Differential sensitivity to perchlorate and caffeine of tetracaine-resistant Ca2+ release in frog skeletal muscle
1. Fluo-3 fluorescence associated with Ca2+ release was recorded with confocal microscopy in single muscle fibres. Clamp depolarization to -65 or -60 mV elicited Ca2+ sparks with amplitudes and spatial widths distributed approximately normally, with mean values of 0.79 of resting fluorescence and 0.8 micron (S.D., 0.17 and 0.2 micron; n = 193), respectively. Given these distributions, events of amplitude less than 0.45 or width less than 0.4 micron are unlikely to be sparks. 2. Low voltage depolarization (-72 mV) elicited only one spark per triad every 6 s, but generated a relative increase in fluorescence at triads of 0.05. This increase must therefore have been due to events smaller than sparks. 3. The variance/mean ratio of triadic fluorescence gradients averaged 0.11 at low voltages and increased severalfold at the higher voltages at which sparks appeared, indicating the existence of at least two event amplitudes. 4. Tetracaine (200 microM) reversibly abolished sparks and the early peak of Ca2+ release at all voltages. In its presence, discrete events were smaller than the spark criterion, and triadic gradients had a variance/mean ratio of 0.11. 5. The phenylalkylamine D600 (2 microM) reduced release at all voltages, abolishing sparks and the peak of Ca2+ release at low but not at high voltages. 6. The parallel abolition of all sparks and the peak of Ca2+ release indicates that both phenomena are activated by Ca2+. The restoration of sparks by voltage in D600 suggests that release in small events provides the trigger Ca2+ for activation of sparks.