Functional Analysis of Ryanodine Receptor Carrying Malignant Hyperthermia Associated Mutations

Abstract

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 200 mutations in the RyR1 gene have been reported in MH patients. Most of those mutations have been found in three “hot spots” regions of RyR1. However, there were only a few experimental results confirming those mutations being responsible for the increment of the CICR sensitivities. 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 mutations 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 were acquired using an inverted microscope equipped with a objective, a cooled CCD camera and a polychromatic illumination system. We characterized the functional mutations on RyR1 in HEK293 cells. It was found that disease-associated 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.