Kreiter 2019
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Kreiter, J; Pohl, EE „A Micro-agar Salt Bridge Electrode for Analyzing the Proton Turnover Rate of Recombinant Membrane Proteins” J Vis Exp. 2019 (143), e58552

In electrophysiological measurements, the presence of a diffusion potential disturbs the precise measurement of the reverse potential by altering the electrode potential. Using a micro-agar salt bridge, the impact of the diffusion potential is minimized, which allows a more precise measurement of substrate turnover numbers of reconstituted recombinant membrane proteins.

Kreiter 2020
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Kreiter, J; Beitz, E; Pohl, EE “A Fluorescence-Based Method to Measure ADP/ATP Exchange of Recombinant Adenine Nucleotide Translocase in Liposomes” Biomolecules. 2020; 10(5):685

Several mitochondrial proteins, such as adenine nucleotide translocase (ANT), aspartate/glutamate carrier, dicarboxylate carrier, and uncoupling proteins 2 and 3, are suggested to have dual transport functions. While the transport of charge (protons and anions) is characterized by an alteration in membrane conductance, investigating substrate transport is challenging. Currently, mainly radioactively labeled substrates are used, which are very expensive and require stringent precautions during their preparation and use. We present and evaluate a fluorescence-based method using Magnesium Green (MgGrTM), a Mg2+-sensitive dye suitable for measurement in liposomes. Given the different binding affinities of Mg2+ for ATP and ADP, changes in their concentrations can be detected. We obtained an ADP/ATP exchange rate of 3.49 ± 0.41 mmol/min/g of recombinant ANT1 reconstituted into unilamellar liposomes, which is comparable to values measured in mitochondria and proteoliposomes using a radioactivity assay. ADP/ATP exchange calculated from MgGrTM fluorescence solely depends on the ANT1 content in liposomes and is inhibited by the ANT-specific inhibitors, bongkrekic acid and carboxyatractyloside. The application of MgGrTM to investigate ADP/ATP exchange rates contributes to our understanding of ANT function in mitochondria and paves the way for the design of other substrate transport assays.

Zuna 2021
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Žuna, K; Jovanović, O; Khailova, LS; Škulj, S; Brkljača, Z; Kreiter, J; Kotova, EA; Vazdar, M; Antonenko, YN; Pohl, EE “Mitochondrial uncoupling proteins (UCP1-UCP3) and adenine nucleotide translocase (ANT1) enhance the protonophoric action of 2,4-dinitrophenol in mitochondria and planar bilayer membranes” Biomolecules. 2021; 11(8):1178

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 mito-
chondrial 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.

Kreiter 2021
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Kreiter, J; Rupprecht, A; Škulj, S; Brkljača, Z; Žuna, K; Knyazev, DG; Bardakji, S; Vazdar, M; Pohl, EE “Ant1 activation and inhibition patterns support the fatty acid cycling mechanism for proton transport” Int J Mol Sci. 2021; 22(5):2490

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 ∆Ψ, 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.

Skulj 2021
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Škulj, S; Brkljača, Z; Kreiter, J; Pohl, EE; Vazdar, M „Molecular dynamics simulations of mitochondrial uncoupling protein 2“. Int J Mol Sci. 2021; 22(3):1214

Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in
the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreement with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further suggest that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes.

Jovanovic 2022
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Jovanović, O; Chekashkina, K; Škulj, S; Žuna, K; Vazdar, M; Bashkirov, PV; Pohl, EE “Membrane Lipid Reshaping Underlies Oxidative Stress Sensing by the Mitochondrial Proteins UCP1 and ANT1” Antioxidants. 2022; 11(12):2314

Oxidative stress and ROS are important players in the pathogenesis of numerous diseases. In addition to directly altering proteins, ROS also affects lipids with negative intrinsic curvature such as phosphatidylethanolamine (PE), producing PE adducts and lysolipids. The formation of PE adducts potentiates the protonophoric activity of mitochondrial uncoupling proteins, but the
molecular mechanism remains unclear. Here, we linked the ROS-mediated change in lipid shape to the mechanical properties of the membrane and the function of uncoupling protein 1 (UCP1) and
adenine nucleotide translocase 1 (ANT1). We show that the increase in the protonophoric activity of both proteins occurs due to the decrease in bending modulus in lipid bilayers in the presence of
lysophosphatidylcholines (OPC and MPC) and PE adducts. Moreover, MD simulations showed that modified PEs and lysolipids change the lateral pressure profile of the membrane in the same direction and by the similar amplitude, indicating that modified PEs act as lipids with positive intrinsic curvature. Both results indicate that oxidative stress decreases stored curvature elastic stress (SCES) in the lipid bilayer membrane. We demonstrated that UCP1 and ANT1 sense SCES and proposed a novel regulatory mechanism for the function of these proteins. The new findings should draw the attention of the scientific community to this important and unexplored area of redox biochemistry.

Kreiter 2023
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J Kreiter, S Škulj, Z Brkljača, S Bardakji, M Vazdar, EE Pohl@ (2023) FA Sliding as the Mechanism for the ANT1-Mediated Fatty Acid Anion Transport in Lipid Bilayers. Int J Mol Sci 24 (18), 13701. DOI: 10.3390/ijms241813701

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.

Zuna 2024
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Žuna K, Tyschuk T, Beikbaghban T, Sternberg F, Kreiter J, Pohl EE. The 2-oxoglutarate/malate carrier extends the family of mitochondrial carriers capable of fatty acid and 2,4-dinitrophenol-activated proton transport. Acta Physiol (Oxf). 2024 Apr 5:e14143. DOI: 10.1111/apha.14143 . Epub ahead of print. PMID: 38577966 .

Aims: Metabolic reprogramming in cancer cells has been linked to mitochondrial dysfunction. The mitochondrial 2-oxoglutarate/malate carrier (OGC) has been suggested as a potential target for preventing cancer progression. Although OGC is involved in the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. We aimed to investigate whether OGC may contribute to the alteration of mitochondrial inner membrane potential by transporting protons.

Methods: The expression of OGC in mouse tissues and cancer cells was investigated by PCR and Western blot analysis. The proton transport function of recombinant murine OGC was evaluated by measuring the membrane conductance (G_m) of planar lipid bilayers. OGC-mediated substrate transport was measured in proteoliposomes using ¹⁴C-malate.

Results: OGC increases proton G_m only in the presence of natural (long-chain fatty acids, FA) or chemical (2,4-dinitrophenol) protonophores. The increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of the FA. OGC substrates and inhibitors compete with FA for the same protein binding site. Arginine 90 was identified as a critical amino acid for the binding of FA, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism.

Conclusion: OGC extends the family of mitochondrial transporters with dual function: (i) metabolite transport and (ii) proton transport facilitated in the presence of protonophores. Elucidating the contribution of OGC to uncoupling may be essential for the design of targeted drugs for the treatment of cancer and other metabolic diseases.

Keywords: SLC25A11; long‐chain fatty acids; mitochondrial transport; planar bilayer membranes; proton transport; total membrane conductance.