Effects of imperatoxin A on local sarcoplasmic reticulum Ca2+ release in frog skeletal muscle

We have investigated the effects of imperatoxin A (IpTx(a)) on local calciu m release events in permeabilized frog skeletal muscle fibers, using laser scanning confocal microscopy in linescan mode. IpTx(a) induced the appearan ce of Ca2+ release events from the sarcoplasmic reticulum that are similar to 2 s and have a smaller amplitude (31 +/- 2%) than the "Ca2+ sparks" norm ally seen in the absence of toxin, The frequency of occurrence of long-dura tion imperatoxin-induced Ca2+ release events increased in proportion to IpT x(a) concentrations ranging from 10 nM to 50 nM. The mean duration of imper atoxin-induced events in muscle fibers was independent of toxin concentrati on and agreed closely with the channel open time in experiments on isolated frog ryanodine receptors (RyRs) reconstituted in planar lipid bilayer, whe re IpTx(a) induced opening of single Ca2+ release channels to prolonged sub conductance states. These results suggest involvement of a single molecule of IpTx(a) in the activation of a single Ca2+ release channel to produce a long-duration event. Assuming the ratio of full conductance to subconductan ce to be the same in the fibers as in bilayer, the amplitude of a spark rel ative to the long event indicates involvement of at most four RyR Ca2+ rele ase channels in the production of short-duration Ca2+ sparks.