Functional Characterization of a Distinct Ryanodine Receptor Mutation in Human Malignant Hyperthermia-susceptible Muscle

Abstract

Malignant hyperthermia is an inherited autosomal disorder of skeletal muscle in which certain volatile anesthetics and depolarizing muscle relaxants trigger an abnormally high release of Ca 2؉ from the intracellular Ca 2؉ store, the sarcoplasmic reticulum. In about 50% of cases, malignant hyperthermia susceptibility is linked to the gene encoding the skeletal muscle ryanodine receptor/Ca 2؉ release channel (RYR1). To date, eight point mutations have been identified in human RYR1. Although these mutations are thought to lead to an increased caffeine and halothane sensitivity in the contractile response of skeletal muscle, their functional consequences have not been investigated on the molecular level. In the present study, we provide the first functional characterization of a point mutation located in the central part of RYR1, Gly 2434 3 Arg. Using high affinity [3 H]ryanodine binding as the experimental approach, we show that this mutation enhances the sensitivity of RYR1 to activating concentrations of Ca 2؉ and to the exogenous and diagnostically used ligands caffeine and 4-chloro-m-cresol. In parallel, the sensitivity to inhibiting concentrations of Ca 2؉ and calmodulin was reduced, transferring the mutant Ca 2؉ release channel into a hyperexcitable state. Malignant hyperthermia (MH)1 is a pharmacogenetic skeletal myopathy of humans and swine and is one of the main causes of death due to anesthesia. Predisposed patients are at high risk for undergoing a fulminant MH crisis when exposed to certain volatile anesthetics and depolarizing muscle relaxants commonly used in anesthesia. A point mutation (Arg 615 3 Cys) in the skeletal muscle ryanodine receptor (RYR1), which functions as the sarcoplasmic reticulum (SR) Ca 2ϩ release channel, has been linked to porcine stress syndrome, an equivalent to human MH (1). Contrary to porcine stress syndrome, human MH is a genetically heterogeneous skeletal muscle disorder. Based on genetic linkage studies, three MH loci are known. The first has been mapped to chromosome 19q12-13.2 encompassing the gene that in homology with the animal model encodes the RYR1 (2, 3), the second has been mapped to chromosome 7q including the gene for the ␣ 2 /␦ subunit of the skeletal muscle dihydropyridine receptor (4), and the third has been mapped to chromosome 3q13.1 (5). To date, mutations have only been identified in RYR1 that count for approximately 50% of human MH cases (reviewed in Ref. 6). MH mutations seem to cluster in two areas of the RYR1 sequence. Six mutations have been localized in the N-terminal sequence of RYR1 containing a homologous mutation to that identified in porcine MH-susceptible (MHS) muscle. Two further mutations have been found in the central amino acid sequence. In vitro, all these mutations induce a hypersensitivity of biopsied muscle to the contracture-triggering agents caffeine and halothane. This enhanced sensitivity is exploited in the diagnostic in vitro contracture test (IVCT). According to the European test protocol (7), dissected muscle fiber bundles are ...

Cardiac and skeletal muscle disorders caused by mutations in the intracellular Ca2+ release channels

“…Richter et al (27) showed that the G2434R mutation identified in patients with MH enhances the sensitivity of RyR1 to Ca 2+ and caffeine. Similar findings were reported by Yang et al (28) when they expressed 6 common RyR1 mutants found in MH patients (R163C, G341R, R614C, R2163C, V2168M, and R2458H) in myotubes differentiated from cells lacking endogenous RyR1, and by Wehner et al (29) when they characterized RyR1 mutants in primary human myotubes cultured from carriers of RyR1 mutations.…”

Identification of a Key Determinant of Ryanodine Receptor Type 1 Required for Activation by 4-Chloro-m-cresol

“…Other Potential 4-CmC Effector Sites-Point mutations in the MH sensitivity region 2 of wtRyR1 (residues 2129 -2458) can either increase (11,17,18) or decrease (32) 4-CmC sensitivity, thus suggesting that this domain might also contain a potential 4-CmC effector site. However, these mutations also alter sensitivity to other RyR agonists such as caffeine and halothane, thus suggesting a more general effect on RyR activity.…”

“…Originally used as a preservative in commercial preparations of some intravenous drugs, including succinylcholine, 4-CmC can directly activate the RyR with 10 -25-fold higher potency compared with the more commonly used RyR activator, caffeine (14 -16). In addition, the sensitivity to activation by 4-CmC is increased for RyR1 channels containing point mutations that have been linked to MH (11,17,18). Indeed, because of its higher potency relative to caffeine, 4-CmC has been recommended as a supplemental test substance in the in vitro contracture test for diagnosing MH in humans (12).…”

Malignant Hyperthermia

“…Once a causative mutation is found in a family member, others may be tested for susceptibility by seeking out that mutation in the DNA. [67,69], and, indirectly, by protein release [70]. Systems using 1B5 dyspedic myotubes are more physiological as they constitutively express all the components of the skeletal muscle with the exception of RYR1 [67].…”