Contact: Dr. Christos Chinopoulos MD, PhD
What can you do with this method

So far, it should be obvious that with this methodology one may do the following:

i) determine ATP efflux rate from energized, isolated or in situ mitochondria of permeabilized cells (or homogenates)

ii) determine ATP influx rate of sufficiently depolarized isolated or in situ mitochondria of permeabilized cells (or homogenates)

iii) determine the extramitochondrial ATP/ADP ratio.

Please keep in mind, that with this method -if performed correctly- you will obtain meaningful results, but by themselves they will not be so informative; this is because ADP-ATP exchange rate is strongly dependent on
DYm. Thus, ADP-ATP exchange rate determination should ALWAYS be performed in parallel to measurements of DYm. Bearing that in mind, the true power of this method appears, which is the determination of ADP-ATP exchange rate as a function of DYm over a range of DYm values. This has been demostrated in Hum Mol Genet. 2011 Aug 1;20(15):2964-74, showing that adPEO mutations in ANT1 impair ADP-ATP translocation in muscle mitochondria. More specifically, it was shown that mutant human ANT1 caused dominant mitochondrial defects characterized by decreased ADP-ATP exchange function and abnormal translocator reversal potential, see figure below.
Legend for figure shown above: ADP-ATP exchange rate/ΔΨm profile of C2C12 myotubes expressing WT and mutant ANT1. Plot of ADP-ATP exchange rate mediated by ANT versus ΔΨm in in situ mitochondria of WT, A114P and V289M C2C12 permeabilized cells depolarized to various voltages by increasing amounts of SF 6847, and normalized to specific citrate synthase activity. Each point in the graph represents the average ADP-ATP exchange rate calculated by linear regression of the MgG fluorescence, calibrated and converted to ATP appearing in the medium, as a function of calibrated safranine O values. Each point is the average of five to seven independent experiments. *: Statistically significant by one-way ANOVA followed by Tukey's post-hoc test versus WT, P = 2.0e−3 (ADP), 4.0e−3 (10 sf), 0.043 (20 sf) and 0.045 (30 sf).

Furthermore, with this methodology one can determine the effective P/O ratio. For a description of such a protocol, click here. That protocol describes the steps needed to extract and process the raw oxygen and fluorescence signals obtained from an O2k oxygraph equipped with an LED Module, as well as the steps required to complete the Excel template file as elaborated in Salin et al. (2016). For any feedbacks or questions, please  contact Dr. Karine Salin: