ANT1 Activation and Inhibition Patterns Support the Fatty Acid Cycling Mechanism for Proton Transport

Abstract

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.

Antioxidant Synergy of Mitochondrial Phospholipase PNPLA8/iPLA2γ with Fatty Acid–Conducting SLC25 Gene Family Transporters

“…However, unlike for UCP1, FAs were suggested to bind within the ANT translocation pathway as cofactors rather than transported species [ 12 ]. However, more recent experimental findings from using purified recombinant ANT1 reconstituted in the planar lipid bilayers reveal that ANT1 mediates H + transport only in the presence of long-chain FAs and the results support the FA cycling mechanism for H + transport [ 83 ].…”

Mitochondrial Uncoupling Proteins (UCP1-UCP3) and Adenine Nucleotide Translocase (ANT1) Enhance the Protonophoric Action of 2,4-Dinitrophenol in Mitochondria and Planar Bilayer Membranes

“…Figure 2A shows that the specific conductance of membranes containing DNP in the presence of ANT1 (ANT1 + DNP, red column; G m = 80.9 ± 9.4 nS/cm 2 ) was approximately double that of membranes containing only DNP at a concentration of 50 µM (DNP, gray column; G m = 42.7 ± 5.9 nS/cm 2 ). Until now, only FAs have been shown to activate ANT1 directly (Figure 2A) [18,26,51,52].…”

“…It was previously shown that ANT1 mediates proton transport in the presence of free long-chain fatty acids (FA), similar to UCPs [26,47,51]. To test whether DNP also activates ANT1, we used a well-defined model of planar bilayer membranes reconstituted with recombinant murine ANT1 (Figure S1) [27].…”

“…Our previous study demonstrated the ability of ANT1 to enhance the proton current across artificial membranes in the presence of weak uncouplers-long-chain fatty acids (FA) [26]. We now hypothesize that DNP's protonophoric function may be also potentiated by mitochondrial proton-transporting proteins, in addition to the DNP-mediated increase in the selective lipid membrane's permeability for protons.…”

ATP-consuming futile cycles as energy dissipating mechanisms to counteract obesity

“…AAC is a member of the solute carrier family (SLC25) that exchanges ATP and ADP between the mitochondria matrix and the cytosol [45]. However, AAC has additional key functions: AAC is a fatty-acid induced proton channel that explains endogenous proton leak, as well as regulating the opening of the permeability transition pore and the removal of mitochondria by mitophagy [45,46]. Remarkably, both AAC-controlled leak and PTP opening can be stimulated by free fatty acids, similarly to how fatty acids activate UCP1 [45].…”