Abstract
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.
Highlights
2,4-Dinitrophenol (DNP), chloroform, dimethyl sulfoxide (DMSO), 1,2-dioleoyl-snglycero-3-phosphotidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cardiolipin (CL), sodium sulfate (Na2 SO4 ), 2-(N-morpholino) ethanesulfonic acid (MES), tris(hydroxymethyl)-aminomethane (Tris), adenosine 50 -triphosphate (ATP), guanosine 50 -triphosphate (GTP), carboxyatractyloside (CATR), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0 -tetraacetic acid (EGTA), Nlauroylsarcosine, Triton X-114, dithiothreitol (DTT), bovine serum albumin (BSA), sucrose, 3-(N-morpholino) propanesulfonic acid (MOPS), rotenone, and safranin O were purchased from Sigma-Aldrich (Vienna, Austria)
adenosine -triphosphate (ATP) was shown to be an inhibitor of fatty acids (FA)-activated ANT1 [26], so we examined its effect on DNP-mediated proton transport as well
Comparing the conductance of the pure lipid bilayer membranes and membranes reconstituted with recombinant ANT1, UCP1, UCP2, or UCP3, we revealed that proteins potentiate DNP-mediated proton transport through the membrane
Summary
Partial suppression of the uncoupling effect was found for other protonophores, including BAM15 [19,20,21,22,23,24] These findings support the hypothesis of ANT’s participation in the uncoupling action of certain protonophores in the mitochondria [25]. We hypothesize that DNP’s protonophoric function may be potentiated by mitochondrial proton-transporting proteins, in addition to the DNP-mediated increase in the selective lipid membrane’s permeability for protons. To test this hypothesis, we used a well-defined model of lipid bilayer membranes reconstituted with recombinant ANT1 or UCPs (1) to investigate the contribution of these proteins to DNP-mediated proton leakage,. (2) to evaluate the efficiency of the inhibitors of proton transport mediated by ANT1/UCP, and (3) to reveal the amino acids crucial for ANT1 activation by DNP
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