Non-Technical Summary  Calcium ions flowing through the type 1 ryanodine receptor (RyR1) calcium channel trigger contraction of skeletal muscle cells. Close to 300 mutations of the gene encoding RyR1 are responsible for several muscular diseases in human. Properties of pathological mutant RyR1s have so far been essentially assessed from studies in cultured cells and in differentiated native muscle fibres from a few available transgenic mouse models. We show that functional properties of mutant RyR1s can be reliably assessed following in vivo expression in adult mouse muscles. The Y523S, R615C and R2163H RyR1 mutants produce a similar over-sensitive activation of the calcium flux whereas I4897T RyR1 mutants are responsible for a depressed Ca2+ flux. The alterations appear to result from inherent modifications of RyR1 channel function and not from indirect changes in the muscle fibre homeostasis. The present strategy will help understand the physio-pathological defects underlying alterations of muscle function in affected patients.

Abstract  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 Ca2+ transients demonstrated alterations of sarcoplasmic reticulum (SR) Ca2+ release spatially correlated with the presence of exogenous RyR1s. The Y523S, R615C and R2163H RyR1 MHS-related mutants were associated with enhanced peak Ca2+ release for low and moderate levels of depolarization, whereas the I4897T CCD mutant produced a chronic reduction of peak SR Ca2+ release. For example, peak Ca2+ 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 Ca2+ 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 Ca2+ content. Furthermore, the results indicate that RyR1 channel density is tightly regulated even under the present conditions of forced exogenous expression.