Voltage modulates halothane-triggered Ca²⁺ release in malignant hyperthermia-susceptible muscle

Malignant hyperthermia (MH) is a fatal hypermetabolic state that may occur during general anesthesia in susceptible individuals. It is often caused by mutations in the ryanodine receptor RyR1 that favor drug-induced release of Ca²⁺ from the sarcoplasmic reticulum. Here, knowing that membrane depolarization triggers Ca²⁺ release in normal muscle function, we study the cross-influence of membrane potential and anesthetic drugs on Ca²⁺ release. We used short single muscle fibers of knock-in mice heterozygous for the RyR1 mutation Y524S combined with microfluorimetry to measure intracellular Ca²⁺ signals. Halothane, a volatile anesthetic used in contracture testing for MH susceptibility, was equilibrated with the solution superfusing the cells by means of a vaporizer system. In the range 0.2 to 3%, the drug causes significantly larger elevations of free myoplasmic [Ca²⁺] in mutant (YS) compared with wild-type (WT) fibers. Action potential-induced Ca²⁺ signals exhibit a slowing of their time course of relaxation that can be attributed to a component of delayed Ca²⁺ release turnoff. In further experiments, we applied halothane to single fibers that were voltage-clamped using two intracellular microelectrodes and studied the effect of small (10-mV) deviations from the holding potential (-80 mV). Untreated WT fibers show essentially no changes in [Ca²⁺], whereas the Ca²⁺ level of YS fibers increases and decreases on depolarization and hyperpolarization, respectively. The drug causes a significant enhancement of this response. Depolarizing pulses reveal a substantial negative shift in the voltage dependence of activation of Ca²⁺ release. This behavior likely results from the allosteric coupling between RyR1 and its transverse tubular voltage sensor. We conclude that the binding of halothane to RyR1 alters the voltage dependence of Ca²⁺ release in MH-susceptible muscle fibers such that the resting membrane potential becomes a decisive factor for the efficiency of the drug to trigger Ca²⁺ release.