BACKGROUND: Mitochondrial energy can be stored as ATP or released as heat by uncoupling protein 1 (UCP1) during non-shivering thermogenesis in brown adipose tissue. UCP1, located in the inner mitochondrial membrane, reduces the proton gradient in the presence of long-chain fatty acids (FA). FA act as weak, protein-independent uncouplers, with the transport of the FA anion across the membrane being the rate-limiting step. According to the fatty acid cycling hypothesis, UCP1 catalyzes this step through an as-yet-undefined mechanism. METHODS: We used computational and experimental techniques, including all-atom molecular dynamics (MD) simulations, membrane conductance measurements, and site-directed mutagenesis. RESULTS: We identified two novel pathways for fatty acid anion translocation (sliding) at the UCP1 protein-lipid interface, ending at key arginine residues R84 and R183 in a nucleotide-binding region. This region forms a stable complex with fatty acid anion, which is crucial for anion transport. Mutations of these two arginines reduced membrane conductance, consistent with the MD simulation prediction that the arachidonic acid anion slides between helices H2-H3 and H4-H5, terminating at R84 and R183. Protonation of the arachidonic acid anion predicts its release from the protein-lipid interface, allowing it to move to either cytosolic or matrix leaflets of the membrane. CONCLUSION: We provide a novel, detailed mechanism by which UCP1 facilitates fatty acid anion transport, as part of the fatty acid cycling process originally proposed by Skulachev. The residues involved in this transport are conserved in other SLC25 proteins, suggesting the mechanism may extend beyond UCP1 to other members of the superfamily.

• Keywords
• anion transporter, cardiolipin, fatty acid cycling, mitochondrial SLC25 family, molecular dynamics simulations, proton transport mechanism,
• MeSH
• Ion Channels * metabolism   chemistry   genetics   MeSH
• Ion Transport physiology   MeSH
• Humans MeSH
• Fatty Acids * metabolism   MeSH
• Mitochondrial Proteins * metabolism   chemistry   genetics   MeSH
• Mutagenesis, Site-Directed MeSH
• Molecular Dynamics Simulation * MeSH
• Uncoupling Protein 1 MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ion Channels * MeSH
• Fatty Acids * MeSH
• Mitochondrial Proteins * MeSH
• Uncoupling Protein 1 MeSH

The mitochondrial ADP/ATP carrier (AAC, ANT), a member of the SLC25 family of solute carriers, plays a critical role in transporting purine nucleotides (ATP and ADP) as well as protons across the inner mitochondrial membrane. However, the precise mechanism and physiological significance of proton transport by ADP/ATP carrier remain unclear. Notably, the presence of uncouplers-such as long-chain fatty acids (FA) or artificial compounds like dinitrophenol (DNP)-is essential for this process. We explore two potential mechanisms that describe ADP/ATP carrier as either (i) a proton carrier that functions in the presence of FA or DNP, or (ii) an anion transporter (FA- or DNP). In the latter case, the proton is translocated by the neutral form of FA, which carries it from the matrix to the intermembrane space (FA-cycling hypothesis). Our recent results support this hypothesis. We describe a four-step mechanism for the "sliding" of the FA anion from the matrix to the mitochondrial intermembrane space and discuss a possible generalization of this mechanism to other SLC25 carriers.

• Keywords
• MD simulations, bilayer lipid membranes, membrane proteins, mitochondrial transporter, reconstituted protein, uncoupling protein,
• MeSH
• 2,4-Dinitrophenol metabolism   MeSH
• Adenosine Triphosphate metabolism   MeSH
• Biological Transport MeSH
• Ion Transport MeSH
• Humans MeSH
• Fatty Acids * metabolism   MeSH
• Mitochondrial ADP, ATP Translocases * metabolism   chemistry   MeSH
• Mitochondria * metabolism   MeSH
• Protons * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• 2,4-Dinitrophenol MeSH
• Adenosine Triphosphate MeSH
• Fatty Acids * MeSH
• Mitochondrial ADP, ATP Translocases * MeSH
• Protons * MeSH

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein-lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane.

• Keywords
• AAC, ADP/ATP carrier, arachidonic acid, fatty acid cycling hypothesis, fatty acids anion transport, proton transport, uncoupling proteins,
• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Anions metabolism   MeSH
• Lipid Bilayers * MeSH
• Fatty Acids metabolism   MeSH
• Mitochondrial ADP, ATP Translocases metabolism   MeSH
• Protons * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Anions MeSH
• Lipid Bilayers * MeSH
• Fatty Acids MeSH
• Mitochondrial ADP, ATP Translocases MeSH
• Protons * MeSH

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.

• Keywords
• ADP/ATP carrier protein, arachidonic acid, fatty acid anion transport, long-chain fatty acids, mitochondrial transporter, proton transport,
• MeSH
• Ion Transport MeSH
• Protein Conformation MeSH
• Fatty Acids metabolism   MeSH
• Membrane Potential, Mitochondrial MeSH
• Mitochondria metabolism   MeSH
• Mice MeSH
• Protons * MeSH
• Adenine Nucleotide Translocator 1 chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fatty Acids MeSH
• Protons * MeSH
• Adenine Nucleotide Translocator 1 MeSH

SIGNIFICANCE: Mitochondria are the energetic, metabolic, redox, and information signaling centers of the cell. Substrate pressure, mitochondrial network dynamics, and cristae morphology state are integrated by the protonmotive force Δp or its potential component, ΔΨ, which are attenuated by proton backflux into the matrix, termed uncoupling. The mitochondrial uncoupling proteins (UCP1-5) play an eminent role in the regulation of each of the mentioned aspects, being involved in numerous physiological events including redox signaling. Recent Advances: UCP2 structure, including purine nucleotide and fatty acid (FA) binding sites, strongly support the FA cycling mechanism: UCP2 expels FA anions, whereas uncoupling is achieved by the membrane backflux of protonated FA. Nascent FAs, cleaved by phospholipases, are preferential. The resulting Δp dissipation decreases superoxide formation dependent on Δp. UCP-mediated antioxidant protection and its impairment are expected to play a major role in cell physiology and pathology. Moreover, UCP2-mediated aspartate, oxaloacetate, and malate antiport with phosphate is expected to alter metabolism of cancer cells. CRITICAL ISSUES: A wide range of UCP antioxidant effects and participations in redox signaling have been reported; however, mechanisms of UCP activation are still debated. Switching off/on the UCP2 protonophoretic function might serve as redox signaling either by employing/releasing the extra capacity of cell antioxidant systems or by directly increasing/decreasing mitochondrial superoxide sources. Rapid UCP2 degradation, FA levels, elevation of purine nucleotides, decreased Mg2+, or increased pyruvate accumulation may initiate UCP-mediated redox signaling. FUTURE DIRECTIONS: Issues such as UCP2 participation in glucose sensing, neuronal (synaptic) function, and immune cell activation should be elucidated. Antioxid. Redox Signal. 29, 667-714.

• Keywords
• UCP2, anion transport, attenuation of superoxide formation, fatty acid cycling, mitochondrial uncoupling proteins, redox signaling,
• MeSH
• Antioxidants metabolism   MeSH
• Humans MeSH
• Mitochondrial Uncoupling Proteins metabolism   MeSH
• Oxidation-Reduction MeSH
• Signal Transduction * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Antioxidants MeSH
• Mitochondrial Uncoupling Proteins MeSH

We reviewed mechanisms that determine reactive oxygen species (redox) homeostasis, redox information signaling and metabolic/regulatory function of autocrine insulin signaling in pancreatic β cells, and consequences of oxidative stress and dysregulation of redox/information signaling for their dysfunction. We emphasize the role of mitochondrion in β cell molecular physiology and pathology, including the antioxidant role of mitochondrial uncoupling protein UCP2. Since in pancreatic β cells pyruvate cannot be easily diverted towards lactate dehydrogenase for lactate formation, the respiration and oxidative phosphorylation intensity are governed by the availability of glucose, leading to a certain ATP/ADP ratio, whereas in other cell types, cell demand dictates respiration/metabolism rates. Moreover, we examine the possibility that type 2 diabetes mellitus might be considered as an inevitable result of progressive self-accelerating oxidative stress and concomitantly dysregulated information signaling in peripheral tissues as well as in pancreatic β cells. It is because the redox signaling is inherent to the insulin receptor signaling mechanism and its impairment leads to the oxidative and nitrosative stress. Also emerging concepts, admiting participation of redox signaling even in glucose sensing and insulin release in pancreatic β cells, fit in this view. For example, NADPH has been firmly established to be a modulator of glucose-stimulated insulin release.

• MeSH
• Insulin-Secreting Cells metabolism   pathology   MeSH
• Homeostasis * MeSH
• Insulin metabolism   MeSH
• Humans MeSH
• Mitochondria metabolism   MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress MeSH
• Insulin Secretion MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Insulin MeSH

Mitochondrial uncoupling proteins (UCPs) are pure anion uniporters, which mediate fatty acid (FA) uniport leading to FA cycling. Protonated FAs then flip-flop back across the lipid bilayer. An existence of pure proton channel in UCPs is excluded by the equivalent flux-voltage dependencies for uniport of FAs and halide anions, which are best described by the Eyring barrier variant with a single energy well in the middle of two peaks. Experiments with FAs unable to flip and alkylsulfonates also support this view. Phylogenetically, UCPs took advantage of the common FA-uncoupling function of SLC25 family carriers and dropped their solute transport function.

• MeSH
• Models, Biological MeSH
• Electrophoresis MeSH
• Ion Channels metabolism   MeSH
• Humans MeSH
• Mitochondrial Proteins metabolism   MeSH
• Protons MeSH
• Uncoupling Protein 1 MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Ion Channels MeSH
• Mitochondrial Proteins MeSH
• Protons MeSH
• Uncoupling Protein 1 MeSH