**1. Introduction**

In mitochondria, oxidative phosphorylation accounts for ATP production by phosphorylating ADP using proton (H+) gradient generated by the respiratory chain proteins (coupling). H+ can return to the matrix by alternative pathways (uncoupling): (i) inhibitor-non sensitive basal H+ leak (JB) and (ii) protein-mediated inhibitor-sensitive proton transport (JH) [1–3]. JB is sensitive to the membrane potential, mitochondrial inner membrane surface area, the composition of phospholipids and free fatty acids (FA) and was observed in

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**Citation:** Kreiter, J.; Rupprecht, A.; Škulj, S.; Brkljaˇca, Z.; Žuna, K.; Knyazev, D.G.; Bardakji, S.; Vazdar, M.; Pohl, E.E. ANT1 Activation and Inhibition Patterns Support the Fatty Acid Cycling Mechanism for Proton Transport. *Int. J. Mol. Sci.* **2021**, *22*, 2490. https://doi.org/10.3390/ ijms22052490

Academic Editor: Masoud Jelokhani-Niaraki

Received: 8 January 2021 Accepted: 24 February 2021 Published: 2 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

mitochondria of all tissues [1]. Uncoupling proteins (UCP) are implicated in the mediation of J H [4–9]. As several tissues such as liver, kidney, skin, and others lack any UCPs under physiological conditions (for review, see [10]), mitochondrial adenine nucleotide translocase (ANT, also cited in the literature as AAC or ADP/ATP carrier) was proposed to provide an alternative pathway for proton transport alongside its well-known function to exchange ADP for ATP [11–15].

The H+ transporting function of ANT in the presence of palmitate has been first observed in experiments with isolated mitochondria [16,17]. The addition of free FA to the proteoliposomes reconstituted with purified ANT caused the transmembrane potential (ΔΦ) decrease, which was restored by carboxyatractyloside (CATR) and bongkrekic acid (BA) [18]. In brown-fat mitochondria from mice knockout for UCP1, fatty-acid-induced uncoupling could also be inhibited by CATR [19]. The H+ conductance of muscle mitochondria from mice knockout for ANT1 was reported to be half that of wild-type controls [20]. Recently, ANT-mediated H+ transport was observed in patched mitoplasts [21]. Although the H+ transporting function of ANT1 seems to be accepted, discrepancies in results obtained in various experimental systems led to different views on the proton transport mechanism. In the 1980–1990s, several groups recognized that the proton transport could occur by the flip-flop of the protonated form of long-chain fatty acid (FA) without membrane proteins' participation [22,23]. However, a fatty acid anion (FA−) transport is a rate-limiting step in FA circulation and has to be accelerated by proteins. In 1991, Skulachev proposed the "fatty acid circuit hypothesis", claiming that proteins such as ANT1 and UCP1 mediate the return of the FA- to the cytosolic side of the membrane, resulting in net proton transport catalyzed by the protein [24]. Our previous results obtained for UCP1-UCP3 can be well described based on the FA cycling model and are consistent with the translocation of FA− at the protein/lipid interface.

In contrast, Bertholet et al. proposed FA to be co-factors in H+ transport by ANT based on patch-clamp experiments [21]. In this model, FA is not translocated but stays in one place as a part of the protein translocating pathway, where it is (de-)protonated. This mechanism differed from the mechanism suggested by the same group for UCP1, in which UCP1 was regarded as a FA–/H+ symporter [25]. Moreover, this model does not necessarily assume direct binding of H+ to the FA anion and allows the proton transport in both directions.

Here, we hypothesized that the H+-transport mediated by ANT has a regulation pattern similar to UCPs and can be explained by the FA cycling concept. The goals of this study were (i) to investigate the dependence of ANT-mediated H+ transport on FA structure, (ii) to estimate ANT-specific H+ turnover number, and (iii) to examine whether the specific ANT substrates inhibit H+ transport.
