Functional analysis of ryanodine receptor type 1 p.R2508C mutation in exon 47.

PURPOSE: Malignant hyperthermia (MH) is a pharmacogenetic disorder of intracellular calcium homeostasis with an autosomal dominant inheritance. Most of the reported mutations in exon 47 were identified in Asian patients. However, no functional analysis of p.R2508C has been performed. We therefore conducted a functional analysis of the mutation by altering calcium homeostasis in human embryonic kidney (HEK) 293 cells transfected with the p.R2508C mutation in exon 47 of the ryanodine receptor 1 (RYR1).

METHODS

The entire RYR1 coding region from genomic DNA, which was extracted from the biopsied muscle specimens of two patients, was sequenced. The p.R2508C mutation was introduced into rabbit RYR1 cDNA, and wild-type or p.R2508C mutant cDNAs were transfected into HEK-293 cells. Using the calcium-sensitive probe Fura 2, we utilized the 340/380 nm ratio to analyze alterations in calcium homeostasis following treatment with caffeine and 4-chloro-m-cresol (4CmC).

RESULTS

Genetic analysis revealed a C-->T point mutation of RYR1 exon 47 at position 7522, resulting in an amino acid exchange of arginine for cysteine at amino acid 2508. The half-maximal activation concentrations (EC(50)) of caffeine and 4CmC for HEK-293 cells transfected with the p.R2508C mutation were 1.86 +/- 0.23 mM and 73.14 +/- 19.44 microM, while those for wild-type RYR1 were 2.62 +/- 0.23 mM and 179.31 +/- 35.23 microM, respectively.

CONCLUSION

We demonstrated that the transfected RYR1 mutant was more sensitive to caffeine and 4CmC than wildtype RYR1. These findings suggest that the p.R2508C mutation may be pathogenetic for susceptibility to MH.