The Spark and Its Ember Separately Gated Local Components of Ca 2 1 Release in Skeletal Muscle

Amplitude, spatial width, and rise time of Ca 2 1 sparks were compared in frog fast-twitch muscle, in three conditions that alter activation of release channels by [Ca 2 1 ]. A total of z 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 [Mg 2 1 ] (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 z 1 m m and amplitude , 0.2, vanishing in z 100 ms. Ridge and ember were absent in caffeine and in permeabilized cells. Exposure of voltage-clamped cells to high internal [Mg 2 1 ] (7 mM) had effects opposite to caffeine, reducing spark width by 26% and amplitude by 27%. In high [Mg 2 1 ], 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 Ca 2 1 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 Mg 2 1 . 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 Ca 2 1 -dependent activation (caffeine, low [Mg 2 1 ]) and became more visible in the presence of its inhibitors, they are probably manifestations of Ca 2 1 release directly operated by voltage sensors. key words: excitation–contraction coupling • calcium sparks • ryanodine receptors • caffeine • magnesium I N T R O D U C T I O N In striated muscle, Ca 2 1 for contractile activation comes largely from the sarcoplasmic reticulum (SR) 1 through ryanodine receptors (Ryr), channel molecules susceptible to activation and inhibition by Ca 2 1 (Franzini-Armstrong and Protasi, 1997; Leong and MacLennan, 1998). In skeletal muscle, a sui-generis Ca 2 1 channel of the L-type, named the dihydropyridine receptor and resident in the transverse-tubule (T-tubule) membrane (Ríos and Brum, 1987; Tanabe et al., 1988), translates membrane depolarization (Hodgkin and Horowicz, 1960) to a mechanical influence that causes opening of Ryrs (Schneider and Chandler, 1973; Nakai et al., 1996). Reasonably, the increased [Ca 2 1 ] i , a consequence of this opening, should interact with the Ca 2 1 -sensitive Ryrs to open them further. To what extent this Ca 2 1 -induced Ca 2 1 release (CICR) mechanism contributes to physiological Ca 2 1 release during an action potential is still debated (Fabiato, 1984; Endo, 1985; Ríos and Pizarro, 1991; Jacquemond et al., 1991; Pape et al., 1993). A substantial evidence of CICR involvement is the existence of Ca 2 1 sparks (Cheng et al., 1993). In skeletal muscle, they are believed to be largely a manifestation of CICR, based on their similarity with sparks of cardiac muscle, where CICR is the main activation mechanism, and their higher frequency upon increasing cytoplasmic [Ca 2 1 ] (Klein et al., 1996). Another indication that CICR underlies sparks is their rapid kinetics, matching those of the initial peak of Ca 2 1 release flux (Baylor et al., 1983; Melzer et al., 1984), which is believed to contain the greatest CICR contribution within the wholecell release waveform (Jacquemond et al., 1991; Ríos and Pizarro, 1991). However attractive, the idea that sparks constitute the “quanta” of release and are activated by CICR brings up new puzzles, including the precise nature of the contribution of voltage to release (whether it consists of sparks as well, or some other form, like the event-less release described by Shirokova and Ríos, 1997), the number of channels involved in a spark Address correspondence to A. González, Department of Molecular Biophysics and Physiology, Rush University, 1750 W. Harrison Street, Suite 1279 JS, Chicago, IL 60612. Fax: 312-942-8711; E-mail: agonzale