Cross-Influence of Halothane and Voltage on Intracellular Ca2+ in Isolated Muscle Fibers of Mice Expressing Human RyR1 Mutation Y522S

Abstract

We studied the effect of halothane and its interference with membrane voltage in malignant hyperthermia (MH) susceptible muscle by using enzymatically isolated single fibers (m. interosseus) of knock-in mice with the RyR1 mutation Y524S (human MH mutation Y522S). To investigate anesthetic- and voltage-triggered changes of Ca2+ concentration, we measured fluorescence from fibers loaded with Fura-2-AM. Similar as in the diagnostic in-vitro contracture test (IVCT) for MH susceptibility, cells were exposed to different halothane-containing solutions using a vaporizer system. The percentage of halothane in the gas phase was determined using infrared photometry. The temporal change of the halothane concentration in the recording chamber was checked using gas chromatography. In Krebs-Ringer's solution at room temperature, a step from 0 to 0.5% halothane in the gas phase caused a clear increase in resting Ca2+ concentration in most of the investigated fibers of mutant mice but very little response in fibers of WT littermates. The Ca2+ response was phasic: after reaching a peak the Ca2+ signal decreased at a rate of about 10%/min indicating either reduced sensitivity to halothane or inhibition of Ca2+ release after activation. A likely explanation is Ca2+-induced inactivation of RyR1. In further experiments, we applied halothane to single fibers which were voltage-clamped using two intracellular microelectrodes. Depolarizing pulses to different membrane potentials from a holding potential of −80 mV revealed a strong shift in the voltage threshold for activation of Ca2+ release to more negative potentials. Correspondingly, hyperpolarizing steps led to a rapid partial recovery from the halothane-induced increase in basal Ca2+ concentration. These results demonstrate reciprocal effects of a volatile anesthetic drug and membrane voltage on Ca2+ release in MH-susceptible skeletal muscle.