The properties of Ca2+ sparks in frog intact skeletal muscle fibers depolarized with 13 mM [K+] Ringer's are well described by a computational model with a Ca2+ source flux of amplitude 2.5 pA (units of current) and duration 4.6 ms (18 °C; Model 2 of Baylor et al., 2002). This result, in combination with the values of single-channel Ca2+ current reported for ryanodine receptors (RyRs) in bilayers under physiological ion conditions, 0.5 pA (Kettlun et al., 2003) to 2 pA (Tinker et al., 1993), suggests that 1–5 RyR Ca2+ release channels open during a voltage-activated Ca2+ spark in an intact fiber. To distinguish between one and greater than one channel per spark, sparks were measured in 8 mM [K+] Ringer's in the absence and presence of tetracaine, an inhibitor of RyR channel openings in bilayers. The most prominent effect of 75–100 μM tetracaine was an approximately sixfold reduction in spark frequency. The remaining sparks showed significant reductions in the mean values of peak amplitude, decay time constant, full duration at half maximum (FDHM), full width at half maximum (FWHM), and mass, but not in the mean value of rise time. Spark properties in tetracaine were simulated with an updated spark model that differed in minor ways from our previous model. The simulations show that (a) the properties of sparks in tetracaine are those expected if tetracaine reduces the number of active RyR Ca2+ channels per spark, and (b) the single-channel Ca2+ current of an RyR channel is ≤1.2 pA under physiological conditions. The results support the conclusion that some normal voltage-activated sparks (i.e., in the absence of tetracaine) are produced by two or more active RyR Ca2+ channels. The question of how the activation of multiple RyRs is coordinated is discussed.
展开▼
