Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA’s transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s−1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion’s transport across the membrane. ANT’s dual function—ADP/ATP and H+ transport in the presence of FA—may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases.

We studied and compared in detail an elusive matrix open (m‐state) and cytoplasmic open (c‐state) state of ADP/ATP carrier (AAC) protein embedded in the DOPC bilayer by microsecond molecular dynamics (MD) simulations. We analyzed both states with and without cardiolipin (CDL) molecules, with a special emphasis on the recently obtained crystallographic structure of the AAC m‐state. The obtained results show that both states of the protein are stable in the DOPC bilayer and impermeable to water. In comparison with the c‐state of AAC, the m‐state is more dynamic, but at the same time possesses a larger occluded area in the protein cavity. Both states of the protein are less flexible in simulations when CDL molecules are present, which is especially visible for the m‐state. Finally, the analysis of the protein conformational changes during MD simulations shows that protein parts at the protein/lipid boundaries are prone to larger conformational changes in contrast to central region of the protein embedded in the bilayer core, thus further supporting the cycling mechanism suggested for ADP/ATP exchange by AAC.

2,4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in “diet pills”, despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP’s uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP’s protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1.

Apparent pKa,app values are calculated for saturated fatty acids of different length embedded in a DOPC bilayer.