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

Abstract

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.

Figure 1.

Changes in Ca²⁺ concentration in response to halothane and temperature elevation. [Ca²⁺] was monitored in 1-min intervals over a period of 29 min. (A) Averaged data of 14 WT and 20 YS fibers. 3% Halothane was applied at minute 2. 10 min later, the temperature was raised from 25°C to 35°C. YS cells showed a significantly larger change than WT cells. The alterations were reversible. Error bars indicate SEM. (B) Representative example of Ca²⁺ removal model fitting (red lines) to a fura-2 fluorescence ratio trace resulting from repetitive field stimulation of action potentials in a WT fiber. (C) Ca²⁺ release flux derived from the measurement in B.

Figure 2.

Halothane slows the relaxation of action potential–induced Ca²⁺ transients. Action potential–triggered Ca²⁺ signals were recorded at the instances indicated by the dashed vertical lines in Fig. 1 A. (A and B) Free Ca²⁺ transient before (continuous line) and after (dashed line) application of 3% halothane. (C and D) Ca²⁺ release flux (normalized to the peak) calculated from the data in A and B. The traces are averaged signals from 14 (WT) and 20 (YS) fibers, respectively. Mean best-fit parameters determined from the WT fibers in halothane-free solution and used for the calculations were as follows: [S]_tot = (2.72 ± 0.30) mM, k_on,S, = (4.80 ± 0.61) · 10⁶ M⁻¹s⁻¹, k_off,S = (0.654 ± 0.020) s⁻¹, and k_NS = (1.20 ± 0.079) · 10⁴ s⁻¹; n = 14.

Figure 3.

Small voltage changes strongly modulate the halothane response in YS fibers. Representative recordings in a WT and a YS fiber showing changes in free [Ca²⁺] in response to a small concentration of halothane (0.2%) and small variations of the membrane potential in voltage-clamped fibers. A 10-mV depolarization and hyperpolarization (for 5 min each) from the holding potential of −80 mV was applied before and during the application of halothane. In the drug-free condition and in the WT fiber after halothane application, the voltage changes had little effect on the [Ca²⁺] level. In contrast, the YS fiber responded strongly to halothane and to the subsequent small voltage alterations. The blue lines show linear fits to three recordings before and after the voltage changes. The lines were subtracted to determine the effect of the voltage change (see Fig. 4).

Figure 4.

Halothane alters the voltage sensitivity of the calcium level near the resting membrane potential. Summarized results of experiments of the kind shown in Fig. 3. In each case, a sloping line was fitted to 3 min before and after the voltage change and subtracted (see blue lines in Fig. 3) to separate the response to voltage from the halothane-induced free [Ca²⁺] level. Data with 0.2 and 0.5% of halothane were pooled. (A and C) YS and WT, respectively, before halothane application. (B and D) Same experiments during halothane application. Averaged data from 13 WT and 12 YS fibers. Error bars indicate SEM.

Figure 5.

Halothane alters the Ca²⁺ response to voltage pulse activation. Averaged free [Ca²⁺] traces of WT (gray shapes) and YS fibers (red shapes) demonstrating changes in baseline [Ca²⁺] and the response to short (50-ms) depolarizations from a holding potential of −80 mV to −50 and −30 mV. Pulses are indicated underneath the traces and were separated by 1-s intervals. The effect of 0.2 and 0.5% halothane is shown.

Figure 6.

Changes in depolarization-activated Ca²⁺ release flux caused by halothane. Mean Ca²⁺ release flux traces from the same set of measurements as in Fig. 5. Note that halothane causes a decrease in the ratio of the respective pulse responses and a marked slowing in the deactivation of release. For quantitative comparison, see Table 2.

Figure 7.

Altered voltage dependence of Ca²⁺ release caused by halothane. (A) Ratio between the activation at V₁ = −50 mV and V₂ = −30 mV assuming a standard Boltzmann-type activation curve [B(V)]: ratio(V_0.5) = B(V₂)/B(V₁) = {1 + exp[(V₁ − V_0.5)/k]}/{1 + exp[(V₂ − V_0.5)/k]}. The vertical lines point to the V_0.5 values estimated by iteratively solving the equation (−11.6, −38.1, and −45.6 mV for WT and −19.7, −49.7, and −50.0 mV for YS assuming a steepness parameter k = 6.6 mV). Black symbols, WT; red symbols, YS. Numbers indicate halothane percentage. (B) Activation as a function of voltage for the six different conditions shown in Fig. 6 derived from the analysis in A.