Figure 6. hUCP2 effects on mitochondrial Ca²⁺ uptake capacity and membrane potential.

(A) Kinetics of Ca²⁺ uptake in brain mitochondria measured by monitoring the change of Fura-6F fluorescence ratio (340/380 nm excitation, 510 nm emission) on Ca²⁺ loading (250 nmol of Ca²⁺/mg protein in each addition, indicated by arrows). The fluorescence peaks corresponds to increase in extra-mitochondrial Ca²⁺, whereas the decreases in fluorescence reflects mitochondrial Ca²⁺ uptake. The sustained increase at the end of trace shows that mitochondria are unable to further accumulate Ca²⁺. In the example, ntg and hUCP2 mitochondria took up six Ca²⁺ additions, whereas G93A and G93A hUCP2 mice mitochondria only took five Ca²⁺ additions. (B) Average brain mitochondrial Ca²⁺ uptake capacity in nmol Ca²⁺/mg of mitochondrial protein. Data are mean ± SEM of n = 5 brain mitochondria preparations per group. (C) Δψm response to 25 nmol Ca²⁺ bolus (250 nmol of Ca²⁺/mg protein) calculated as [(1/FL_{N nmol Ca}²⁺)/(1/FL_{0 nmol Ca}²⁺) × 100], where FL_N is fluorescence of safranin-O at N nmol Ca²⁺. Safranin-O fluorescence inversely correlates with Δψm. Data are mean of n = 4 brain mitochondria preparations per group. (D) Δψm response to respiratory chain uncoupler SF6847 calculated as in C. Data are presented as mean of n =3 for hUCP2 and n = 4 for hUCP2 G93A. IC₅₀values in C and D were calculated from the fitted curves of the Hill equation y=xⁿ/(kⁿ+xⁿ), where x is Ca²⁺ or SF6847 concentration, n is the Hill coefficient, and k is the IC₅₀ value