Assessing the relative role of adenine nucleotide translocator and ATP synthase in the permeability transition

In response to high Ca2+, the inner mitochondrial membrane can undergo a process defined as the permeability transition (PT), that leads to matrix swelling and eventually culminates in cell death initiation. The PT is due to the opening of the PT pore (PTP), a Ca2+-activated and high-conductance channel. Among PTP modulators, thiol reagents (e.g. phenylarsine oxide, PAO) are inducers, while acidic pH acts as inhibitor. The molecular nature of the PTP was highly debated. The adenine nucleotide translocator (ANT) was considered a key mediator, although genetic studies revealed that other components are likely involved. Recently, the ATP synthase was shown to generate Ca2+-dependent channels perfectly matching those of the PTP. Whether these two permeation pathways act independently responding to specific PT modulators or cooperate is not known. Here, we evaluated the relative contribution of ANT and ATP synthase in the PT, by analyzing the effect of ANT specific ligands bongkrekic acid (BKA) and atractylate (ATR), which inhibits and activates the ANT channel, respectively, on Ca2+-dependent swelling at different pH. At pH 7.4, BKA and ATR showed only partial effects on PT occurrence. Moreover, PAO does not induce the PT through the ANT as ANTs knock-out fibroblasts are still sensitive to the thiol reagent. Thus, at pH 7.4, ATP synthase likely predominates. Conversely, at pH 6.5 (a condition that prevents opening of the ATP synthase channel), the PT can be activated by ATR and fully prevented by BKA, suggesting a primary role of the ANT. Remarkably, at pH 6.5, benzodiazepine (Bz)-423, which binds OSCP subunit of the ATP synthase, triggers mitochondrial swelling that can be prevented by BKA, suggesting an intimate connection between ATP synthase and ANT. Consistently, co-immunoprecipitation experiments revealed an interaction between ANT and pore-forming subunits c and g of the ATP synthase, hinting at a possible cross-talk between the two candidates in the PT process.