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.