Fig. 6. Properties of the PT in yeast mitochondria. A) Time of PTP opening after calcium stimulation based on the CRC assay. Five hundred micrograms of mitochondria was suspended in CRC buffer, and 50 µM CaCl₂ was added at time 0 and immediately taken up by mitochondria. The time of yPTP opening was determined by a rapid increase in Calcium Green fluorescence due to calcium release into the buffer. Traces are representative of at least 5 independent experiments, and the histogram refers to the time of Ca²⁺ release. B) Mitochondrial swelling assay. Swelling was induced by the addition of 140 µM CaCl₂ to mitochondria suspended in CRC buffer and measured as the decrease in absorbance at 540 nm. Alamethicin (10 µM) was added after 15 minutes of swelling for normalization of the experiment. The fraction of swollen mitochondria after yPTP opening was calculated using maximal swelling induced by alamethicin as 1. Traces are representative of at least 5 independent experiments, and the histogram refers to the fraction of swollen mitochondria 15 minutes after CaCl₂ addition. Statistical significance is indicated.