Mitochondrial Ca2+ Clearance Measurement

Fluorometric measurements of mitochondrial Ca²⁺ clearance were performed as previously described (20 (link)). Briefly, 300 μg (for PTP experiment) and 750 μg (for uptake study) of isolated mouse brain cortical mitochondrial protein were resuspended in 1.5 ml of intracellular medium [120 mM KCl, 10 mM NaCl, 1 mM KH₂PO₄, 20 mM tris-Hepes (pH 7.2)] supplemented with proteases inhibitors (leupetin, antipain, and pepstatin, 1 μg/ml each), 2 mM Mg–adenosine 5′-triphosphate (ATP), and 2 μM thapsigargin (Enzo) and maintained in a stirred thermostated cuvette at 35°C. For PTP experiments, the intracellular medium was used without Mg-ATP. Assays were performed in the presence of 20 μM CGP-37157 (Enzo) and 1 mM malate/pyruvate using a DeltaRAM fluorimeter (Photon Technology International). The extramitochondrial Ca²⁺ concentration [Ca²⁺]_c was measured by the ratiometric Ca²⁺ probe furaFF (1 μM) using 340- to 380-nm excitation and 500-nm emission. Calibration of the furaFF signal was carried out at the end of each measurement, adding 1 mM CaCl₂, followed by 10 mM EGTA/tris (pH 8.5).