Time course of individual Ca2+ sparks in frog skeletal muscle recorded at high time resolution

Discrete Ca2+ release events (Ca2+ "sparks") were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning con focal microscope operating in line-scan mode (63 mu s per line). Fibers loa ded with the Ca2+ indicator fluo-3 were voltage clamped a a holding potenti al of 0 mV, briefly reprimed at -90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this hig h time resolution system, it was possible to record individual Ca2+ sparks at similar to 30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5-2 ms per li ne confocal systems. Analysis of the time course of individual identified e vents indicates that fluorescence begins to rise rather abruptly at the sta rt of the spark, continues to rise at a slightly decreasing rate to a relat ively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 +/- 0.1 ms, and there was no corre lation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual spa rks having similar rise times gave a lower noise representation of the spar ks, consistent with the time course of individual events. In theory, the ri sing phase of a spark provides a lower bound estimation of the time that Ca 2+ ions are being released by the sarcoplasmic reticulum Ca2+ channel(s) ge nerating the spark. The observed time course of fluorescence suggests that the Ca2+ release underlying a spark could continue at a fairly constant rat e throughout the rising phase of the spark, and then stop rather abruptly a t the time of the peak.