Rapid kinetic analysis of the calcium-release channels of skeletal muscle sarcoplasmic reticulum: the effect of inhibitors.

During excitation of skeletal muscle fibers, Ca ions stored in the cisternal compartments of the sarcoplasmic reticulum (SR) are released to the cytosol within milliseconds. In this study, the kinetics of the fast release of Ca were analyzed by means of a newly developed rapid filtration apparatus. Isolated SR vesicles of cisternal origin were preloaded with 1 mM 45CaCl2, and Ca efflux was studied (between 20 and 1000 ms) after dilution into media of various composition. The effect of extravesicular Ca on the gating of the Ca-release channels and its susceptibility to the influence of drugs were thoroughly investigated. In the presence of 1 mM MgCl2 and 3 mM ATP, highest rates of Ca release were observed at a free Ca concentration between 1 and 50 microM. In the lower micromolar Ca range, compounds such as neomycin and FLA 365 inhibited the release monophasically and with an IC50 of 0.37 and 3.4 microM, respectively. At Ca concentrations between 10 and 50 microM, the inhibitors could not block Ca release effectively. Close analysis of the dose-response curves revealed a biphasic pattern, indicative of the presence of two substrates of the Ca-release channel, displaying high- and low-affinity binding sites for the inhibitors. Interestingly, neomycin (or ruthenium red) and FLA 365 at low concentrations acted synergistically and blocked release completely. The results indicate the existence of various open substates of the Ca channels that can be distinguished pharmacologically. Effective blockade of rapid Ca release requires inhibition of all substates coexisting under a given condition.