Involvement of multiple intracellular release channels in calcium sparks of skeletal muscle

In many types of muscle, intracellular Ca²⁺ release for contraction consists of brief Ca²⁺ sparks. Whether these result from the opening of one or many channels in the sarcoplasmic reticulum is not known. Examining massive numbers of sparks from frog skeletal muscle and evaluating their Ca²⁺ release current, we provide evidence that they are generated by multiple channels. A mode is demonstrated in the distribution of spark rise times in the presence of the channel activator caffeine. This finding contradicts expectations for single channels evolving reversibly, but not for channels in a group, which collectively could give rise to a stereotyped spark. The release channel agonists imperatoxin A, ryanodine, and bastadin 10 elicit fluorescence events that start with a spark, then decay to steady levels roughly proportional to the unitary conductances of 35%, 50%, and 100% that the agonists, respectively, promote in bilayer experiments. This correspondence indicates that the steady phase is produced by one open channel. Calculated Ca²⁺ release current decays 10- to 20-fold from spark to steady phase, which requires that six or more channels be open during the spark.