Fig. 6. BAPTA (AM form) prevents the acceleration of Ins(1,4,5)P₃-mediated Ca²⁺ release and limits the rise in [Ca²⁺]_c. Depolarization (–70 to +10 mV, 3 seconds) (B) activated I_Ca (C) and increased [Ca²⁺]_c (A). At –70 mV (B), local photolysis of Ins(1,4,5)P₃ [; concentrations producing maximum (red) or submaximum (blue) responses] increased [Ca²⁺]_c (A). Prior (7 minutes) introduction of BAPTA AM (50 µM) to the bathing solution reduced the Ins(1,4,5)P₃-evoked [Ca²⁺]_c rise (A, unfilled bar) owing to increased cytoplasmic Ca²⁺ buffering, as revealed by the reduced [Ca²⁺]_c rise for a similar Ca²⁺ influx (A,C). Note the smaller rise in measured [Ca²⁺]_c (from the fluorescence measurements) for a given calculated [Ca²⁺]_c increase (from the Ca²⁺ current) in BAPTA (D) compared with that of controls. Scaling the [Ca²⁺]_c transients obtained in BAPTA so that the depolarization-evoked transients in the presence and absence of the chelator are of comparable size allowed a compensation for the increased buffer capacity of the cell to be made (E; left-hand panel). Application of the same scaling factor to the Ins(1,4,5)P₃-evoked [Ca²⁺]_c increases revealed that the Ins(1,4,5)P₃-evoked [Ca²⁺]_c transients were substantially reduced in the presence of the chelator (E; middle and right panels). Significantly, when the cytoplasmic Ca²⁺ buffer capacity had been increased (with BAPTA), the increase in velocity of the [Ca²⁺]_c rise (d[Ca²⁺]_c/dt) seen in control was substantially reduced (F). Thus the rate of Ca²⁺ release was largely constant rather than increased (F) during the release process in the presence of the chelator – that is, the velocity increase arose as a result of a Ca²⁺-dependent positive feedback acting at the cytoplasmic aspect of the Ins(1,4,5)P₃R. BAPTA in its Ca²⁺-free form (BAPTA_Cafree) might itself directly inhibit Ins(1,4,5)P₃R (Richardson and Taylor, 1993). However, high concentrations of BAPTA are required – for example, increasing BAPTA_Cafree from 90 µM to 9 mM reduced Ins(1,4,5)P₃-mediated Ca²⁺ release by 8% (Bootman et al., 1995).