Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B‐lymphocytes |
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Authors: | Alberto Zullo Werner Klingler Claudia De Sarno Marina Ferrara Giuliana Fortunato Giuseppa Perrotta Elvira Gravino Rosella Di Noto Frank Lehmann‐Horn Werner Melzer Francesco Salvatore Antonella Carsana |
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Affiliation: | 1. CEINGE Biotecnologie Avanzate, Naples, Italy;2. Department of Biochemistry and Medical Biotechnology, University of Naples “Federico II”;3. Institute of Applied Physiology, Ulm University, Germany;4. A. Zullo and W. Klingler contributed equally to this work.;5. Department of Anesthesiology, Ulm University, Germany;6. Communicated by William S. Oetting;7. Department of Surgical, Anesthesiological, Resuscitation and Emergency Sciences, University of Naples “Federico II”, Italy;8. Clinica Pineta Grande, Castelvolturno, Caserta, Italy |
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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 RYR1Cys4664Arg 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 Ca2+‐induced metabolic changes in cells harboring mutant RYR1 channels. © 2009 Wiley‐Liss, Inc. |
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Keywords: | RYR1 malignant hyperthermia proton release B‐lymphocytes calcium channel |
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