH and Ca2+ sensitivity of Ca2+ release channel Po of MHS muscle.

scholarly journals Increased sensitivity of the ryanodine receptor to halothane-induced oligomerization in malignant hyperthermia-susceptible human skeletal muscle

2004 ◽  
Vol 96 (1) ◽  
pp. 11-18 ◽  
Author(s):  
Louise Glover ◽  
James J. A. Heffron ◽  
Kay Ohlendieck
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Human Skeletal Muscle ◽  
Dihydropyridine Receptor ◽  
Threshold Concentration ◽  
Release Channel ◽  
Functional Dynamics ◽  
Increased Sensitivity

Mutations in the skeletal muscle RyR1 isoform of the ryanodine receptor (RyR) Ca2+-release channel confer susceptibility to malignant hyperthermia, which may be triggered by inhalational anesthetics such as halothane. Using immunoblotting, we show here that the ryanodine receptor, calmodulin, junctin, calsequestrin, sarcalumenin, calreticulin, annexin-VI, sarco(endo)plasmic reticulum Ca2+-ATPase, and the dihydropyridine receptor exhibit no major changes in their expression level between normal human skeletal muscle and biopsies from individuals susceptible to malignant hyperthermia. In contrast, protein gel-shift studies with halothane-treated sarcoplasmic reticulum vesicles from normal and susceptible specimens showed a clear difference. Although the α2-dihydropyridine receptor and calsequestrin were not affected, clustering of the Ca2+-ATPase was induced at comparable halothane concentrations. In the concentration range of 0.014–0.35 mM halothane, anesthetic-induced oligomerization of the RyR1 complex was observed at a lower threshold concentration in the sarcoplasmic reticulum from patients with malignant hyperthermia. Thus the previously described decreased Ca2+-loading ability of the sarcoplasmic reticulum from susceptible muscle fibers is probably not due to a modified expression of Ca2+-handling elements, but more likely a feature of altered quaternary receptor structure or modified functional dynamics within the Ca2+-regulatory apparatus. Possibly increased RyR1 complex formation, in conjunction with decreased Ca2+ uptake, is of central importance to the development of a metabolic crisis in malignant hyperthermia.

scholarly journals Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum

2010 ◽  
Vol 137 (1) ◽  
pp. 43-57 ◽  
Author(s):  
Ryan E. Loy ◽  
Murat Orynbayev ◽  
Le Xu ◽  
Zoita Andronache ◽  
Simona Apostol ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Ryanodine Receptor ◽  
Muscle Weakness ◽  
Dominant Negative ◽  
Release Mechanism ◽  
Ion Permeation ◽  
Release Channel ◽  
Ec Coupling

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation–contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1I4898T mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4–6-mo-old IT/+ mice. The sensitivity of the SR Ca2+ release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca2+ permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation.

Reconstitution of abnormalities in the malignant hyperthermia-susceptible pig ryanodine receptor

1993 ◽  
Vol 264 (1) ◽  
pp. C125-C135 ◽  
Author(s):  
N. H. Shomer ◽  
C. F. Louis ◽  
M. Fill ◽  
L. A. Litterer ◽  
J. R. Mickelson
Keyword(s):  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Ryanodine Receptors ◽  
Primary Defect ◽  
Open Probability ◽  
Release Channel ◽  
Malignant Hyperthermia Susceptible ◽  
Channel Opening ◽  
Kd Values

Malignant hyperthermia-susceptible (MHS) pigs homozygous for the Cys615 ryanodine receptor allele demonstrate altered sarcoplasmic reticulum (SR) ryanodine binding and Ca2+ release channel regulatory properties when compared with normal pigs homozygous for the Arg615 allele. While solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified MHS and normal ryanodine receptors had a similar dissociation constant (Kd) for ryanodine, maximum binding, and Ca2+ concentration for half-maximal stimulation and inhibition of ryanodine binding (Ca2+(0.5)); however, after reconstitution into proteoliposomes, the purified MHS and normal receptors had Kd values for ryanodine of 75 and 150 nM, respectively, which were significantly different. The purified MHS and normal porcine ryanodine receptors also had similar single-channel Cs+ conductance, optimal cis-Ca2+ for channel opening, and cis-Ca2+(0.5) for channel activation. Significantly, at inactivating levels of cis-Ca2+ (> 0.1 mM), MHS channels had a greater open probability, a higher cis-Ca2+(0.5) for inhibition of channel opening (250 vs. 75 microM for MHS and normal, respectively), longer mean open times, and shorter mean closed times than did normal channels. We conclude that the mutation at residue 615 causes a detectable alteration in ryanodine receptor/Ca2+ channel activity and thus may represent the primary defect responsible for the altered SR Ca2+ regulation characteristic of MHS porcine muscle.

Altered binding site for Ca2+ in the ryanodine receptor of human malignant hyperthermia

1991 ◽  
Vol 261 (2) ◽  
pp. C237-C245 ◽  
Author(s):  
H. H. Valdivia ◽  
K. Hogan ◽  
R. Coronado
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Binding Site ◽  
Ryanodine Receptor ◽  
Human Skeletal Muscle ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Binding Properties ◽  
Release Channel ◽  
Planar Bilayers

The binding properties of [3H]ryanodine, a specific ligand of the receptor complex that forms the Ca2+ release channel of sarcoplasmic reticulum, were studied in normal (N) and malignant hyperthermia-susceptible (MH) human skeletal muscle. Integrity of the solubilized ryanodine receptor was demonstrated by single-channel recordings in planar bilayers and by the changes produced by activators and inhibitors of the Ca2+ release channel on the binding properties of [3H]ryanodine. N and MH receptors were capable of binding [3H]ryanodine in a Ca(2+)-dependent manner. Scatchard analysis showed that a single binding site for [3H]ryanodine was present in either N or MH muscle. Binding affinity was approximately the same in N and MH (Kd approximately 7 nM), when the Ca2+ concentration was greater than 30 microM. At 0.3 microM Ca2+, MH receptors displayed a higher affinity for [3H]ryanodine (Kd = 4.1 +/- 1.0 nM) than N receptors (Kd = 7.1 +/- 0.8 nM). The presence of a single Kd for [3H]ryanodine in MH muscle, distinct from that of N muscle, indicated that MH muscle does not have detectable levels of N receptors. Ca2+ dependence of [3H]ryanodine binding further suggested that MH receptors had a higher affinity for Ca2+ (Kd[Ca2+] = 120 +/- 50 nM) than N receptors (Kd[Ca2+] = 250 +/- 80 nM). Caffeine increased [3H]ryanodine binding at submicromolar [Ca2+], and the effect was larger in MH. Apparent affinity constants for caffeine were 13 +/- 1.8 mM in N and 6 +/- 0.8 mM in MH receptors. Evidently, the ryanodine receptor of MH-susceptible human skeletal muscle has an unusually high sensitivity to Ca2+ which is augmented by caffeine.(ABSTRACT TRUNCATED AT 250 WORDS)

Skeletal muscle junctional membrane protein content in pigs with different ryanodine receptor genotypes

1994 ◽  
Vol 267 (1) ◽  
pp. C282-C292 ◽  
Author(s):  
J. R. Mickelson ◽  
J. M. Ervasti ◽  
L. A. Litterer ◽  
K. P. Campbell ◽  
C. F. Louis
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Membrane Protein ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Muscle Development ◽  
Relative Content ◽  
Junctional Proteins ◽  
Malignant Hyperthermia Susceptible ◽  
Total Muscle

The content of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, transverse tubule dihydropyridine receptor (DHPR), and SR ryanodine receptor (RyR) was determined in muscle of pigs homozygous for the normal RyR allele and homozygous or heterozygous for the malignant hyperthermia-susceptible (MHS) RyR allele. Total muscle membranes isolated from 1-day-old pigs of the three different genotypes did not differ in the content of any of these proteins. However, at 28 days of age, crude membranes and total muscle homogenates from homozygous MHS pigs exhibited only 61-81% of the [3H]PN 200-110 or [3H]ryanodine binding of identical preparations isolated from normal pigs; these MHS membranes also contained only 50% of the normal content of each of the DHPR subunits. The crude membranes and muscle homogenates from heterozygous pigs were intermediate to both types of homozygotes in terms of [3H]PN 200-110 binding, [3H]ryanodine binding, and the content of the DHPR subunits. However, membrane preparations enriched in triadic junctional proteins isolated from 3- to 4-mo-old pigs of the three different genotypes did not differ in their [3H]PN 200-110 binding, [3H]ryanodine binding, or Ca(2+)-ATPase activities. We conclude that, although the stoichiometry of the RyR to DHPR is not altered, the presence of the MHS RyR allele during muscle development results in a decreased relative content of these two proteins. This is probably due to a lower junctional membrane content and may be an important ultrastructural consequence of the altered sarcoplasmic Ca2+ regulation in MHS muscle.

Mg2+Dependence of Halothane-induced Ca2+Release from the Sarcoplasmic Reticulum in Skeletal Muscle from Humans Susceptible to Malignant Hyperthermia

2004 ◽  
Vol 101 (6) ◽  
pp. 1339-1346 ◽  
Author(s):  
Adrian M. Duke ◽  
Philip M. Hopkins ◽  
Jane P. Halsal ◽  
Derek S. Steele
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Process Approach ◽  
Major Influence ◽  
Clinical Implications ◽  
Vastus Medialis ◽  
Release Process ◽  
Halothane Concentration ◽  
Malignant Hyperthermia Susceptible

Background Recent work suggests that impaired Mg(2+) regulation of the ryanodine receptor is a common feature of both pig and human malignant hyperthermia. Therefore, the influence of [Mg(2+)] on halothane-induced Ca(2+) release from the sarcoplasmic reticulum was studied in malignant hyperthermia-susceptible (MHS) or -nonsusceptible (MHN) muscle. Methods Vastus medialis fibers were mechanically skinned and perfused with solutions containing physiologic (1 mm) or reduced concentrations of free [Mg(2+)]. Sarcoplasmic reticulum Ca(2+) release was detected using fura-2 or fluo-3. Results In MHN fibers, 1 mm halothane consistently did not induce sarcoplasmic reticulum Ca(2+) release in the presence of 1 mm Mg(2+). It was necessary to increase the halothane concentration to 20 mm or greater before Ca release occurred. However, when [Mg(2+)] was reduced below 1 mm, halothane became an increasingly effective stimulus for Ca(2+) release; e.g., at 0.4 mm Mg(2+), 58% of MHN fibers responded to halothane. In MHS fibers, 1 mm halothane induced Ca(2+) release in 57% of MHS fibers at 1 mm Mg(2+). Reducing [Mg(2+)] increased the proportion of MHS fibers that responded to 1 mm halothane. Further experiments revealed differences in the characteristics of halothane-induced Ca(2+) release in MHS and MHN fibers: In MHN fibers, at 1 mm Mg(2+), halothane induced a diffuse increase in [Ca(2+)], which began at the periphery of the fiber and spread slowly inward. In MHS fibers, halothane induced a localized C(2+)a release, which then propagated along the fiber. However, propagated Ca(2+) release was observed in MHN fibers when halothane was applied at an Mg(2+) concentration of 0.4 mm or less. Conclusions When Mg(2+) inhibition of the ryanodine receptor is reduced, the halothane sensitivity of MHN fibers and the characteristics of the Ca release process approach that of the MHS phenotype. In MHS fibers, reduced Mg(2+) inhibition of the ryanodine receptor would be expected to have a major influence on halothane sensitivity. The Mg dependence of the halothane response in MHN and MHS may have important clinical implications in circumstances where intracellular [Mg(2+)] deviates from normal physiologic concentrations.

Effects of Propofol on Calcium sup 2+ Regulation by Malignant Hyperthermia-susceptible Muscle Membranes 

1995 ◽  
Vol 82 (5) ◽  
pp. 1274-1282 ◽  
Author(s):  
Bradley R. Fruen ◽  
James R. Mickelson ◽  
Timothy J. Roghair ◽  
Lynn A. Litterer ◽  
Charles F. Louis
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Membrane Vesicles ◽  
Dihydropyridine Receptor ◽  
Muscle Membrane ◽  
Inhalation Anesthetics ◽  
Transverse Tubule ◽  
Malignant Hyperthermia Susceptible

Background The effects of inhalation anesthetics on Ca2+ regulation in malignant hyperthermia-susceptible skeletal muscle are considered to be responsible for triggering malignant hyperthermia. The intravenous anesthetic propofol does not trigger malignant hyperthermia in susceptible patients or experimental animals, suggesting that there are important differences between the effects of propofol and the effects of inhalation anesthetics on Ca2+ regulation in malignant hyperthermia-susceptible muscle. Understanding these differences may help to clarify the mechanisms responsible for triggering malignant hyperthermia. Methods To investigate the effects of propofol on Ca2+ regulation by malignant hyperthermia-susceptible skeletal muscle, we determined its effects on the membrane channels and pumps that control myoplasmic Ca2+ concentrations: the sarcoplasmic reticulum ryanodine receptor, the transverse tubule dihydropyridine receptor, and the sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase (Ca(2+)-ATPase). Terminal cisternae-derived sarcoplasmic reticulum vesicles enriched in the junctional proteins of the sarcoplasmic reticulum and the transverse tubule membranes were isolated from the muscle of malignant hyperthermia-susceptible and normal pigs. Ca2+ flux, Ca(2+)-ATPase, and ligand binding measurements on these isolated vesicle preparations were performed in the presence of varying propofol concentrations. Results Propofol (10-500 microM) had no effect on ryanodine receptor-mediated Ca2+ efflux from muscle membrane vesicles. Propofol (1-100 microM) also had no effect on sarcoplasmic reticulum vesicle [3H]ryanodine binding, whereas higher concentrations (200-300 microM) slightly inhibited [3H]ryanodine binding. Binding of the dihydropyridine receptor Ca2+ channel blocker [3H]PN200-110 to these preparations was inhibited by propofol (10-300 microM). Ca(2+)-ATPase activity was stimulated by 10-100 microM propofol but was inhibited by higher concentrations. In all cases, the effects of propofol on malignant hyperthermia-susceptible and normal membrane preparations were similar. Conclusions In contrast to malignant hyperthermia-triggering inhalation anesthetics, propofol does not stimulate malignant hyperthermia-susceptible or normal ryanodine receptor channel activity, even at > 100 times clinical concentrations. Effects on dihydropyridine receptor and Ca(2+)-ATPase function, however, are similar to the effects of inhalation anesthestics and require much lower concentrations of propofol. These findings, demonstrating that propofol does not activate ryanodine receptor Ca2+ channels, suggest a plausible explanation for why propofol does not trigger malignant hyperthermia in susceptible persons.

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