The Spark and Its Ember: Separately Gated Local Components of Ca²⁺ Release in Skeletal Muscle

Amplitude, spatial width, and rise time of Ca²⁺ sparks were compared in frog fast-twitch muscle, in three conditions that alter activation of release channels by [Ca²⁺]. A total of ∼17,000 sparks from 30 cells were evaluated. In cells under voltage clamp, caffeine (0.5 or 1 mM) increased average spark width by 28%, rise time by 18%, and amplitude by 7%. Increases in width were significant even among events of the same rise time. Spontaneous events recorded in permeabilized fibers with low internal [Mg²⁺] (0.4 mM), had width and rise times greater than in reference, and not significantly different than those in caffeine. The spark average in reference rides on a continuous fluorescence “ridge” and is continued by an “ember,” a prolongation of width ∼1 μm and amplitude <0.2, vanishing in ∼100 ms. Ridge and ember were absent in caffeine and in permeabilized cells. Exposure of voltage-clamped cells to high internal [Mg²⁺] (7 mM) had effects opposite to caffeine, reducing spark width by 26% and amplitude by 27%. In high [Mg²⁺], the ember was visible in individual sparks as a prolongation of variable duration and amplitude up to 1.2. Based on simulations and calculation of Ca²⁺ release flux from averaged sparks, the increase in spark width caused by caffeine was interpreted as evidence of an increase in radius of the release source—presumably by recruitment of additional channels. Conversely, spark narrowing suggests loss of contributing channels in high Mg²⁺. Therefore, these changes in spark width at constant rise times are evidence of a multichannel origin of sparks. Because ridge and ember were reduced by promoters of Ca²⁺-dependent activation (caffeine, low [Mg²⁺]) and became more visible in the presence of its inhibitors, they are probably manifestations of Ca²⁺ release directly operated by voltage sensors.