Table 1.

Fractional Release of [^Ca2+]_SR

Stimulation	Fractional release	[Ca2+]SR, free (μM)
1 Hz	0.195 ± 0.029	261 ± 15.8
10 Hz	0.734 ± 0.049	101 ± 11.1
50 Hz	0.882 ± 0.028	79 ± 8.4
1 mM 4CmC	1	62 ± 4.3 (raw)8 ± 1.6 (corrected)

FDB fibers were loaded with fluo-5N and exposed to trains of electrical field stimulation at frequencies of 1, 10, or 50 Hz for 10 s. Changes in fluo-5N fluorescence were monitored under confocal optics. After stimulation, fluorescence returned to baseline and the fibers were treated with 1 mM 4-CmC to induce maximal SR Ca²⁺ release. Fractional release was calculated by comparing the electrically induced fluorescence change to the 4-CmC-induced change. Using the K_D and resting [Ca²⁺]_SR,free values calculated by in situ calibration of fluo-5N (see Fig. 5) and applying equations derived by Cannell et al. (15), we calibrated the new [Ca²⁺]_SR,free after steady-state activation at each stimulation frequency. As described in Fig. 6, 4-CmC induced transients in intact fibers appears to reduce [Ca²⁺]_SR,free to ∼60 μM. Calibration of the [Ca²⁺]_{SR, free} after 4-CmC treatment by permeabilization demonstrates that 4-CmC actually reduces [Ca²⁺]_SR,free to ∼8 μM. The background correction made to 4-CmC-induced [Ca²⁺]_SR,free (see Fig. 6) suggests that [Ca²⁺]_SR,free calculated for near tetanic stimulation rates, where fractional release approaches 1, is likely to be overestimated.