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 ...
The Ca(2+)-gated Ca2+ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulphonate (CHAPS) in the presence and absence of 3[H]-ryanodine and centrifuged through linear sucrose gradients. A single 3[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca(2+)- and monovalent-ion-conducting channel (110 pS in 100 mM Ca2+, 360 pS in 250 mM K+). The channel was activated by micromolar Ca2+, modulated by millimolar adenosine triphosphate, Mg2+ and the Ca(2+)-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca(2+)-gated Ca2+ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca2+ release channel complexes.
The Ca" release channel of rabbit skeletal muscle sarcoplasmic reticulum (SR) can be phosphorylated by membrane associated protein kinase(s) utilizing endogenously synthesized or exogenously added ATP. The channel protein has been enriched in non-phosphorylated and phosphorylated form from heavy SR following solubilization with CHAPS (3-[(3cholamidopropyI)dimethylammonio-l-propane-sulfonate) and ultracentrifugation on a linear sucrose/CHAPS gradient. Reconstitution of the isolated channels into planar bilayers shows that phosphorylation enhances the open probability by increasing the sensitivity towards micromolar Ca" and ATP. The phosphorylation induced enhancement of the channel activity can be reversed by purified protein phosphatase 2A.
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