title: Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B-lymphocytes.
abstract: Mutations in the RYR1 gene are linked to malignant hyperthermia (MH), central core disease and multi-minicore disease. We screened by DHPLC the RYR1 gene in 24 subjects for mutations, and characterized functional alterations caused by some RYR1 variants. Three novel sequence variants and twenty novel polymorphisms were identified. Immortalized lymphoblastoid cell lines from patients with RYR1 variants and from controls were stimulated with 4-chloro-m-cresol (4-CmC) and the rate of extracellular acidification was recorded. We demonstrate that the increased acidification rate of lymphoblastoid cells in response to 4-CmC is mainly due to RYR1 activation. Cells expressing RYR1 variants in the N-terminal and in the central region of the protein (p.Arg530His, p.Arg2163Pro, p.Asn2342Ser, p.Glu2371Gly and p.Arg2454His) displayed higher activity compared with controls; this could account for the MH-susceptible phenotype. Cell lines harboring RYR1(Cys4664Arg) were significantly less activated by 4-CmC. This result indicates that the p.Cys4664Arg variant causes a leaky channel and depletion of intracellular stores. The functional changes detected corroborate the variants analyzed as disease-causing alterations and the acidification rate measurements as a means to monitor Ca(2+)-induced metabolic changes in cells harboring mutant RYR1 channels.
authors.affiliation: CEINGE Biotecnologie Avanzate, Naples, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Zullo
authors.forename: Werner
authors.initials: W
authors.lastname: Klingler
authors.forename: Claudia
authors.initials: C
authors.lastname: De Sarno
authors.forename: Marina
authors.initials: M
authors.lastname: Ferrara
authors.forename: Giuliana
authors.initials: G
authors.lastname: Fortunato
authors.forename: Giuseppa
authors.initials: G
authors.lastname: Perrotta
authors.forename: Elvira
authors.initials: E
authors.lastname: Gravino
authors.forename: Rosella
authors.initials: R
authors.lastname: Di Noto
authors.forename: Frank
authors.initials: F
authors.lastname: Lehmann-Horn
authors.forename: Werner
authors.initials: W
authors.lastname: Melzer
authors.forename: Francesco
authors.initials: F
authors.lastname: Salvatore
authors.forename: Antonella
authors.initials: A
authors.lastname: Carsana
mesh_terms: D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003408:Cresols / Q000494:pharmacology; D004252:DNA Mutational Analysis; D005110:Extracellular Space / Q000737:chemistry / Q000187:drug effects; D005192:Family Health; D005260:Female; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing; D014644:Genetic Variation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008305:Malignant Hyperthermia / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D009154:Mutation; D020914:Myopathies, Structural, Congenital / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D010375:Pedigree; D011110:Polymorphism, Genetic; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000502:physiology
chemical_list: D003408:Cresols; D019837:Ryanodine Receptor Calcium Release Channel; C006984:chlorocresol