Modulation of DHPR Inactivation by RyR1 Activity in Mouse Skeletal Muscle Fibers

Malignant hyperthermia is a potentially fatal hypermetabolic state originating from excessive release of calcium stored in the sarcoplasmic reticulum (SR) of skeletal muscle. In most cases, MH susceptibility results from mutations in the type 1 ryanodine receptor (RyR1). In previous work (Andronache et al., PNAS 2009) we reported that heterozygous murine carriers of MH mutation Y524S (human Y522S) exhibit changes in steady state inactivation of the dihydropyridine receptor (DHPR), the sensor of the transverse tubular (TT) membrane potential. Availability curves were left shifted along the voltage axis suggesting that a feedback signal from RyR1 modulates DHPR inactivation. In the present study we investigated the hypothesis that junctional fluctuations of free Ca2+ concentration are involved in the feedback mechanism. We performed two-electrode voltage clamp experiments on enzymatically isolated toe muscle fibers of both WT and mutant mice (Y524S+/−) and measured L-type Ca2+ current and optical signals from fluorescent Ca2+ indicators. To test the hypothesis we applied conditions that would modify junctional Ca2+ levels. Millimolar concentrations of caffeine led to a left shift in the availability curve for L-type current indicating that drug-induced RyR1 hyperactivity can mimic the effect of the mutation. On the other hand, internal dialysis with an artificial solution containing 10 mM of BAPTA to effectively reduce the local Ca2+ transients near open RyR1 channels had little effect on the difference in steady state inactivation between WT and mutant fibers. We conclude that the altered inactivation depends on RyR1 hyperactivity but does not require the continuous presence of local Ca2+ fluctuations within the junctional gap separating TT and SR.