Effects of Dantrolene on Steps of Excitation-Contraction Coupling in Mammalian Skeletal Muscle Fibers

Abstract

The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca2+]i) by inhibiting the release of calcium from the SR. The suppression of [Ca2+]i was dose-dependent, with no effect at or below 1 μM and a 53 ± 8% (mean ± SEM, n = 9, cut fibers) attenuation at 0 mV with 25 μM of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 ± 8%) and of the early peak component (46 ± 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (ICa) were left, essentially, unaltered. However, the inactivation of ICa was slowed fourfold, and the conductance was reduced from 200 ± 16 to 143 ± 8 SF−1 (n = 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 ± 10% (n = 3) at 12 μM. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30–50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.

Dantrolene requires Mg 2+ to arrest malignant hyperthermia

“…13). This is necessary in bilayer studies to remove the resting inhibition on the RyR and induce a measureable opening probability in the isolated channel.We hypothesized that the lack of dantrolene action on single RyRs was due to the fact that the experiments were carried out either in the absence of or in low free [Mg 2+ ] cyto (26,30,31). In contrast, the intact fiber experiments, where dantrolene inhibited the RyR, maintained the endogenous level of free [Mg 2+ ] cyto (26).…”

“…Functional studies on muscle fibers suggested the RyR as the potential target for the action of dantrolene (25,26), and respective binding sites were mapped to the protein (27)(28)(29). However, no action of dantrolene was found in most studies on single RyRs incorporated into lipid bilayers (26,30,31).…”

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

“…Although dantrolene has been shown to suppress the ryanodine binding activity of the SR (13,14), this finding is not universal (15). Dantrolene (1-5 M) has been reported to have a biphasic effect on the open probability of RyR1 channels in lipid bilayers, first activating and then blocking at nanomolar concentrations (16), but others have not been able to see any effect of this drug on single channels (3). Importantly, dantrolene has been shown to at least partially restore the normal properties of RyR1 Ca 2ϩ channels in SR isolated from MH-susceptible pigs (1,17,18).…”

“…1 MH is a potentially deadly, pharmacogenetically mediated, hypermetabolic response to volatile anesthetics that results from unregulated intramyoplasmic Ca 2ϩ release (1). The drug is known to inhibit excitation-contraction coupling of skeletal muscle (2) by suppressing depolarization-induced sarcoplasmic reticulum (SR) Ca 2ϩ release in normal and MH-susceptible skeletal muscle without affecting voltage sensor activation (3). In MH, the voltage dependence of contractile activation is shifted to lower voltages (4), whereas in the presence of clinical concentrations of dantrolene, i.e.…”

Targeting ryanodine receptors for anti-arrhythmic therapy

“…Dantrolene has been shown to directly bind to the N-terminus of RyR1 and to prevent SR Ca 2+ leak in skeletal muscle, thereby improving clinical outcomes [29] . Dantrolene is believed to stabilize interdomain interactions between the N-terminal and central domains of RyR1 [30] and RyR2 [31] , although the effects of dantrole on single RyR channels remains controversial [32] .…”