Figure 2.

Ca²⁺retention capacities and rates of Ca²⁺uptake by brain nonsynaptic and synaptic mitochondria isolated from CD1, MCU-KO, C57BL/6, CyD-KO, and MCU/CyD-DKO mice. Ca²⁺ uptake was assessed in brain nonsynaptic (A, C, E, G, I, red traces) and synaptic (B, D, F, H, J, black traces) mitochondria incubated at 37 °C in the standard incubation medium supplemented with the Complex I substrates pyruvate (3 mM) plus malate (1 mM). Where indicated nonsynaptic or synaptic mitochondria (Mtc), both 60 μg of protein, were added. Where indicated, 10 μM Ca²⁺ (delivered as CaCl₂) was applied to brain mitochondria from CD1 (A and B), MCU-KO (C and D), C57BL/6 (E and F), CyD-KO (G and H), and MCU/CyD-DKO (I and J) mice. In all experiments, incubation medium was additionally supplemented with 100 μM ADP and 1 μM oligomycin (65). K and L, Ca²⁺ retention capacity data are summarized for experiments with nonsynaptic (K) and synaptic (L) mitochondria, respectively. Data are mean ± SD from five separate experiments. ∗∗∗p < 0.001 comparing Ca²⁺ retention capacity of MCU-KO and CD1 Mtc, for both nonsynaptic and synaptic Mtc (ANOVA p < 0.0001, F = 246.9 for nonsynaptic Mtc and F = 266.0 for synaptic Mtc); ^###p < 0.001 comparing Ca²⁺ retention capacity of CyD-KO and C57BL/6 Mtc, for both nonsynaptic and synaptic Mtc (ANOVA p < 0.0001, F = 246.9 for nonsynaptic Mtc and F = 266.0 for synaptic Mtc); ^@@@p < 0.001 comparing Ca²⁺ retention capacity of MCU/CyD-DKO and CyD-KO Mtc, for both nonsynaptic and synaptic Mtc (ANOVA p < 0.0001, F = 246.9 for nonsynaptic Mtc and F = 266.0 for synaptic Mtc). M and N, rates of Ca²⁺ uptake by brain nonsynaptic and synaptic mitochondria, respectively, isolated from CD1, MCU-KO, C57BL/6, CyD-KO, and MCU/CyD-DKO mice. Ca²⁺ uptake rates were quantified from experiments shown in Figure 2 as Ca²⁺ uptake per minute per mg of mitochondrial protein. Data are mean ± S.D. from five separate experiments. ^###p < 0.001 comparing rates of Ca²⁺ uptake by mitochondria from CD1 and MCU-KO mice (ANOVA p < 0.0001, F = 128.9 for nonsynaptic Mtc and F = 143.6 for synaptic Mtc); ∗∗∗p < 0.001 comparing rates of Ca²⁺ uptake by mitochondria from CD1, C57BL/6, and CyD-KO mice and by mitochondria from MCU/CyD-DKO mice (ANOVA p < 0.0001, F = 128.9 for nonsynaptic Mtc and F = 143.6 for synaptic Mtc). CyD, cyclophilin D; MCU, mitochondrial calcium uniporter; MCU/CyD-DKO, MCU/CyD-double KO.