Fig. 2

Overview of protocols. After loading SR with Ca²⁺ at pCa 6.5 in the solution containing 4.1 mM ATP (CaG1M1.5, see Table 1) for 2 min at 24°C, Ca²⁺ release was triggered by rapid cooling (protocol A), 50 mM caffeine (protocol B), and rapid cooling followed by 50 mM caffeine (protocol C). Ca²⁺ released from the SR was bound to fluo-3 in a glass capillary lumen, and the resultant fluorescence signal was measured as described in the text. Protocol A Temperature of a solution was lowered from 24 to 3°C within 3 s with simultaneous application of 4.6 mM ATP and 20 μM fluo-3 (G0M1.5F, see Table 1). Protocol B A solution that contained 50 mM caffeine, 25 mM AMP and 20 μM fluo-3 (G0RM0CafF, see Table 1) was applied at 3°C. Protocol C Rapid cooling (temperature of G0M1.5F lowered to 3°C) and 50 mM caffeine (G0RM0CafF) were sequentially applied. The meanings of dark and shaded trapezoids are full Ca²⁺ and decreased Ca²⁺ in SR, respectively