[Ca²⁺]_mito (black), [Ca²⁺]_out (green), and ΔΨ_m (red) responses to large or small Ca²⁺ additions in isolated guinea pig heart mitochondria. (A–D) Ca²⁺ additions were as follows: 15.3 µM Ca²⁺ first addition, 5.7 µM for each subsequent addition. (E–H) Ca²⁺ additions were as follows: 0.2 µM Ca²⁺ first addition, 0.011 µM for each subsequent addition. Corresponding calculated values of free Ca²⁺_mito expressed in nmol/mg, total ∑Ca²⁺ taken up, and the bound/free Ca²⁺ ratio are shown for large (A–D) or small (E–H) additions. [Ca²⁺]_mito and [Ca²⁺]_out were monitored with Calcium green and fura-FF simultaneously. Experimental solution was the standard KCl-based buffer with 5 mM NaCl, 5 mM G/M, 1 mM KH₂PO₄, 20 µM EGTA, and 1 µM CsA. 300 nM TMRM was used to monitor ΔΨ_m ratiometrically.