Fig. 4. Submaximal [Ins(1,4,5)P₃] does not deplete the SR store of Ca²⁺. At –70 mV, high [Ins(1,4,5)P₃] producing maximal responses (pink area) and lower [Ins(1,4,5)P₃] producing submaximal responses (blue area; produced by lowering the flash-lamp energy), each evoked approximately reproducible increases in [Ca²⁺]_c. In a Ca²⁺-free solution (containing 1 mM EGTA and 3 mM MgCl₂; for the duration of the filled bar), the submaximal [Ca²⁺]_c increases declined, then disappeared. The absence of a response to [Ins(1,4,5)P₃] was not due to depletion of the store. Increasing Ins(1,4,5)P₃ (pink; right side) evoked further Ca²⁺ release. Another mechanism, other than depletion of the store of Ca²⁺, for example `lumenal' regulation of Ins(1,4,5)P₃R, might have accounted for the loss of response to Ins(1,4,5)P₃ (see main text). The time between each Ins(1,4,5)P₃ challenge was 1 minute, except after the introduction of the Ca²⁺-free solution, which took 3 minutes to equilibrate.