Para- and ortho-substitutions are key determinants of polybrominated diphenyl ether activity toward ryanodine receptors and neurotoxicity.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants that bioaccumulate in human tissues. Their neurotoxicity involves dysregulation of calcium ion (Ca(2+))signaling; however, specific mechanisms have yet to be defined. OBJECTIVE: We aimed to define the structure-activity relationship (SAR) for PBDEs and their metabolites toward ryanodine receptors type 1 (RyR1) and type 2 (RyR2) and to determine whether it predicts neurotoxicity. METHODS: We analyzed [3H]ryanodine binding, microsomal Ca(2+) fluxes, cellular measurements of Ca(2+) homeostasis, and neurotoxicity to define mechanisms and specificity of PBDE-mediated Ca(2+) dysregulation. RESULTS: PBDEs possessing two ortho-bromine substituents and lacking at least one para-bromine substituent (e.g., BDE-49) activate RyR1 and RyR2 with greater efficacy than corresponding congeners with two para-bromine substitutions (e.g., BDE-47). Addition of a methoxy group in the free para position reduces the activity of parent PBDEs. The hydroxylated BDEs 6-OH-BDE-47 and 4´-OH-BDE-49 are biphasic RyR modulators. Pretreatment of HEK293 cells (derived from human embryonic kidney cells) expressing either RyR1 or RyR2 with BDE-49 (250 nM) sensitized Ca2+ flux triggered by RyR agonists, whereas BDE-47 (250 nM) had negligible activity. The divergent activity of BDE-49, BDE-47, and 6-OH-BDE-47 toward RyRs predicted neurotoxicity in cultures of cortical neurons. CONCLUSIONS: We found that PBDEs are potent modulators of RyR1 and RyR2. A stringent SAR at the ortho and para position determined whether a congener enhanced, inhibited, or exerted nonmonotonic actions toward RyRs. These results identify a convergent molecular target of PBDEs previously identified for noncoplanar polychlorinated biphenyls (PCBs) that predicts their cellular neurotoxicity and therefore could be a useful tool in risk assessment of PBDEs and related compounds.

Pharmacologic and functional characterization of malignant hyperthermia in the R163C RyR1 knock-in mouse.

BACKGROUND: Malignant hyperthermia is a pharmacogenetic disorder affecting humans, dogs, pigs, and horses. In the majority of human cases and all cases in animals, malignant hyperthermia has been associated with missense mutations in the skeletal ryanodine receptor (RyR1). METHODS: The authors used a "knock-in" targeting vector to create mice carrying the RyR1 R163C malignant hyperthermia mutation. RESULTS: Validation of this new mouse model of human malignant hyperthermia susceptibility includes (1) proof of transcription of the R163C allele and expression of ryanodine receptor protein in R163C heterozygous and R163C homozygous animals; (2) fulminant malignant hyperthermia episodes in R163C heterozygous mice after exposure to 1.25-1.75% halothane or an ambient temperature of 42 degrees C characterized by increased rectal temperature, respiratory rate, and inspiratory effort, with significant blood biochemical changes indicating metabolic acidosis, ending in death and hyperacute rigor mortis; (3) intraperitoneal pretreatment with dantrolene provided 100% protection from the halothane-triggered fulminant malignant hyperthermia episode; (4) significantly increased sensitivity (decreased effective concentration causing 50% of the maximal response) of R163C heterozygous and homozygous myotubes to caffeine, 4-chloro-m-cresol, and K-induced depolarization; (5) R163C heterozygous and homozygous myotubes have a significantly increased resting intracellular Ca concentration compared with wild type; (6) R163C heterozygous sarcoplasmic reticulum membranes have a twofold higher affinity (Kd = 35.4 nm) for [H]ryanodine binding compared with wild type (Kd = 80.1 nm) and a diminished inhibitory regulation by Mg. CONCLUSIONS: Heterozygous R163C mice represent a valid model for studying the mechanisms that cause the human malignant hyperthermia syndrome.

Heat- and anesthesia-induced malignant hyperthermia in an RyR1 knock-in mouse.

Malignant hyperthermia (MH) is a life-threatening disorder characterized by skeletal muscle rigidity and elevated body temperature in response to halogenated anesthetics such as isoflurane or halothane. Mutation of tyrosine 522 of RyR1 (the predominant skeletal muscle calcium release channel) to serine has been associated with human malignant hyperthermia. In the present study, mice created harboring this mutation were found to represent the first murine model of human malignant hyperthermia. Mice homozygous for the Y522S mutation exhibit skeletal defects and die during embryonic development or soon after birth. Heterozygous mice, which correspond to the human occurrence of this mutation, are MH susceptible, experiencing whole body contractions and elevated core temperatures in response to isoflurane exposure or heat stress. Skeletal muscles from heterozygous mice exhibit increased susceptibility to caffeine- and heat-induced contractures in vitro. In addition, the heterozygous expression of the mutation results in enhanced RyR1 sensitivity to activation by temperature, caffeine, and voltage but not uncompensated sarcoplasmic reticulum calcium leak or store depletion. We conclude that the heterozygous expression of the Y522S mutation confers susceptibility to both heat- and anesthetic-induced MH responses.

Association of a mutation in the ryanodine receptor 1 gene with equine malignant hyperthermia.

Equine malignant hyperthermia MH has been suspected but never genetically confirmed. In this study, we investigated whether mutations in a candidate gene, RyR1, were associated with MH in two clinically affected horses. RyR1 gene sequences revealed polymorphisms in exons 15, 17, and 46 in WTRyR1 and MHRyR1 horses with one derived amino acid change in MHRyR1 exon 46, R2454G. The MHRyR1 horses were genetically heterozygous for this mutation, but presented an MH phenotype with halothane challenge. Skeletal sarcoplasmic reticulum from a R2454G heterozygote collected during a fulminant MH episode showed significantly higher affinity and density of [3H]ryanodine-binding sites compared to WTRyR1, but no differences in Ca2+, Mg2+, and caffeine modulation. In conclusion, an autosomal missense mutation in RyR1 is associated with MH in the horse, providing a screening test for susceptible individuals. [3H]ryanodine-binding analysis suggests that long-lasting changes in RyR1 conformation persists in vitro after the triggering event.