Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia.

Malignant hyperthermia (MH) causes neurological, liver, and kidney damage and death in humans and major economic losses in the swine industry. A single point mutation in the porcine gene for the skeletal muscle ryanodine receptor (ryr1) was found to be correlated with MH in five major breeds of lean, heavily muscled swine. Haplotyping suggests that the mutation in all five breeds has a common origin. Assuming that this is the causal mutation for MH, the development of a noninvasive diagnostic test will provide the basis for elimination of the MH gene or its controlled inclusion in swine breeding programs.

Intracellular calcium homeostasis in human primary muscle cells from malignant hyperthermia-susceptible and normal individuals. Effect Of overexpression of recombinant wild-type and Arg163Cys mutated ryanodine receptors.

Malignant hyperthermia (MH) is a hypermetabolic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. Nine point mutations in the skeletal muscle ryanodine receptor (RYR) gene have so far been identified and shown to correlate with the MH-susceptible phenotype, yet direct evidence linking abnormal Ca2+ homeostasis to mutations in the RYR1 cDNA has been obtained for few mutations. In this report, we show for the first time that cultured human skeletal muscle cells derived from MH-susceptible individuals exhibit a half-maximal halothane concentration causing an increase in intracellular Ca2+ concentration which is twofold lower than that of cells derived from MH-negative individuals. We also present evidence demonstrating that overexpression of wild-type RYR1 in cells obtained from MH-susceptible individuals does not restore the MH-negative phenotype, as far as Ca2+ transients elicited by halothane are concerned; on the other hand, overexpression of a mutated RYR1 Arg163Cys Ca2+ channel in muscle cells obtained from MH-negative individuals conveys hypersensitivity to halothane. Finally, our results show that the resting Ca2+ concentration of cultured skeletal muscle cells from MH-negative and MH-susceptible individuals is not significantly different.

Malignant hyperthermia domain in the regulation of ca(2+) release channel (ryanodine receptor).

Malignant hyperthermia (MH) is a potentially lethal condition that is manifested in humans as an acute increase of body temperature in response to stress and exposure to volatile anaesthetics (halothane, enflurane) and muscle relaxants. To date, eight point mutations in the ryanodine receptor gene, the Ca(2+) release channel of the skeletal muscle sarcoplasmic reticulum, segregate with the MH phenotype, yet direct evidence linking altered [Ca(2+)](i) homeostasis to mutation in recombinant RYR has been obtained only for one such mutation. Most of these mutations appear in an "MH domain" that is localized at the NH(2) terminus of the skeletal muscle ryanodine receptor Ca(2+) channel. In this review, we summarize the available data concerning the role of the MH domain in the altered functions of the ryanodine receptor Ca(2+) channel. (Trends Cardiovasc Med 1997;7:312-316). © 1997, Elsevier Science Inc.

Functional properties of EGFP-tagged skeletal muscle calcium-release channel (ryanodine receptor) expressed in COS-7 cells: sensitivity to caffeine and 4-chloro-m-cresol.

We constructed and expressed in COS-7 cells, three E-green fluorescent protein (EGFP) tagged recombinant skeletal muscle ryanodine receptors (RYR). EGFP was tagged to (i) the NH2-terminus (nEGFP-RYR(FL)) and to (ii) the COOH-terminus (cRYR(FL)-EGFP) of the full length RYR; we also tagged the EGFP to (iii) the NH2-terminus of a truncated version of the RYR (nEGFP-RYR(Bhat)) lacking the bulk of the protein. The fluorescent pattern EGFP with all three constructs colocalize with that of an endoplasmic reticulum (ER) membrane tracker fluorescent dye, indicating that the RYR constructs are targeted to ER membranes. Our results show that: (i) COOH-terminal tagging abolishes the sensitivity of the RYR to caffeine, whereas the presence of EGFP at the NH2-terminus does not affect caffeine sensitivity and (ii) 4-Cl-m-cresol sensitivity is lost both with the truncated nEGFP-RYR(Bhat) and the nEGFP-RYR(FL), while COOH-terminal tagging does not affect sensitivity to 4-chloro-m-cresol. The dose-response curves of caffeine-induced calcium release of nEGFP-RYR(FL) differ from those of the truncated nEGFP-RYR(Bhat). Maximal calcium release was approached at 10 mM caffeine with the nEGFP-RYR(FL), while cells expressing the nEGFP-RYR(Bhat) construct displayed a bell shaped curve and the maximal concentration for caffeine-induced calcium release was 5 mM. Equilibrium [3H]-ryanodine binding confirmed the calcium photometry data. Our results demonstrate that EGFP tagging modifies the pharmacological properties of RYR, and suggest that 4-chloro-m-cresol and caffeine act through different mechanisms and probably interact with different sites on the RYR calcium release channel.

Genotype-phenotype comparison of the Swiss malignant hyperthermia population.

Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disease, triggered by inhalative anesthetics or depolarizing muscle relaxants in genetically predisposed individuals. Linkage analysis have revealed MH to be a heterogenetic disease with about 50% of MH families linked to the locus of the ryanodine receptor calcium channel (RYR1). We investigated the frequency of the 23 published MH linked RYR1 gene mutations in the Swiss MH population and compared our findings to the results of the in vitro contracture test (IVCT). IVCT was performed following the protocol of the European MH Group and mutation screening was done by PCR amplification of genomic DNA followed by restriction enzyme digestion or SSCP. We identified RYR1 gene mutations in 40% of unrelated MH families (19/48) with a high incidence of the mutation V2168M (27%). IVCT results revealed a significantly stronger functional effect of mutations R614C and V2168M as compared to mutations G2434R and R2458C. This is the first time that such a high incidence of RYR1 gene mutations in an MH population has been found, supporting the use of molecular genetic testing for the diagnosis of MH susceptibility in suitable families. In addition our data show that different RYR1 gene mutations are associated with different IVCT phenotypes.

Ca2+ channel agonist BAY-k 8644 does not elicit Ca2+ release from skeletal muscle sarcoplasmic reticulum.

BAY-k 8644, a nifedipine analogue, promotes Ca2+ influx into excitable cells via plasma membrane voltage-sensitive Ca2+ channels. We report here that sarcoplasmic reticulum (SR) Ca2+ release channels are insensitive to BAY-k 8644, as studied in highly purified isolated fractions and in chemically skinned fibers of rabbit skeletal muscle. This result suggests that a subcellular heterogeneity exists among Ca2+ channels, at least with respect to drug-receptor sites. In the course of this study, however we found that BAY-k 8644 reversibly inhibits the SR Ca2+ pump, i.e., it decreases Ca2+ influx into the SR lumen, although at concentrations (IC50 = 3-5 X 10(-5) M) much higher than those effective on voltage-sensitive Ca2+ channels.

Autoantibodies in thymoma-associated myasthenia gravis with myositis or neuromyotonia.

BACKGROUND: About 50% of patients with thymoma have paraneoplastic myasthenia gravis (MG). Myositis and myocarditis or neuromyotonia (NMT) will also develop in some. Patients with thymoma-associated MG produce autoantibodies to a variety of neuromuscular antigens, particularly acetylcholine receptor (AChR), titin, skeletal muscle calcium release channel (ryanodine receptor [RyR]), and voltage-gated potassium channels (VGKC). OBJECTIVE: To examine whether neuromuscular autoantibodies in patients with thymoma correlate with specific clinical syndromes. METHODS: Serum and plasma samples from 19 patients with thymoma-associated MG, of whom 5 had myositis and 6 had NMT, underwent testing for antibodies to AChR, titin, RyR, and VGKC. RESULTS: Antibodies to AChR and titin were found in 19 and 17 patients, respectively. Antibodies to RyR correlated with the presence of myositis (P = .03); they were found in all 5 patients with myositis and in only 1 patient with NMT, but also in 4 of 8 patients with neither disease. Antibodies to VGKC were found in 4 patients with NMT, 1 of 3 patients undergoing testing for myositis, and 2 of 7 patients undergoing testing with neither comorbidity. Presence of RyR antibodies correlated with high levels of titin antibodies. CONCLUSIONS: The results appear to distinguish partially between 3 groups of patients with thymoma-associated MG: the first with RyR antibodies and myositis or myocarditis, the second with NMT without RyR antibodies, and the third without RyR antibodies, myositis, or NMT. Differences in the thymoma may underlie these pathologic associations.

Complement activation by titin and ryanodine receptor autoantibodies in myasthenia gravis. A study of IgG subclasses and clinical correlations.

To elucidate the mechanism of immune damage caused by titin and ryanodine receptor (RyR) autoantibodies in myasthenia gravis (MG), we studied the complement-activating capacity and the IgG subclass distribution of these autoantibodies in sera from 49 MG patients. Complement activation occurred in 38 out of 49 titin antibody positive sera, and in 14 out of 21 RyR antibody positive sera. The titin antibodies occurred only in the IgG 1 and IgG 4 subclasses, whereas the RyR antibodies occurred in all four IgG subclasses but with IgG 1 predominance. Complement-activating RyR antibodies occurred with higher frequency in sera of thymoma MG than of late-onset MG. RyR IgG 1 antibodies occurred more often in severe MG than in mild and moderate disease groups. Mean total IgG and IgG 1 titin and RyR antibody titers fell during long-time patient observation together with an improvement of the MG symptoms.

Methyl p-hydroxybenzoate (E-218) a preservative for drugs and food is an activator of the ryanodine receptor Ca(2+) release channel.

1. Haloperidol is a drug used in the management of several psychotic disorders and its use has been linked to Neuroleptic Malignant Syndrome. In the present study we have investigated the effect of a commercial preparation of haloperidol, Serenase, on skeletal muscle sarcoplasmic reticulum. 2. Addition of Serenase to isolated terminal cisternae caused a rapid release of calcium. We tested whether the active Ca(2+)-releasing substance was haloperidol or another compound present in the preparation. 3. Our results show that methyl p-hydroxybenzoate, one of the preservatives and a commonly used anti-microbial agent (E-218) is an activator of Ca(2+) release (E.C. 50=2.0 mM), mediated by a ruthenium red-sensitive Ca(2+) release channel present in skeletal muscle terminal cisternae.

Surface plasmon resonance for real-time detection of molecular interactions between chromomycin and target DNA sequences.

DNA-binding drugs interfere with the activity of a large variety of transcription factors, leading to an alteration of transcription. This and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases. The analysis of sequence selectivity of DNA-binding drugs by footprinting, gel retardation studies, polymerase chain reaction and in vitro transcription does not allow an easy study of kinetics of binding and dissociation. The recent development of biosensor technologies for biospecific interaction analysis (BIA) enables the monitoring of a variety of molecular reactions in real-time by surface plasmon resonance (SPR). In this report we demonstrate that molecular interactions between the DNA-binding drug chromomycin and a biotinylated GC-rich Ha-ras oligonucleotide probe immobilized on a sensor chip is detectable by SPR technology using the BIAcore(TM) biosensor. This approach appears of interest in the development of drugs exhibiting differential affinity for target DNA sequences for the following reasons: a) results are obtained within one hour; b) unlike footprinting and gel retardation studies, this technology does not require P-32-labelled probes; c) BIA allows kinetic studies of both association and dissociation.

A possible role of the junctional face protein JP-45 in modulating Ca2+ release in skeletal muscle.

We investigated the functional role of JP-45, a recently discovered protein of the junctional face membrane (JFM) of skeletal muscle. For this purpose, we expressed JP-45 C-terminally tagged with the fluorescent protein DsRed2 by nuclear microinjection in myotubes derived from the C2C12 skeletal muscle cell line and performed whole-cell voltage-clamp experiments. We recorded in parallel cell membrane currents and Ca(2+) signals using fura-2 during step depolarization. It was found that properties of the voltage-activated Ca(2+) current were not significantly changed in JP-45-DsRed2-expressing C2C12 myotubes whereas the amplitude of depolarization-induced Ca(2+) transient was decreased compared to control myotubes expressing only DsRed2. Converting Ca(2+) transients to Ca(2+) input flux using a model fit approach to quantify Ca(2+) removal, the change could be attributed to an alteration in voltage-activated Ca(2+) permeability rather than to altered removal properties or a lower Ca(2+) content of the sarcoplasmic reticulum (SR). Determining non-linear capacitive currents revealed a reduction of Ca(2+) permeability per voltage-sensor charge. The results may be explained by a modulatory effect of JP-45 related to its reported in vitro interaction with the dihydropyridine receptor and the SR Ca(2+) binding protein calsequestrin (CSQ).

Novel sarco(endo)plasmic reticulum proteins and calcium homeostasis in striated muscles.

The impact of calcium signaling on many cellular functions is reflected by the tight regulation of the intracellular Ca(2+) concentration that is ensured by diverse pumps, channels, transporters and Ca(2+) binding proteins. In this review, we present recently identified novel sarco(endo)plasmic reticulum proteins that may have a potential involvement in the regulation of Ca(2+) homeostasis in striated muscles.

Calcium binding proteins of junctional sarcoplasmic reticulum: detection by 45Ca ligand overlay.

In skeletal muscle, the junctional sarcoplasmic reticulum (JFM) plays a crucial role in excitation-contraction coupling and Ca2+ release. In the present report, the sarcoplasmic reticulum (SR) was fractionated into longitudinal SR (LSR), terminal cisternae (TC), and JFM. Each fraction had a unique protein profile as detected by SDS-polyacrylamide gel electrophoresis as well as specific Ca2+ binding proteins as judged by 45Ca ligand overlay of nitrocellulose blots. Ca2+ binding proteins of LSR were the Ca2+ ATPase (Mr of 115K), an 80K polypeptide, and the intrinsic glycoprotein (Mr of 160K); Ca2+ binding proteins of JFM were polypeptides with the following Mr values: 350K and 325K (feet components), 200K, 170K, a doublet of 140K, 118K, 65K (calsequestrin), and 52K. Measurements of Ca2+ binding to SR fractions by equilibrium dialysis indicated that 8-17 nmol Ca2+/mg of protein was specifically bound. After EDTA extraction of calsequestrin, JFM still bound Ca2+ (5-6 nmol/mg of protein), suggesting the existence of specific Ca2+ binding sites. The Ca2+ binding sites of Ca2+-gated Ca2+ release channels might be on two JFM polypeptides (Mr's of 350K and 170K) which are putative channel constituents (F. Zorzato, A. Margreth, and P. Volpe (1986) J. Biol. Chem. 261, 13252-13257).

Direct photoaffinity labeling of junctional sarcoplasmic reticulum with [14C]doxorubicin.

Doxorubicin, an anticancer drug, induces Ca2+ release from the terminal cisternae (TC) of skeletal muscle (Zorzato, F., Salviati, G., Facchinetti, T., and Volpe, P. (1985) J. Biol. Chem. 260, 7349-7355). Long wave ultraviolet irradiation of a TC fraction with morphologically intact feet structures (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885) in the presence of [14C]doxorubicin, led to covalent photolabeling of two proteins that exhibited apparent Mr values of 350,000 and 170,000. Such proteins were found to be absent in a fraction of longitudinal sarcoplasmic reticulum but enriched in junctional face membranes obtained by Triton X-100 treatment of the TC fraction. Three additional proteins with Mr values of 80,000, 60,000, and 30,000 were also faintly labeled in the junctional face membrane fraction. On a molar basis the highest level of incorporation was found in the 170,000-Da protein, probably a Ca2+-binding protein (Campbell, K. P., MacLennan, D. H., and Jorgensen, A. O. (1983) J. Biol. Chem. 258, 11267-11273). A lower level of labeling was observed in the 350,000-Da protein, tentatively identified as a component of the feet structures (Cadwell, J. J. S., and Caswell, A. H. (1982) J. Cell Biol. 93, 543-550). Photolabeling of junctional TC proteins did not occur if a 10-50-fold excess cold doxorubicin was included in the assay medium, indicating that it was displaceable and specific, and if ultraviolet irradiation was omitted. Photolabeling was inhibited by caffeine or ruthenium red, i.e. by an activator and an inhibitor of Ca2+ release from TC, respectively. Furthermore, photolabeling was prevented by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid suggesting that doxorubicin binding is Ca2+-dependent. Doxorubicin-binding proteins are constituents of the junctional sarcoplasmic reticulum and might be involved in modulating Ca2+ release from TC.

Antibodies to junctional sarcoplasmic reticulum proteins: probes for the Ca2+-release channel.

The junctional face membrane plays a key role in excitation-contraction coupling in skeletal muscle. A protein of 350 kDa, tentatively identified as a component of the junctional feet, connects transverse tubules to terminal cisternae of sarcoplasmic reticulum [Kawamoto, Brunschwig, Kim & Caswell (1986) J. Cell Biol. 103, 1405-1414]. The membrane topology and protein composition of sarcoplasmic reticulum Ca2+-release channels of rabbit skeletal muscle were investigated using an immunological approach, with anti-(junctional face membrane) and anti-(350 kDa protein) polyclonal antibodies. Upon preincubation of the terminal cisternae with anti-(junctional face membrane) antibodies, Ca2+-ATPase and Ca2+-loading activities were not affected, whereas anti-(350 kDa protein) antibodies stimulated Ca2+-ATPase activity by 25% and inhibited Ca2+-loading activity by 50% (at an antibody/terminal cisternae protein ratio of 1:1). Specific photolabelling of terminal cisternae proteins with [14C]doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Stimulation of Ca2+ release by doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Half-maximal inhibition was obtained at an antibody/terminal cisternae protein ratio of 1:1. Kinetic measurements of Ca2+ release indicated that anti-(350 kDa protein) antibodies prevented Ca2+-induced Ca2+ release, whereas the ATP-stimulation and the inhibition by Mg2+ were not affected. These results suggest that: (i) Ca2+- and doxorubicin-induced Ca2+ release is mediated by Ca2+ channels which are selectively localized in the junctional face membrane; (ii) the 350 kDa protein is a component of the Ca2+-release channel in native terminal cisternae vesicles; and (iii) the Ca2+-activating site of the channel is separate from other allosteric sites.

Identification of calreticulin isoforms in the central nervous system.

In the present paper we report the cloning and sequencing of the cDNA encoding two calreticulin isoforms from Xenopus laevis central nervous system. The two isoforms display 93% identity at the amino acid level. The predicted amino acid sequences of the amphibian calreticulins are very similar (76%) to those of mammalian liver and skeletal muscle. Xenopus laevis calreticulins are characterized by a very acidic c-terminal domain endowed with the endoplasmic-reticulum retention signal KDEL. The cDNAs of both clones encode an N-glycosylation consensus sequence. A third clone of calreticulin was also identified. The restriction map of this clone was clearly distinct from that of the two sequenced clones. These results indicate the existence of multiple calreticulin isoforms in the central nervous system and open questions about their functional role in different cells and/or subcellular compartments.

Identification of two ryanodine receptor transcripts in neonatal, slow-, and fast-twitch rabbit skeletal muscles.

Analysis of the primary structure of the rabbit skeletal muscle ryanodine receptor led to the identification of two molecules of 5032 and 5037 residues, respectively. Such a sequence discrepancy is likely to be due to the alternative splicing of a 15 bp exon (1) encoding a 5 amino acid insertion (Ala-Gly-Asp-Ala-Gln) after residue 3479. By using PCR on first strand cDNA, we searched for the 15 base pair insertion in the ryanodine receptor mRNA from adult slow- and fast-twitch skeletal muscle, as well as from fast-muscles, at various stages of post-natal development. All rabbit skeletal muscle mRNAs, regardless of their developmental stage and twitch properties, contain two RYR transcripts, suggesting the coexistence of two RYR isoforms in mammalian skeletal muscle.

Identification of the domain recognized by anti-(ryanodine receptor) antibodies which affect Ca(2+)-induced Ca2+ release.

In the present paper we have defined putative functional domains of the ryanodine receptor Ca2+ channel. cDNA fragments of the skeletal muscle ryanodine receptor were fused in-frame with the Escherichia coli trpe protein and the resulting fusion proteins were evaluated for their ability to react with anti-(ryanodine receptor) antibodies, which are known to block Ca(2+)-dependent activation of the Ca(2+)-release channel. Anti-(ryanodine receptor) antibodies react with epitopes lying within a 245-amino-acid-long polypeptide which is located in a region (residues 4380-4625) encompassing most of myoplasmic loop 2, the predicted transmembrane segment M5 and part of the next lumenal loop (45 residues). Purification of the anti-(ryanodine receptor) antibodies by affinity chromatography led to the isolation of a population of antibodies which was capable of decreasing (by > 30%) the doxorubicin-induced Ca2+ release from isolated terminal cisternae. Polyclonal antibodies raised against a ryanodine receptor fusion encompassing part (198 out of 245 residues) of the immunopositive polypeptide decreased by 2-fold the first-order rate constant of Ca(2+)-induced 45Ca2+ efflux from isolated terminal cisternae. These results suggest strongly that the Ca(2+)-activating domain of the skeletal muscle Ca(2+)-release channel is close to, or associated with, myoplasmic loop 2.

Characteristics of skeletal muscle calsequestrin: comparison of mammalian, amphibian and avian muscles.

Calsequestrin was identified in the isolated sarcoplasmic reticulum from skeletal muscle of three mammalian species (man, rat and rabbit) and from frog and chicken muscle, using electrophoretic and immunoblot techniques. It was further characterized in sarcoplasmic reticulum protein mixtures and at several stages of purification, following extraction with EDTA. We found extensive similarities in apparent molecular weight values, Stains All staining properties and in Cleveland's peptide maps, between mammalian calsequestrins, and no detectable difference within a species between fast and slow muscle. Human calsequestrin, with an apparent molecular weight of 60,000 when measured at alkaline pH and of 41,000 when measured at neutral pH, appears to be the smallest in size. Frog calsequestrin, although weakly cross-reactive with rabbit calsequestrin and having a relatively higher apparent molecular weight at alkaline pH (72,000), shares several significant properties with mammalian calsequestrins. It bound calcium with a high capacity (1300 nmol per mg protein), it contained about 32% acidic amino acid residues and focused at closely similar pI values. We observed the formation of a complex with Stains All absorbing maximally at 535 nm, rather than at 600 nm, and an even more marked shift in apparent molecular weight at neutral pH. We found distinct differences in the case of chicken calsequestrin, in addition to those previously reported. It is a highly acidic, calcium-precipitable protein, but its amino acid composition is contradistinguished by a higher ratio of glutamate to aspartate and its rate of electrophoretic mobility is minimally affected by changes in pH. It stained deep bluish with Stains All after gel electrophoresis and yielded a protein-dye complex in aqueous solution, absorbing maximally at 560 nm, and finally, it bound fluorescent Concanavalin A.