Mitochondrial uncoupling proteins (UCP1-UCP3) and adenine nucleotide translocase (ANT1) enhance protonophoric action of 2,4-dinitrophenol in mitochondria and planar bilayer membranes

2, 4-dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria and directly increases the metabolic rate by increasing the body’s heat generation. It is still used as a “diet pill” despite its high toxicity and lack of antidotes. DNP increases the proton current through the pure lipid membranes similar to other chemical uncouplers. However, the molecular mechanism of its action in mitochondria is far from being understood. Since DNP´s protonophoric function in mitochondria is known to be sensitive to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), we hypothesized that DNP modifies the activity of ANT and other mitochondrial proton-transporting proteins. To test this hypothesis, we investigated the contribution of recombinant ANT1 or uncoupling proteins UCP2 and UCP3 to the DNP-mediated proton leak using the well-defined model of the planar bilayer membranes. All three proteins significantly enhanced the DNP-mediated proton transport, an activity previously known to happen only in the presence of long-chain free fatty acids. Using site-directed mutagenesis and molecular dynamic simulations we have shown that arginine 79 of ANT1 is crucial for the DNP-mediated increase of the membrane conductance, implying that this amino acid participates in DNP binding to ANT1.