Fig. 4. Ca²⁺accumulated by mitochondria during Ins(1,4,5)P₃- or I_Ca-evoked [Ca²⁺]_c transients can be released by mitochondrial depolarization in the 15 seconds after [Ca²⁺]_c elevation. Local photolysis of caged Ins(1,4,5)P₃ (UV-flash release at , A) or plasmalemmal depolarization (–70 to +10 mV, 500 ms, C) each transiently increased [Ca²⁺]_c, as indicated by an increase in Fluo-4 fluorescence (controls, black line). In a subsequent activation by Ins(1,4,5)P₃ (A) or plasmalemmal depolarization (C) in the same cells, rapid _m depolarization (A,C, as shown by a decrease in TMRE fluorescence, red line) by CCCP (20 µM, by pressure-ejection puffer pipette for 2 seconds as shown), caused a transient increase in [Ca²⁺]_c (blue line). The magnitude of mitochondrial Ca²⁺ release was measured from the time-integrated Fluo-4 signal for a 10-second period immediately after CCCP application minus the time-integrated signal for the control (hashed area, A,C, shown in panels B,D, n=3 individual cells for each data point).