Novel Mutations in C-terminal Channel Region of the Ryanodine Receptor in Malignant Hyperthermia Patients

Abstract

ABSTRACT-Malignant hyperthermia (MH) is a pharmacogenetical complication of general anesthesia resulting from abnormal Ca 2+ -induced Ca 2+ release (CICR) via the type 1 ryanodine receptor (RyR1) in skeletal muscles. In this study, we analyzed the genomic DNAs prepared for determination of all the 106 exons of the RyR1 gene from blood samples donated by two MH patients with extremely high CICR rates in their biopsied skeletal muscles and a clear history of MH incidence. Two novel point mutations were found in the exons 96 and 101 with alterations in the coded amino acids within the C-terminal channel region, i.e., Pro4668 to Ser and Leu4838 to Val. The latter mutation was found in both MH patients. Rabbit RyR1 channels carrying corresponding mutations were expressed in CHO cells for functional assay. It was found that the L to V but not the P to S mutation of the RyR1 resulted in enhanced Ca 2+ release activity. These results indicate that the L4838V mutation is responsible for the MH incidence. The L4838V mutation is unique because it is the mutation first found within a hydrophobic transmembrane segment of the channel region and should provide further information on the function of the RyR1 as well as for genetic diagnosis of MH.Keywords: Calcium, Sarcoplasmic reticulum, Skeletal muscle, Mutation, General anesthesia Malignant hyperthermia (MH) is a serious complication of general anesthesia triggered by commonly used inhalational anesthetics (1 -3). The typical symptoms include a rapid increase in body temperature and induction of a hypermetabolic state with skeletal muscle rigidity. These symptoms, if not immediately reversed, result in death of the patient. The predisposition to MH is inherited as an autosomal dominant trait. The first hint of the nature of the abnormality in MH patients was obtained when Kalow et al. (4) demonstrated that the patients' muscle has a higher sensitivity than normal muscle to the contracture-inducing action of caffeine applied with or without halothane as a potentiator. It has been shown that caffeine or halothane accelerates the Ca 2+ -induced Ca 2+ release (CICR) mechanism in the sarcoplasmic reticulum (SR) of striated muscle (5, 6). Endo et al. (6) demonstrated that both Ca 2+ sensitivity and the maximum rate of CICR in an MH patient were much higher than those in control human muscles. Thus, induction of muscle contracture due to pharmacological enhancement of CICR by inhalational anesthetics in addition to a genetic basis of CICR acceleration was considered to be the primary cause of elevated body temperature in MH episodes. Furthermore, a cluster analysis based on the rates of CICR in 84 patients suspected of having MH objectively classified the patients into three groups, and a strong correlation was identified between the clinical signs of MH during anesthesia and the acceleration of CICR (7).The channel protein responsible for the CICR was isolated through its high-affinity binding of the plant alkaloid ryanodine; hence it is called the ryanodine receptor (RyR). The...

The Role of a Brain-specific Splice Variant of Ryanodine Receptor Type 1

“…Using showing much stronger uorescent signal in the cytoplasm than in the nucleus, as previously reported [12][13][14] . These results indicated highly reproducible expression of exogenous RyR1 at the expected location from cell to cell and from clone to clone using this experimental system.…”

“…View, CA or pBI bidirectional expression vector Clontech 13 . However, the nal size of these expression vectors 250,000 bp was too large for the ef cient expression of both recombinant proteins.…”

Ryanodine receptor type 1 gene mutations found in the Canadian malignant hyperthermia population

“…10 The p.Val4847Leu that co-segregated well with an MHS phenotype within a large French-Canadian family, C-27, is positioned within the putative transmembrane segment and maps to the HS3b ''mutation hot spot'' region in RyR1. 13,38 At least three other closely spaced MH/CCD mutations are located within the same transmembrane segment 33,39,40 (Fig. 3).…”

Calcium-Induced Calcium Release in Skeletal Muscle

“…Certain MH mutations are believed to cause partial domain unzipping and to destabilize the channel (4,183). However, mutations in the COOH-terminal region, which is probably not directly related to the domain switch, also cause MH (17,179,208).…”