ANT activity depends on many parameters, to an unequal extent. The most important parameter is the mitochondrial membrane potential, ΔΨm, see The ADP-ATP Translocation in mitochondria, a membrane potential controlled transport. Because of this, ANT activity should ALWAYS be measured together with ΔΨm. What can be even more informative, is the determination of ANT activity within a range of ΔΨm values. That can be achieved by 'clamping' DYm with small amounts of an uncoupler, and measure in parallel ANT activity and ΔΨm. These data can be later combined, and presented as 'exchange rate activity as a function of voltage', just like a typical I/V curve of a channel or a transporter. Such an example is given below (adapted from here):
In the figures above, the estimation of ADP-ATP exchange rates and ΔΨm are shown, in permeabilized cells. A: Time course of [ATP]e in the medium, calculated from [Mg2+]free. Effect of membrane depolarization to various voltages by stepwise addition of 10 nM SF 6847. B: Reconstructed time course of ΔΨm, calculated from safranin O fluorescence. Permeabilized cells were challenged initially by 2 mM ADP, followed by stepwise additions of 10 nM SF 6847. By combining the data obtained from these two experiments, the correlation of ADP-ATP exchange rate as a function of ΔΨm can be depicted:
Note how steeply dependent is the ADP-ATP exchange rate to ΔΨm in the interval -145 mV - -130 mV. The choice of substrates, quality of mitochondria, buffer composition, to name a few, will all affect ΔΨm, and as extension of this, ADP-ATP exchange rate. ALWAYS measure ADP-ATP exchange rate as a function of ΔΨm.
Also, from such experiments, the membrane potential value at which there is no net flux of ADP nor ATP across the inner mitochondrial membrane through the ANT, termed the reversal potential of the ANT (Erev_ANT), can be determined. See more about Erev_ANT.