Human mitochondrial carriers of the SLC25 family function as monomers exchanging substrates with a ping-pong kinetic mechanism.

Members of the SLC25 mitochondrial carrier family link cytosolic and mitochondrial metabolism and support cellular maintenance and growth by transporting compounds across the mitochondrial inner membrane. Their monomeric or dimeric state and kinetic mechanism have been a matter of long-standing debate. It is believed by some that they exist as homodimers and transport substrates with a sequential kinetic mechanism, forming a ternary complex where both exchanged substrates are bound simultaneously. Some studies, in contrast, have provided evidence indicating that the mitochondrial ADP/ATP carrier (SLC25A4) functions as a monomer, has a single substrate binding site, and operates with a ping-pong kinetic mechanism, whereby ADP is imported before ATP is exported. Here we reanalyze the oligomeric state and kinetic properties of the human mitochondrial citrate carrier (SLC25A1), dicarboxylate carrier (SLC25A10), oxoglutarate carrier (SLC25A11), and aspartate/glutamate carrier (SLC25A13), all previously reported to be dimers with a sequential kinetic mechanism. We demonstrate that they are monomers, except for dimeric SLC25A13, and operate with a ping-pong kinetic mechanism in which the substrate import and export steps occur consecutively. These observations are consistent with a common transport mechanism, based on a functional monomer, in which a single central substrate-binding site is alternately accessible.

The organic anion transporter (OAT) family: a systems biology perspective.

The organic anion transporter (OAT) subfamily, which constitutes roughly half of the SLC22 (solute carrier 22) transporter family, has received a great deal of attention because of its role in handling of common drugs (antibiotics, antivirals, diuretics, nonsteroidal anti-inflammatory drugs), toxins (mercury, aristolochic acid), and nutrients (vitamins, flavonoids). Oats are expressed in many tissues, including kidney, liver, choroid plexus, olfactory mucosa, brain, retina, and placenta. Recent metabolomics and microarray data from Oat1 [Slc22a6, originally identified as NKT (novel kidney transporter)] and Oat3 (Slc22a8) knockouts, as well as systems biology studies, indicate that this pathway plays a central role in the metabolism and handling of gut microbiome metabolites as well as putative uremic toxins of kidney disease. Nuclear receptors and other transcription factors, such as Hnf4α and Hnf1α, appear to regulate the expression of certain Oats in conjunction with phase I and phase II drug metabolizing enzymes. Some Oats have a strong selectivity for particular signaling molecules, including cyclic nucleotides, conjugated sex steroids, odorants, uric acid, and prostaglandins and/or their metabolites. According to the "Remote Sensing and Signaling Hypothesis," which is elaborated in detail here, Oats may function in remote interorgan communication by regulating levels of signaling molecules and key metabolites in tissues and body fluids. Oats may also play a major role in interorganismal communication (via movement of small molecules across the intestine, placental barrier, into breast milk, and volatile odorants into the urine). The role of various Oat isoforms in systems physiology appears quite complex, and their ramifications are discussed in the context of remote sensing and signaling.

Structures suggest a mechanism for energy coupling by a family of organic anion transporters.

Members of the solute carrier 17 (SLC17) family use divergent mechanisms to concentrate organic anions. Membrane potential drives uptake of the principal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use proton cotransport to drive efflux from the lysosome. To delineate the divergent features of ionic coupling by the SLC17 family, we determined the structure of Escherichia coli D-galactonate/H+ symporter D-galactonate transporter (DgoT) in 2 states: one open to the cytoplasmic side and the other open to the periplasmic side with substrate bound. The structures suggest a mechanism that couples H+ flux to substrate recognition. A transition in the role of H+ from flux coupling to allostery may confer regulation by trafficking to and from the plasma membrane.

Engineering energetically efficient transport of dicarboxylic acids in yeast Saccharomyces cerevisiae.

Biobased C4-dicarboxylic acids are attractive sustainable precursors for polymers and other materials. Commercial scale production of these acids at high titers requires efficient secretion by cell factories. In this study, we characterized 7 dicarboxylic acid transporters in Xenopus oocytes and in Saccharomyces cerevisiae engineered for dicarboxylic acid production. Among the tested transporters, the Mae1(p) from Schizosaccharomyces pombe had the highest activity toward succinic, malic, and fumaric acids and resulted in 3-, 8-, and 5-fold titer increases, respectively, in S. cerevisiae, while not affecting growth, which was in contrast to the tested transporters from the tellurite-resistance/dicarboxylate transporter (TDT) family or the Na+ coupled divalent anion-sodium symporter family. Similar to SpMae1(p), its homolog in Aspergillus carbonarius, AcDct(p), increased the malate titer 12-fold without affecting the growth. Phylogenetic and protein motif analyses mapped SpMae1(p) and AcDct(p) into the voltage-dependent slow-anion channel transporter (SLAC1) clade of transporters, which also include plant Slac1(p) transporters involved in stomata closure. The conserved phenylalanine residue F329 closing the transport pore of SpMae1(p) is essential for the transporter activity. The voltage-dependent SLAC1 transporters do not use proton or Na+ motive force and are, thus, less energetically expensive than the majority of other dicarboxylic acid transporters. Such transporters present a tremendous advantage for organic acid production via fermentation allowing a higher overall product yield.

Molecular nature and regulation of the mitochondrial permeability transition pore(s), drug target(s) in cardioprotection.

The mitochondrial permeability transition, an established mechanism for heart diseases, is a long-standing mystery of mitochondrial biology and a prime drug target for cardioprotection. Several hypotheses about its molecular nature have been put forward over the years, and the prevailing view is that permeabilization of the inner mitochondrial membrane follows opening of a high-conductance channel, the permeability transition pore, which is also called mitochondrial megachannel or multiconductance channel. The permeability transition strictly requires matrix Ca2+ and is favored by the matrix protein cyclophilin D, which mediates the inhibitory effects of cyclosporin A. Here we provide a review of the field, with specific emphasis on the possible role of the adenine nucleotide translocator and of the F-ATP synthase in channel formation, and on currently available small molecule inhibitors. While the possible mechanisms through which the adenine nucleotide translocator and the F-ATP synthase might form high-conductance channels remain unknown, reconstitution experiments and site-directed mutagenesis combined to electrophysiology have provided important clues. The hypothesis that more than one protein may act as a permeability transition pore provides a reasonable explanation for current controversies in the field, and holds great promise for the solution of the mystery of the permeability transition.

The malate-activated ALMT12 anion channel in the grass Brachypodium distachyon is co-activated by Ca2+/calmodulin.

In plants, strict regulation of stomatal pores is critical for modulation of CO2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca2+) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminum-activated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate- and voltage-dependent. However, this was shown to be true only in the presence of Ca2+ Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca2+-dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca2+ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca2+/CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.

Two- and three-dimensional QSAR studies on hURAT1 inhibitors with flexible linkers: topomer CoMFA and HQSAR.

hURAT1 (human urate transporter 1) is a successful target for hyperuricemia. Recently, the modification work on hURAT1 inhibitors showed that the flexible linkers would benefit biological activity. The study aimed to investigate the contribution of the linkers and give modification strategies on this kind of structures based on QSAR models (HQSAR and topomer CoMFA). The most effective HQSAR and topomer CoMFA models were generated by applying the training set containing 63 compounds, with the cross-validated q2 values of 0.869/0.818 and the non-cross-validated correlation coefficients r2 of 0.951/0.978, respectively. The Y-randomization test was applied to ensure the robustness of the models. The external predictive correlation coefficient (rpred2) grounded on the external test set (21 compounds) of two models was 0.910 and 0.907, respectively. In addition, the models were validated by Golbraikh-Tropsha and Roy methods, as well as other statistical metrics. The results showed that both models were reliable. Topomer CoMFA steric/electrostatic contours and HQSAR atomic contribution maps illustrated the structural features which governed their inhibitory potency. The dependable results could provide important insights to guide the designing of more potential hURAT1 inhibitors.

Stimuli-Responsive Cycloaurated "OFF-ON" Switchable Anion Transporters.

Anion transporters have shown potential application as anti-cancer agents that function by disrupting homeostasis and triggering cell death. In this research article we report switchable anion transport by gold complexes of anion transporters that are "switched on" in situ in the presence of the reducing agent GSH by decomplexation of gold. GSH is found in higher concentrations in tumors than in healthy tissue and hence this approach offers a strategy to target these systems to tumors.

Crystal structure of the bacterial acetate transporter SatP reveals that it forms a hexameric channel.

Acetate is found ubiquitously in the natural environment and can be used as an exogenous carbon source by bacteria, fungi, and mammalian cells. A representative member of the acetate uptake transporter (AceTr) family named SatP (also yaaH) has been preliminarily identified as a succinate-acetate/proton symporter in Escherichia coli However, the molecular mechanism of acetate uptake by SatP still remains elusive. Here, we report the crystal structure of SatP from E. coli at 2.8 Å resolution, determined with a molecular replacement approach using a previously developed predicted model algorithm, which revealed a hexameric UreI-like channel structure. Structural analysis identified six transmembrane (TM) helices surrounding the central channel pore in each protomer and three conserved hydrophobic residues, FLY, located in the middle of the TM region for pore constriction. According to single-channel conductance recordings, performed with purified SatP reconstituted into lipid bilayer, three conserved polar residues in the TM1 facing to the periplasmic side are closely associated with acetate translocation activity. These analyses provide critical insights into the mechanism of acetate translocation in bacteria and a first glimpse of a structure of an AceTr family transporter.

Genetic variability and population diversity of the human SLCO (OATP) transporter family.

Organic anion transporting polypeptides (OATP) encoded by the SLCO gene family constitute clinically important transporters involved in the disposition of endogenous compounds and many commonly prescribed drugs, including statins, methotrexate and antihypertensive medications. Common genetic polymorphisms in SLCO genes are known to affect OATP function and modulate efficacy and safety of OATP substrates. However, current frequency data of these variants and haplotypes is generally based on few rather heterogenous populations of relatively small sample size. Furthermore, the genetic variability beyond these selected pharmacogenetic biomarkers has not been systematically analyzed. Here, we provide a global consolidated map of SLCO variability by leveraging fully compatible Next Generation Sequencing data from 138,632 unrelated individuals across seven major human populations. Overall, we find 9811 exonic single nucleotide variants and 155 copy number variations of which 99.3% were rare with frequencies <1%. Using orthogonal computational functionality predictors optimized for pharmacogenetic assessments, we find that four out of five individuals carry at least one deleterious variant in an SLCO transporter gene and rare variants contribute 23% to the genetically encoded functional variability. Moreover, 74.9% of all variants were found to be population-specific with important consequences for population-specific genotyping strategies and precision public health approaches. Combined, our analyses provide the most comprehensive data set of SLCO variability published to date and incentivize the integration of comprehensive NGS-based genotyping into personalized predictions of OATP substrate disposition.

Molecular Mechanism of Acetate Transport through the Acetate Channel SatP.

Acetate is a central metabolite that plays a key role in almost all organisms, and acetate channels are often essential for their survival. Recently solved structures of the acetate channel Succinate-Acetate Permease (SatP) provide an atomic view of its closed state. However, the open state of the channel, the key residue conformational changes that trigger the channel to open, and the free energy barrier of acetate transportation remain elusive. To address these questions, we performed microsecond time scale molecular dynamics (MD) simulations and umbrella sampling. Several acetate passing events were observed in the MD trajectories with the application of an external electric field. Further analyses reveal the molecular mechanism of the channel opening, which results from the repacking of key residues, such as Gln50 and Phe17, as well as the subsequent outward movement of all transmembrane helices. Our simulations show that acetate is always surrounded by several water molecules when passing through the channel. Furthermore, a high energy barrier of 15 kcal/mol was observed from the free energy profile generated by umbrella sampling on the closed state of the channel. Our study deepens the understanding of the molecular mechanism of acetate transport through the channel SatP and is expected to facilitate the drug discovery on this target.

Integrative Data Mining, Scaffold Analysis, and Sequential Binary Classification Models for Exploring Ligand Profiles of Hepatic Organic Anion Transporting Polypeptides.

Hepatocellular organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1) are important for proper liver function and the regulation of the drug elimination process. Understanding their roles in different conditions of liver toxicity and cancer requires an in-depth investigation of hepatic OATP-ligand interactions and selectivity. However, such studies are impeded by the lack of crystal structures, the promiscuous nature of these transporters, and the limited availability of reliable bioactivity data, which are spread over different data sources in the open domain. To this end, we integrated ligand bioactivity data for hepatic OATPs from five open data sources (ChEMBL, the UCSF-FDA TransPortal database, DrugBank, Metrabase, and IUPHAR) in a semiautomatic KNIME workflow. Highly curated data sets were analyzed with respect to enriched scaffolds, and their activity profiles and interesting scaffold series providing indication for selective, dual-, or pan-inhibitory activity toward hepatic OATPs could be extracted. In addition, a sequential binary modeling approach revealed common and distinctive ligand features for inhibitory activity toward the individual transporters. The workflows designed for integrating data from open sources, data curation, and subsequent substructure analyses are freely available and fully adaptable. The new data sets for inhibitors and substrates of hepatic OATPs as well as the insights provided by the feature and substructure analyses will guide future structure-based studies on hepatic OATP-ligand interactions and selectivity.

The mechanism of Oatp1a5-mediated microcystin-leucine arginine entering into GnRH neurons.

Microcystin-leucine arginine (MC-LR) enters into gonadotropin-releasing hormone (GnRH) neurons and induces decline of serum GnRH levels resulting in male reproductive toxicity via hypothalamic-pituitary-testis axis. The organic anion transporting polypeptide 1a5 (Oatp1a5) is a critical transporter for the uptake of MC-LR by GnRH neurons. However, the underlying molecular mechanisms of the transport process are still elusive. In this study, we found that the transmembrane domains 2, 8, and 9 played important roles in transporting function of Oatp1a5. In addition, our data demonstrated that N-linked glycosylation was involved in the transport of MC-LR by Oatp1a5. Moreover, we showed that N-linked glycosylation sites Asn483 and Asn492 were vital for the transport function of Oatp1a5. In summary, the study furthered our understanding of mechanisms that the uptake of MC-LR by GnRH neurons and laid a theoretical foundation for preventing MC-LR from injuring male reproductive health.

Transmembrane Domain 1 of Human Organic Anion Transporting Polypeptide 2B1 Is Essential for Transporter Function and Stability.

Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are important membrane transporter proteins that mediate the uptake of wide ranges of endogenous and exogenous compounds. OATP2B1 has been found in multiple organs and tissues, including the liver, small intestine, kidney, brain, placenta, heart, skin, as well as skeletal muscle, and is proposed to be involved in the uptake of orally administered drugs. Quite a few reports have demonstrated that transmembrane domains (TMs) are crucial for proper functions of OATP family members. Comparative modeling proposed that TM1, along with TM2, 4, and 5 of the N-terminal half of OATP2B1, may be localized within the substrate interaction pocket and are important for uptake function of the transporter. Alanine scanning of the putative transmembrane domain 1 of OATP2B1 revealed that substitution of L58 with alanine dramatically altered the Km value, and mutation of V52, H55, Q59, and L69 resulted in significantly reduced substrate turnover number, whereas A61V, Q62A, and S66A exhibited significant change in both Km and Vmax values. In addition, phenylalanine at position 51 seems to play an important role in maintaining proper folding of OATP2B1 because alanine replacement of F51 caused accelerated degradation of the transporter protein. Although proteasome and lysosome inhibitors could partially recover protein level, the mutant transporter remained nonfunctional. Taken together, the identification of nine essential amino acid residues within TM1 of OATP2B1 suggested that the transmembrane domain is important for maintaining proper function of the transporter.

Effect of Rifampicin on the Distribution of [11C]Erlotinib to the Liver, a Translational PET Study in Humans and in Mice.

Organic anion-transporting polypeptides (OATPs) mediate the uptake of various drugs from blood into the liver in the basolateral membrane of hepatocytes. Positron emission tomography (PET) is a potentially powerful tool to assess the activity of hepatic OATPs in vivo, but its utility critically depends on the availability of transporter-selective probe substrates. We have shown before that among the three OATPs expressed in hepatocytes (OATP1B1, OATP1B3, and OATP2B1), [11C]erlotinib is selectively transported by OATP2B1. In contrast to OATP1B1 and OATP1B3, OATP2B1 has not been thoroughly explored yet, and no specific probe substrates are currently available. To assess if the prototypical OATP inhibitor rifampicin can inhibit liver uptake of [11C]erlotinib in vivo, we performed [11C]erlotinib PET scans in six healthy volunteers without and with intravenous pretreatment with rifampicin (600 mg). In addition, FVB mice underwent [11C]erlotinib PET scans without and with concurrent intravenous infusion of high-dose rifampicin (100 mg/kg). Rifampicin caused a moderate reduction in the liver distribution of [11C]erlotinib in humans, while a more pronounced effect of rifampicin was observed in mice, in which rifampicin plasma concentrations were higher than in humans. In vitro uptake experiments in an OATP2B1-overexpressing cell line indicated that rifampicin inhibited OATP2B1 transport of [11C]erlotinib in a concentration-dependent manner with a half-maximum inhibitory concentration of 72.0 ± 1.4 µM. Our results suggest that rifampicin-inhibitable uptake transporter(s) contributed to the liver distribution of [11C]erlotinib in humans and mice and that [11C]erlotinib PET in combination with rifampicin may be used to measure the activity of this/these uptake transporter(s) in vivo. Furthermore, our data suggest that a standard clinical dose of rifampicin may exert in vivo a moderate inhibitory effect on hepatic OATP2B1.

Anion transport and supramolecular medicinal chemistry.

New approaches to the transmembrane transport of anions are discussed in this review. Advances in the design of small molecule anion carriers are reviewed in addition to advances in the design of synthetic anion channels. The application of anion transporters to the potential future treatment of disease is discussed in the context of recent findings on the selectivity of anion transporters.

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution.

The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to available crystal structures. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. The results are in excellent agreement with previous in vivo sialylation experiments. The structure of the spin-labeled Q54R1/L173R1 R125A mutant was solved at 2.1 Å resolution, revealing no significant changes in the protein structure. Thus, the loss of domain closure appears to be solely due to loss of binding. In conclusion, these data are consistent with TRAP SBPs undergoing a simple two-state transition from an open-unliganded to closed-liganded state during the transport cycle.

Computational Discovery and Experimental Validation of Inhibitors of the Human Intestinal Transporter OATP2B1.

Human organic anion transporters (OATPs) are vital for the uptake and efflux of drugs and endogenous compounds. Current identification of inhibitors of these transporters is based on experimental screening. Virtual screening remains a challenge due to a lack of experimental three-dimensional protein structures. Here, we describe a workflow to identify inhibitors of the OATP2B1 transporter in the DrugBank library of over 5,000 drugs and druglike molecules. OATP member 2B1 transporter is highly expressed in the intestine, where it participates in oral absorption of drugs. Predictions from a Random forest classifier, prioritized by docking against multiple comparative protein structure models of OATP2B1, indicated that 33 of the 5,000 compounds were putative inhibitors of OATP2B1. Ten predicted inhibitors that are prescription drugs were tested experimentally in cells overexpressing the OATP2B1 transporter. Three of these ten were validated as potent inhibitors of estrone-3-sulfate uptake (defined as more than 50% inhibition at 20 µM) and tested in multiple concentrations to determine exact IC50. The IC50 values of bicalutamide, ticagrelor, and meloxicam suggest that they might inhibit intestinal OATP2B1 at clinically relevant concentrations and therefore modulate the absorption of other concomitantly administered drugs.

Tryptophan Residue Located at the Middle of Putative Transmembrane Domain 11 Is Critical for the Function of Organic Anion Transporting Polypeptide 2B1.

Organic anion transporting polypeptide 2B1 (OATP2B1), which is highly expressed in enterocytes and hepatocytes could be a key determinant for the intestinal absorption and hepatic uptake of its substrates, most of which are amphipathic organic anions. Tryptophan residues may possess a multitude of functions for a transport protein through aromatic interactions, such as maintaining the proper protein structure, guiding the depth of membrane insertion, or interacting directly with substrates. There are totally six tryptophan residues in OATP2B1. However, little is known about their role in the function and expression of OATP2B1. Our results show that, while W272, W276, and W277 located at the border of extracellular loop 3 and transmembrane domain 6 exhibit a moderate effect on the surface expression of OATP2B1, W611 located at the middle of transmembrane domain 11 plays a critical role in the function of OATP2B1. The tryptophan-to-alanine mutation of W611 changes the kinetic characteristics of OATP2B1-mediated estrone-3-sulfate (E3S) transport radically, from a monophasic saturation curve (with Km and Vmax values being of 7.1 ± 1.1 µM and 182 ± 7 pmol/normalized mg/min, respectively) to a linear curve. Replacing alanine with a phenylalanine will rescue most of OATP2B1's function, suggesting that the aromatic side chain of residue 611 is very important. However, hydrogen-bond forming and positively charged groups at this position are not favorable. The important role of W611 is not substrate-dependent. Molecular modeling indicates that the side chain of W611 faces toward the substrate translocation pathway and might interact with substrates directly. Taken together, our findings reveal that W611 is critical for the function of OATP2B1.

Discovery of potent and orally bioavailable inhibitors of Human Uric Acid Transporter 1 (hURAT1) and binding mode prediction using homology model.

This Letter describes the discovery of a series of potent inhibitors of Human Uric Acid Transporter 1 (hURAT1). Lead generation and optimization via 3D pharmacophore analysis resulted in compound 41. With an IC50 of 33.7nM, 41 also demonstrated good oral bioavailability in rat (74.8%) and displayed a consistent PK profile across all species tested (rat, dog and monkey).