Calcium release flux underlying Ca2+ sparks of frog skeletal muscle

An algorithm for the calculation of Ca2+ release flux underlying Ca2+ spark s (Blatter, L.A., j. Huser, and E. Rios. 1997. Proc. Natl. Acad. Sci. USA. 94:4176-4181) was modified and applied to sparks obtained by confocal micro scopy in single frog skeletal muscle fibers, which were voltage clamped in a two-Vaseline gap chamber of permeabilized and immersed in fluro-3-contain ing internal solution. The performance of the algorithm was characterized o n sparks obtained by simulation of fluorescence due to the release of Ca2from a spherical source, in a homogeneous three-dimensional space that cont ained components representing cytoplasmic molecules and Ca2+ removal proces ses. Total release flux of current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simu lation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0.7 mu m. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically i n a representative voltage clamp experiment, the algorithm reported a relea se current of 16.9 pA, coming from a source of 0.5 mu m, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers. so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current re ported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concent ration, was 8 pA. Such a current of the level recorded in bilayer experimen ts. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the sourc e for real sparks. This may again indicate a multichannel origin of sparks, of could reflect limitations in spatial resolution.