Human mitochondrial ADP/ATP carrier SLC25A4 operates with a ping-pong kinetic mechanism.

The mitochondrial ADP/ATP carrier (SLC25A4), also called the adenine nucleotide translocase, imports ADP into the mitochondrial matrix and exports ATP, which are key steps in oxidative phosphorylation. Historically, the carrier was thought to form a homodimer and to operate by a sequential kinetic mechanism, which involves the formation of a ternary complex with the two exchanged substrates bound simultaneously. However, recent structural and functional data have demonstrated that the mitochondrial ADP/ATP carrier works as a monomer and has a single substrate binding site, which cannot be reconciled with a sequential kinetic mechanism. Here, we study the kinetic properties of the human mitochondrial ADP/ATP carrier by using proteoliposomes and transport robotics. We show that the Km/Vmax ratio is constant for all of the measured internal concentrations. Thus, in contrast to earlier claims, we conclude that the carrier operates with a ping-pong kinetic mechanism in which substrate exchange across the membrane occurs consecutively rather than simultaneously. These data unite the kinetic and structural models, showing that the carrier operates with an alternating access mechanism.

Metabolic impact of genetic and chemical ADP/ATP carrier inhibition in renal proximal tubule epithelial cells.

Mitochondrial dysfunction is pivotal in drug-induced acute kidney injury (AKI), but the underlying mechanisms remain largely unknown. Transport proteins embedded in the mitochondrial inner membrane form a significant class of potential drug off-targets. So far, most transporter-drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC). Since it remains unknown to what extent AAC contributes to drug-induced mitochondrial dysfunction in AKI, we here aimed to better understand the functional role of AAC in the energy metabolism of human renal proximal tubular cells. To this end, CRISPR/Cas9 technology was applied to generate AAC3-/- human conditionally immortalized renal proximal tubule epithelial cells. This AAC3-/- cell model was characterized with respect to mitochondrial function and morphology. To explore whether this model could provide first insights into (mitochondrial) adverse drug effects with suspicion towards AAC-mediated mechanisms, wild-type and knockout cells were exposed to established AAC inhibitors, after which cellular metabolic activity and mitochondrial respiratory capacity were measured. Two AAC3-/- clones showed a significant reduction in ADP import and ATP export rates and mitochondrial mass, without influencing overall morphology. AAC3-/- clones exhibited reduced ATP production, oxygen consumption rates and metabolic spare capacity was particularly affected, mainly in conditions with galactose as carbon source. Chemical AAC inhibition was stronger compared to genetic inhibition in AAC3-/-, suggesting functional compensation by remaining AAC isoforms in our knockout model. In conclusion, our results indicate that ciPTEC-OAT1 cells have a predominantly oxidative phenotype that was not additionally activated by switching energy source. Genetic inhibition of AAC3 particularly impacted mitochondrial spare capacity, without affecting mitochondrial morphology, suggesting an important role for AAC in maintaining the metabolic spare respiration.

KH-17, a simplified derivative of bongkrekic acid, weakly inhibits the mitochondrial ADP/ATP carrier from both sides of the inner mitochondrial membrane.

Two natural products, bongkrekic acid and carboxyatractyloside, are known to specifically inhibit the mitochondrial ADP/ATP carrier from its matrix side and cytosolic side, respectively, in concentration ranges of 10-6  M. In the present study, we investigated the manner of action of a synthetic bongkrekic acid derivative, KH-17, lacking three methyl groups, one methoxy group, and five internal double bonds, on the mitochondrial ADP/ATP carrier. At slightly acidic pH, KH-17 inhibited mitochondrial [3 H]ADP uptake, but its inhibitory action was about 10 times weaker than that of its parental compound, bongkrekic acid. The main site of action of KH-17 was confirmed as the matrix side of the ADP/ATP carrier by experiments using submitochondrial particles, which have an inside-out orientation of the inner mitochondrial membrane. However, when we added KH-17 to mitochondria at neutral pH, it had a weak inhibitory effect on [3 H]ADP uptake, and its inhibitory strength was similar to that of bongkrekic acid. These results indicated that KH-17 weakly inhibits the ADP/ATP carrier not only from the matrix side but also from the cytosolic side. To ascertain whether this interpretation was correct, we examined the effects of KH-17 and carboxyatractyloside on mitochondrial [3 H]ADP uptake at two [3 H]ADP concentrations. We found that both KH-17 and carboxyatractyloside showed a stronger inhibitory effect at the lower [3 H]ADP concentration. Therefore, we concluded that the bongkrekic acid derivative, KH-17, weakly inhibits the mitochondrial ADP/ATP carrier from both sides of the inner mitochondrial membrane. These results suggested that the elimination of three methyl groups, one methoxy group, and five internal double bonds present in bongkrekic acid altered its manner of action towards the mitochondrial ADP/ATP carrier. Our data will help to improve our understanding of the interaction between bongkrekic acid and the mitochondrial ADP/ATP carrier.

Substrate binding in the mitochondrial ADP/ATP carrier is a step-wise process guiding the structural changes in the transport cycle.

Mitochondrial ADP/ATP carriers import ADP into the mitochondrial matrix and export ATP to the cytosol to fuel cellular processes. Structures of the inhibited cytoplasmic- and matrix-open states have confirmed an alternating access transport mechanism, but the molecular details of substrate binding remain unresolved. Here, we evaluate the role of the solvent-exposed residues of the translocation pathway in the process of substrate binding. We identify the main binding site, comprising three positively charged and a set of aliphatic and aromatic residues, which bind ADP and ATP in both states. Additionally, there are two pairs of asparagine/arginine residues on opposite sides of this site that are involved in substrate binding in a state-dependent manner. Thus, the substrates are directed through a series of binding poses, inducing the conformational changes of the carrier that lead to their translocation. The properties of this site explain the electrogenic and reversible nature of adenine nucleotide transport.

The effects of cardiolipin on the structural dynamics of the mitochondrial ADP/ATP carrier in its cytosol-open state.

Cardiolipin (CL) has been shown to play a crucial role in regulating the function of proteins in the inner mitochondrial membrane. As the most abundant protein of the inner mitochondrial membrane, the ADP/ATP carrier (AAC) has long been the model of choice to study CL-protein interactions, and specifically bound CLs have been identified in a variety of crystal structures of AAC. However, how CL binding affects the structural dynamics of AAC in atomic detail remains largely elusive. Here we compared all-atom molecular dynamics simulations on bovine AAC1 in lipid bilayers with and without CLs. Our results show that on the current microsecond simulation time scale: 1) CL binding does not significantly affect overall stability of the carrier or structural symmetry at the matrix-gate level; 2) pocket volumes of the carrier and interactions involved in the matrix-gate network become more heterogeneous in parallel simulations with membranes containing CLs; 3) CL binding consistently strengthens backbone hydrogen bonds within helix H2 near the matrix side; and 4) CLs play a consistent stabilizing role on the domain 1-2 interface through binding with the R30:R71:R151 stacking structure and fixing the M2 loop in a defined conformation. CL is necessary for the formation of this stacking structure, and this structure in turn forms a very stable CL binding site. Such a delicate equilibrium suggests the strictly conserved R30:R71:R151stacking structure of AACs could function as a switch under regulation of CLs. Taken together, these results shed new light on the CL-mediated modulation of AAC function.

Novel US-CpHMD Protocol to Study the Protonation-Dependent Mechanism of the ATP/ADP Carrier.

We have designed a protocol combining constant-pH molecular dynamics (CpHMD) simulations with an umbrella sampling (US) scheme (US-CpHMD) to study the mechanism of ADP/ATP transport (import and export) by their inner mitochondrial membrane carrier protein [ADP/ATP carrier (AAC)]. The US scheme helped overcome the limitations of sampling the slow kinetics involved in these substrates' transport, while CpHMD simulations provided an unprecedented realism by correctly capturing the associated protonation changes. The import of anionic substrates along the mitochondrial membrane has a strong energetic disadvantage due to a smaller substrate concentration and an unfavorable membrane potential. These limitations may have created an evolutionary pressure on AAC to develop specific features benefiting the import of ADP. In our work, the potential of mean force profiles showed a clear selectivity in the import of ADP compared to ATP, while in the export, no selectivity was observed. We also observed that AAC sequestered both substrates at longer distances in the import compared to the export process. Furthermore, only in the import process do we observe transient protonation of both substrates when going through the AAC cavity, which is an important advantage to counteract the unfavorable mitochondrial membrane potential. Finally, we observed a substrate-induced disruption of the matrix salt-bridge network, which can promote the conformational transition (from the C- to M-state) required to complete the import process. This work unraveled several important structural features where the complex electrostatic interactions were pivotal to interpreting the protein function and illustrated the potential of applying the US-CpHMD protocol to other transport processes involving membrane proteins.

Conformational change of adenine nucleotide translocase-1 mediates cisplatin resistance induced by EBV-LMP1.

Adenine nucleotide translocase-1 (ANT1) is an ADP/ATP transporter protein located in the inner mitochondrial membrane. ANT1 is involved not only in the processes of ADP/ATP exchange but also in the composition of the mitochondrial membrane permeability transition pore (mPTP); and the function of ANT1 is closely related to its own conformational changes. Notably, various viral proteins can interact directly with ANT1 to influence mitochondrial membrane potential by regulating the opening of mPTP, thereby affecting tumor cell fate. The Epstein-Barr virus (EBV) encodes the key tumorigenic protein, latent membrane protein 1 (LMP1), which plays a pivotal role in promoting therapeutic resistance in related tumors. In our study, we identified a novel mechanism for EBV-LMP1-induced alteration of ANT1 conformation in cisplatin resistance in nasopharyngeal carcinoma. Here, we found that EBV-LMP1 localizes to the inner mitochondrial membrane and inhibits the opening of mPTP by binding to ANT1, thereby favoring tumor cell survival and drug resistance. The ANT1 conformational inhibitor carboxyatractyloside (CATR) in combination with cisplatin improved the chemosensitivity of EBV-LMP1-positive cells. This finding confirms that ANT1 is a novel therapeutic target for overcoming cisplatin resistance in the future.

A Single Cysteine Residue in the Translocation Pathway of the Mitosomal ADP/ATP Carrier from Cryptosporidium parvum Confers a Broad Nucleotide Specificity.

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.

Cardiolipin, conformation, and respiratory complex-dependent oligomerization of the major mitochondrial ADP/ATP carrier in yeast.

The phospholipid cardiolipin has pleiotropic structural and functional roles that are collectively essential for mitochondrial biology. Yet, the molecular details of how this lipid supports the structure and function of proteins and protein complexes are poorly understood. To address this property of cardiolipin, we use the mitochondrial adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier (Aac) as a model. Here, we have determined that cardiolipin is critical for both the tertiary and quaternary assembly of the major yeast Aac isoform Aac2 as well as its conformation. Notably, these cardiolipin-provided structural roles are separable. In addition, we show that multiple copies of Aac2 engage in shared complexes that are largely dependent on the presence of assembled respiratory complexes III and IV or respiratory supercomplexes. Intriguingly, the assembly state of Aac2 is sensitive to its transport-related conformation. Together, these results expand our understanding of the numerous structural roles provided by cardiolipin for mitochondrial membrane proteins.

Molecular dynamics simulations on apo ADP/ATP carrier shed new lights on the featured motif of the mitochondrial carriers.

The ADP/ATP carrier (AAC) is a transporter responsible for the equal molar exchange of cytosolic ADP and ATP synthesized within mitochondrial matrix across the mitochondrial membrane. Its primary structure consists of three homologous repeats, and each repeat contains a conserved motif that is shared by all members of the mitochondrial carrier family (MCF). Although these MCF motif residues cluster together in the crystal structure of AAC, detailed analyses on the interactions among the motif residues are still limited. In the present study, all-atom molecular dynamics (MD) simulations of up to 10 µs have been carried out on AAC, and interactions and structural dynamics of the MCF motif residues have been specifically investigated. Our simulations have revealed: i) a very asymmetrical electrostatic network at the bottom of the pocket of apo AAC, ii) the asymmetrical interactions between the Pro kink region and the [YWF][KR] G motif in three repeats, iii) the role of the conserved Arg residues in stabilizing the C-ends of the odd-numbered helices, iv) the structural change of the [YWF][KR] G motif and its potential involvement in substrate translocation process. Our results highlight the asymmetry of the MCF residues in the three repeats, which might contribute to the ability of the carriers to transport the asymmetrical substrates. Our observations provide microscopic basis for further research on the translocation mechanism of mitochondrial carriers.

Calcium Induces Mitochondrial Oxidative Stress Because of its Binding to Adenine Nucleotide Translocase.

Several studies have demonstrated that the mitochondrial membrane switches from selective to non-selective permeability because of its improved matrix Ca2+ accumulation and oxidative stress. This process, known as permeability transition, evokes severe dysfunction in mitochondria through the opening of a non-specific pore, whose chemical nature is still under discussion. There are some proposals regarding the components of the pore structure, e.g., the adenine nucleotide translocase and dimers of the F1 Fo-ATP synthase. Our results reveal that Ca2+ induces oxidative stress, which not only increases lipid peroxidation and ROS generation but also brings about both the collapse of the transmembrane potential and the membrane release of cytochrome c. Additionally, it is shown that Ca2+ increases the binding of the probe eosin-5-maleimide to adenine nucleotide translocase. Interestingly, these effects are diminished after the addition of ADP. It is suggested that pore opening is caused by the binding of Ca2+ to the adenine nucleotide translocase.

Chemoproteomic Profiling Reveals Ethacrynic Acid Targets Adenine Nucleotide Translocases to Impair Mitochondrial Function.

Ethacrynic acid (EA) is a diuretic drug that is widely used to treat high-blood pressure and swelling caused by congestive heart failure or kidney failure. It acts through noncovalent inhibition of the Na+-K+-2Cl- cotransporter in the thick ascending limb of Henle's loop. Chemically, EA contains a Michael acceptor group that can react covalently with nucleophilic residues in proteins; however, the proteome reactivity of EA remains unexplored. Herein, we took a quantitative chemoproteomic approach to globally profile EA's targets in cancer cells. We discovered that EA induces impaired mitochondrial function accompanied by increased ROS production. Our profiling revealed that EA targets functional proteins on mitochondrial membranes, including adenine nucleotide translocases (ANTs). Site-specific mapping identified that EA covalently modifies a functional cysteine in ANTs, a mutation of which resulted in the rescuing effect on EA-induced mitochondrial dysfunction. The newly discovered modes of action offer valuable information to repurpose EA for cancer treatment.

Atomistic modeling of alternating access of a mitochondrial ADP/ATP membrane transporter with molecular simulations.

The mitochondrial ADP/ATP carrier (AAC) is a membrane transporter that exchanges a cytosolic ADP for a matrix ATP. Atomic structures in an outward-facing (OF) form which binds an ADP from the intermembrane space have been solved by X-ray crystallography, and revealed their unique pseudo three-fold symmetry fold which is qualitatively different from pseudo two-fold symmetry of most transporters of which atomic structures have been solved. However, any atomic-level information on an inward-facing (IF) form, which binds an ATP from the matrix side and is fixed by binding of an inhibitor, bongkrekic acid (BA), is not available, and thus its alternating access mechanism for the transport process is unknown. Here, we report an atomic structure of the IF form predicted by atomic-level molecular dynamics (MD) simulations of the alternating access transition with a recently developed accelerating technique. We successfully obtained a significantly stable IF structure characterized by newly formed well-packed and -organized inter-domain interactions through the accelerated simulations of unprecedentedly large conformational changes of the alternating access without a prior knowledge of the target protein structure. The simulation also shed light on an atomistic mechanism of the strict transport selectivity of adenosine nucleotides over guanosine and inosine ones. Furthermore, the IF structure was shown to bind ATP and BA, and thus revealed their binding mechanisms. The present study proposes a qualitatively novel view of the alternating access of transporters having the unique three-fold symmetry in atomic details and opens the way for rational drug design targeting the transporter in the dynamic functional cycle.

Modelling the free energy profile of the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier catalyses the equimolar exchange of adenosine di- and tri-phosphates. It operates by an alternating access mechanism in which a single substrate-binding site is made available either to the mitochondrial matrix or the intermembrane space through conformational changes. These changes are prevented in the absence of substrate by a large energy barrier due to the need for sequential disruption and formation of a matrix and cytoplasmic salt bridge network that are located on either side of the central cavity. In analogy to enzyme catalysis, substrate lowers the energy barrier by binding tighter in the intermediate state. Here we provide an in-silico kinetic model that captures the free energy profile of these conformational changes and treats the carrier as a nanomachine moving stochastically from the matrix to cytoplasmic conformation under the influence of thermal energy. The model reproduces the dependency of experimentally determined kcat and KM values on the cytoplasmic network strength with good quantitative accuracy, implying that it captures the transport mechanism and can provide a framework to understand the structure-function relationships of this class of transporter. The results show that maximum transport occurs when the interaction energies of the cytoplasmic network, matrix network and substrate binding are approximately equal such that the energy barrier is minimized. Consequently, the model predicts that there will be other interactions in addition to those of the cytoplasmic network that stabilise the matrix conformation of the ADP/ATP carrier.

New insights about the structural rearrangements required for substrate translocation in the bovine mitochondrial oxoglutarate carrier.

The oxoglutarate carrier (OGC) belongs to the mitochondrial carrier family and plays a key role in important metabolic pathways. Here, site-directed mutagenesis was used to conservatively replace lysine 122 by arginine, in order to investigate new structural rearrangements required for substrate translocation. K122R mutant was kinetically characterized, exhibiting a significant Vmax reduction with respect to the wild-type (WT) OGC, whereas Km value was unaffected, implying that this substitution does not interfere with 2-oxoglutarate binding site. Moreover, K122R mutant was more inhibited by several sulfhydryl reagents with respect to the WT OGC, suggesting that the reactivity of some cysteine residues towards these Cys-specific reagents is increased in this mutant. Different sulfhydryl reagents were employed in transport assays to test the effect of the cysteine modifications on single-cysteine OGC mutants named C184, C221, C224 (constructed in the WT background) and K122R/C184, K122R/C221, K122R/C224 (constructed in the K122R background). Cysteines 221 and 224 were more deeply influenced by some sulfhydryl reagents in the K122R background. Furthermore, the presence of 2-oxoglutarate significantly enhanced the degree of inhibition of K122R/C221, K122R/C224 and C224 activity by the sulfhydryl reagent 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), suggesting that cysteines 221 and 224, together with K122, take part to structural rearrangements required for the transition from the c- to the m-state during substrate translocation. Our results are interpreted in the light of the homology model of BtOGC, built by using as a template the X-ray structure of the bovine ADP/ATP carrier isoform 1 (AAC1).

The transport mechanism of the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

The switching mechanism of the mitochondrial ADP/ATP carrier explored by free-energy landscapes.

The ADP/ATP carrier (AAC) of mitochondria has been an early example for elucidating the transport mechanism alternating between the external (c-) and internal (m-) states (M. Klingenberg, Biochim. Biophys. Acta 1778 (2008) 1978-2021). An atomic resolution crystal structure of AAC is available only for the c-state featuring a three repeat transmembrane domain structure. Modeling of transport mechanism remained hypothetical for want of an atomic structure of the m-state. Previous molecular dynamics studies simulated the binding of ADP or ATP to the AAC remaining in the c-state. Here, a full description of the AAC switching from the c- to the m-state is reported using well-tempered metadynamics simulations. Free-energy landscapes of the entire translocation from the c- to the m-state, based on the gyration radii of the c- and m-gates and of the center of mass, were generated. The simulations revealed three free-energy basins attributed to the c-, intermediate- and m-states separated by activation barriers. These simulations were performed with the empty and with the ADP- and ATP-loaded AAC as well as with the poorly transported AMP and guanine nucleotides, showing in the free energy landscapes that ADP and ATP lowered the activation free-energy barriers more than the other substrates. Upon binding AMP and guanine nucleotides a deeper free-energy level stabilized the intermediate-state of the AAC2 hampering the transition to the m-state. The structures of the substrate binding sites in the different states are described producing a full picture of the translocation events in the AAC.

Identification of new highly selective inhibitors of the human ADP/ATP carriers by molecular docking and in vitro transport assays.

Mitochondrial carriers are proteins that shuttle a variety of metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. The mitochondrial ADP/ATP carriers (AACs) specifically translocate the ATP synthesized within mitochondria to the cytosol in exchange for the cytosolic ADP, playing a key role in energy production, in promoting cell viability and regulating mitochondrial permeability transition pore opening. In Homo sapiens four genes code for AACs with different tissue distribution and expression patterns. Since AACs are dysregulated in several cancer types, the employment of known and new AAC inhibitors might be crucial for inducing mitochondrial-mediated apoptosis in cancer cells. Albeit carboxyatractyloside (CATR) and bongkrekic acid (BKA) are known to be powerful and highly selective AAC inhibitors, able to induce mitochondrial dysfunction at molecular level and poisoning at physiological level, we estimated here for the first time their affinity for the human recombinant AAC2 by in vitro transport assays. We found that the inhibition constants of CATR and BKA are 4 nM and 2.0 µM, respectively. For finding new AAC inhibitors we also performed a docking-based virtual screening of an in-house developed chemical library and we identified about 100 ligands showing high affinity for the AAC2 binding region. By testing 13 commercially available molecules, out of the 100 predicted candidates, we found that 2 of them, namely suramin and chebulinic acid, are competitive AAC2 inhibitors with inhibition constants 0.3 µM and 2.1 µM, respectively. We also demonstrated that chebulinic acid and suramin are "highly selective" AAC2 inhibitors, since they poorly inhibit other human mitochondrial carriers (namely ORC1, APC1 and AGC1).

An adenine nucleotide translocase (ANT) gene from Apostichopus japonicus; molecular cloning and expression analysis in response to lipopolysaccharide (LPS) challenge and thermal stress.

The adenine nucleotide translocases (ANTs) play a vital role in energy metabolism via ADP/ATP exchange in eukaryotic cells. Apostichopus japonicus (Echinodermata: Holothuroidea) is an important economic species in China. Here, a cDNA representing an ANT gene of A. japonicus was isolated and characterized from respiratory tree and named AjANT. The full-length AjANT cDNA is 1924 bp, including a 5'-untranslated region (UTR) of 38 bp, 3'-UTR of 980 bp and an open reading frame (ORF) of 906 bp encoding a polypeptide of 301 amino acids. The protein contains three homologous repeat Mito_carr domains (Pfam00153). The deduced AjANT protein sequence has 49-81% in comparison to ANT proteins from other individuals. The predicted tertiary structure of AjANT protein is highly similar to animal ANT proteins. Phylogenetic analysis showed that the AjANT is closely related to Holothuroidea ANT genes. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that AjANT expression is higher in the respiratory tree than in other examined tissues. After thermal stress or LPS challenge, expression of AjANT was significantly fluctuant compared to the control. These results suggested that changes in the expression of ANT gene might be involved in immune defense and in protecting A. japonicus against thermal stress.

Diamide accelerates opening of the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

Opening of the mitochondrial permeability transition pore (MPTP) in the inner membrane is due to matrix Ca(2+) overload and matrix glutathione loss. Fixing the 'm' conformation of the adenine nucleotide translocase (ANT) by ADP or N-ethylmaleimide (NEM) inhibits opening of the MPTP. Oxidants (diamide or tert-butylhydroperoxide (tBHP)) fix the ANT in 'c' conformation, and the ability of ADP to inhibit the MPTP is thus attenuated. Earlier we found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria resulted in Tl(+)-induced MPTP opening, which was accompanied by a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration, as well as increased swelling and membrane potential dissipation. These effects, which were increased by diamide and tBHP, were visibly reduced in the presence of the MPTP inhibitors (ADP, NEM, and cyclosporine A). Our data suggest that conformational changes of the ANT and matrix glutathione loss may be directly involved in opening the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria.