Effects of dantrolene on steps of excitation-contraction coupling in mammalian skeletal muscle fibers

The effects of the muscle relaxant dantrolene on steps of excitation-contra ction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurement s, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the do uble Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium conc entration ([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 muM and it 53 +/-8% (mean +/- SEM, n=9, cut fibers) attenuation at 0 mV wit h 25 muM of extracellularly applied dantrolene. The drug was not found to b e more effective if injected than if applied extracellularly. Calculating t he 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 wi th the activation of the voltage sensor in as much as the voltage dependenc e of both intramembrane charge movements and the L-type calcium currents (k a) were left, essentially, unaltered. However, the inactivation of 1,, 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 ef flux from heave, SR vesicles by 44 +/- 10% (n=3) at 12 muM. On the other ha nd, dantrolene failed to affect the isolated RYR incorporated into lipid bi layers. 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 si te for dantrolene to be on the SR membrane, but be distinct from the purifi ed RYR itself.