Ryanodine receptors, located in the sarcoplasmic/endoplasmic reticulum (SR/ER) membrane, are required for intracellular Ca2+ release that is involved in a wide range of cellular functions. Malignant hyperthermia (MH) is a pharmacogenetical complication of general anesthesia resulting from abnormal Ca2+-induced Ca2+ release (CICR) via the type 1 ryanodine receptor (RyR1) in skeletal muscles. The typical symptoms include a rapid increase in body temperature and induction of a hypermetabolic state with skeletal muscle rigidity. More than 100 mutations in the RyR1 gene have been reported in MH patients. Most of those mutations have been found in three “hot spots” regions (#1-614, #2129-2458 and #4637-4973 as the amino acid numbers) of RyR1. However, there were only a few experimental results confirming those mutations being responsible for the increment of the CICR sensitivities, since such a long cDNA of RyR1 not only required much complicated procedures for making desired mutations but also caused its low transfection efficiency of the mutant DNAs. We improved the method for making MH mutants in the cDNA of RyR1. We characterized the functional mutations on RyR1 in non-muscle cells, specifically HEK293 cells with tetracycline-regulated RyR1 expression. Rabbit RyR1 channels carrying corresponding Japanese mutations (L13R, Q155K, R163C, D166G, R533H, etc.) were expressed in HEK293 cells for functional assay. HEK293 cells were loaded at room temperature with fura-2 AM in physiological salt solution. Fluorescence images at >420nm were acquired using an inverted microscope equipped with a objective, a cooled CCD camera and a polychromatic illumination system at a rate of one frame every 1 or 2 s. It was found that R163C and Q155K mutations of the RyR1 resulted in enhanced Ca2+ release activity, therefore these mutations would be responsible for the MH incidence. These results suggest that exploration of the functional mutations of RyR1 is probably effective in preventive diagnosis of patients associated with MH disease.
37th Congress of IUPS (Birmingham, UK) (2013) Proc 37th IUPS, PCD246
Poster Communications: Exploration of functional mutations of ryanodine receptor in malignant hyperthermia
T. Yamazawa1,4, H. Oyamada2, T. Murayama3, N. Kurebayashi3, K. Oguchi2, T. Sakurai3, M. Iino4, S. Takemori1
1. Department of Molecular Physiology, The Jikei University School of Medicine, Minato-ku, Tokyo, Japan. 2. Department of Pharmacology, School of Medicine, Showa University, Shinagawa-ku, Tokyo, Japan. 3. Department of Pharmacology, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan. 4. Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
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Where applicable, experiments conform with Society ethical requirements.