Defects in Ca2+ release associated with local expression of pathological ryanodine receptors in mouse muscle fibres.

Mutations of the gene encoding the type 1 ryanodine receptor (RyR1) are associated with skeletal muscle disorders including malignant hyperthermia susceptibility (MHS) and central core disease (CCD). We used in vivo expression of EGFP-RyR1 constructs in fully differentiated mouse muscle fibres to characterize the function of several RyR1 mutants. Wild-type and Y523S, R615C, R2163H and I4897T mutants of RyR1 were separately expressed and found to be present within restricted regions of fibres with a pattern consistent with triadic localization. Confocal measurements of voltage-clamp-activated myoplasmic Ca(2+) transients demonstrated alterations of sarcoplasmic reticulum (SR) Ca(2+) release spatially correlated with the presence of exogenous RyR1s. The Y523S, R615C and R2163H RyR1 MHS-related mutants were associated with enhanced peak Ca(2+) release for low and moderate levels of depolarization, whereas the I4897T CCD mutant produced a chronic reduction of peak SR Ca(2+) release. For example, peak Ca(2+) release in response to a depolarization to -20 mV in regions of fibres expressing Y523S and I4897T was 2.0 ± 0.3 (n = 9) and 0.46 ± 0.1 (n = 5) times the corresponding value in adjacent, non-expressing regions of the same fibre, respectively. Interestingly no significant change in the estimated total amount of Ca(2+) released at the end of large depolarizing pulses was observed for any of the mutant RyR1 channels. Overall, results are consistent with an 'inherent' increase in RyR1 sensitivity to activation by the voltage sensor for the MHS-related RyR1 mutants and a partial failure of voltage-gated release for the CCD-related I4897T mutant, that occur with no sign of change in SR Ca(2+) content. Furthermore, the results indicate that RyR1 channel density is tightly regulated even under the present conditions of forced exogenous expression.