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Original Article

^a From the Department of Molecular Biophysics and Physiology, Rush University, Chicago, Illinois 60612
^b Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224
^c Departamento de Biofísica, Facultad de Medicina, Universidad de la República, G. Flores 2125, Montevideo, Uruguay
Department of Molecular Biophysics and Physiology, Rush University, 1750 W. Harrison Street, Chicago, IL 60612.Fax: 312-942-8711;

erios{at}rush.edu

An algorithm for the calculation of Ca²⁺ release flux underlying Ca²⁺ sparks (Blatter, L.A., J. Hüser, and E. Ríos. 1997. Proc. Natl. Acad. Sci. USA. 94:4176–4181) was modified and applied to sparks obtained by confocal microscopy in single frog skeletal muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3–containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca²⁺ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca²⁺ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0.7 µm. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 µm, 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 reported 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⁺ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca²⁺ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.

Key Words: sarcoplasmic reticulum • excitation–contraction coupling • confocal microscopy • computer algorithm

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