The ergothioneine transporter (ETT): substrates and locations, an inventory.

In all vertebrates including mammals, the ergothioneine transporter ETT (obsolete name OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). ETT is not expressed ubiquitously and only cells with high ETT cell-surface levels can accumulate ET to high concentration. Without ETT, there is no uptake because the plasma membrane is essentially impermeable to this hydrophilic zwitterion. Here, we review the substrate specificity and localization of ETT, which is prominently expressed in neutrophils, monocytes/macrophages, and developing erythrocytes. Most sites of strong expression are conserved across species, but there are also major differences. In particular, we critically analyze the evidence for the expression of ETT in the brain as well as recent data suggesting that the transporter SLC22A15 may also transport ET. We conclude that, to date, ETT remains the only well-defined biomarker for intracellular ET activity. In humans, the ability to take up, distribute, and retain ET depends principally on this transporter.

Engineered MATE multidrug transporters reveal two functionally distinct ion-coupling pathways in NorM from Vibrio cholerae.

Multidrug and toxic compound extrusion (MATE) transport proteins confer multidrug resistance on pathogenic microorganisms and affect pharmacokinetics in mammals. Our understanding of how MATE transporters work, has mostly relied on protein structures and MD simulations. However, the energetics of drug transport has not been studied in detail. Many MATE transporters utilise the electrochemical H+ or Na+ gradient to drive substrate efflux, but NorM-VC from Vibrio cholerae can utilise both forms of metabolic energy. To dissect the localisation and organisation of H+ and Na+ translocation pathways in NorM-VC we engineered chimaeric proteins in which the N-lobe of H+-coupled NorM-PS from Pseudomonas stutzeri is fused to the C-lobe of NorM-VC, and vice versa. Our findings in drug binding and transport experiments with chimaeric, mutant and wildtype transporters highlight the versatile nature of energy coupling in NorM-VC, which enables adaptation to fluctuating salinity levels in the natural habitat of V. cholerae.

Principles of Alternating Access in Multidrug and Toxin Extrusion (MATE) Transporters.

The multidrug and toxin extrusion (MATE) transporters catalyze active efflux of a broad range of chemically- and structurally-diverse compounds including antimicrobials and chemotherapeutics, thus contributing to multidrug resistance in pathogenic bacteria and cancers. Multiple methodological approaches have been taken to investigate the structural basis of energy transduction and substrate translocation in MATE transporters. Crystal structures representing members from all three MATE subfamilies have been interpreted within the context of an alternating access mechanism that postulates occupation of distinct structural intermediates in a conformational cycle powered by electrochemical ion gradients. Here we review the structural biology of MATE transporters, integrating the crystallographic models with biophysical and computational studies to define the molecular determinants that shape the transport energy landscape. This holistic analysis highlights both shared and disparate structural and functional features within the MATE family, which underpin an emerging theme of mechanistic diversity within the framework of a conserved structural scaffold.

Omega-3 Polyunsaturated Fatty Acids Inhibit the Function of Human URAT1, a Renal Urate Re-Absorber.

The beneficial effects of fatty acids (FAs) on human health have attracted widespread interest. However, little is known about the impact of FAs on the handling of urate, the end-product of human purine metabolism, in the body. Increased serum urate levels occur in hyperuricemia, a disease that can lead to gout. In humans, urate filtered by the glomerulus of the kidney is majorly re-absorbed from primary urine into the blood via the urate transporter 1 (URAT1)-mediated pathway. URAT1 inhibition, thus, contributes to decreasing serum urate concentration by increasing net renal urate excretion. Here, we investigated the URAT1-inhibitory effects of 25 FAs that are commonly contained in foods or produced in the body. For this purpose, we conducted an in vitro transport assay using cells transiently expressing URAT1. Our results showed that unsaturated FAs, especially long-chain unsaturated FAs, inhibited URAT1 more strongly than saturated FAs. Among the tested unsaturated FAs, eicosapentaenoic acid, α-linolenic acid, and docosahexaenoic acid exhibited substantial URAT1-inhibitory activities, with half maximal inhibitory concentration values of 6.0, 14.2, and 15.2 µM, respectively. Although further studies are required to investigate whether the ω-3 polyunsaturated FAs can be employed as uricosuric agents, our findings further confirm FAs as nutritionally important substances influencing human health.

Molecular Mechanisms of Amphetamines.

There is a plethora of amphetamine derivatives exerting stimulant, euphoric, anti-fatigue, and hallucinogenic effects; all structural properties allowing these effects are contained within the amphetamine structure. In the first part of this review, the interaction of amphetamine with the dopamine transporter (DAT), crucially involved in its behavioral effects, is covered, as well as the role of dopamine synthesis, the vesicular monoamine transporter VMAT2, and organic cation 3 transporter (OCT3). The second part deals with requirements in amphetamine's effect on the kinases PKC, CaMKII, and ERK, whereas the third part focuses on where we are in developing anti-amphetamine therapeutics. Thus, treatments are discussed that target DAT, VMAT2, PKC, CaMKII, and OCT3. As is generally true for the development of therapeutics for substance use disorder, there are multiple preclinically promising specific compounds against (meth)amphetamine, for which further development and clinical trials are badly needed.

Functional Expression of Choline Transporters in the Blood-Brain Barrier.

Cholinergic neurons in the central nervous system play a vital role in higher brain functions, such as learning and memory. Choline is essential for the synthesis of the neurotransmitter acetylcholine by cholinergic neurons. The synthesis and metabolism of acetylcholine are important mechanisms for regulating neuronal activity. Choline is a positively charged quaternary ammonium compound that requires transporters to pass through the plasma membrane. Currently, there are three groups of choline transporters with different characteristics, such as affinity for choline, tissue distribution, and sodium dependence. They include (I) polyspecific organic cation transporters (OCT1-3: SLC22A1-3) with a low affinity for choline, (II) high-affinity choline transporter 1 (CHT1: SLC5A7), and (III) choline transporter-like proteins (CTL1-5: SLC44A1-5). Brain microvascular endothelial cells, which comprise part of the blood-brain barrier, take up extracellular choline via intermediate-affinity choline transporter-like protein 1 (CTL1) and low-affinity CTL2 transporters. CTL2 is responsible for excreting a high concentration of choline taken up by the brain microvascular endothelial cells on the brain side of the blood-brain barrier. CTL2 is also highly expressed in mitochondria and may be involved in the oxidative pathway of choline metabolism. Therefore, CTL1- and CTL2-mediated choline transport to the brain through the blood-brain barrier plays an essential role in various functions of the central nervous system by acting as the rate-limiting step of cholinergic neuronal activity.

Elucidation of N 1-methyladenosine as a Potential Surrogate Biomarker for Drug Interaction Studies Involving Renal Organic Cation Transporters.

Endogenous substrates are emerging biomarkers for drug transporters, which serve as surrogate probes in drug-drug interaction (DDI) studies. In this study, the results of metabolome analysis using wild-type and Oct1/2 double knockout mice suggested that N 1-methyladenosine (m1A) was a novel organic cation transporter (OCT) 2 substrate. An in vitro transport study revealed that m1A is a substrate of mouse Oct1, Oct2, Mate1, human OCT1, OCT2, and multidrug and toxin exclusion protein (MATE) 2-K, but not human MATE1. Urinary excretion accounted for 77% of the systemic elimination of m1A in mice. The renal clearance (46.9 ± 4.9 ml/min per kilogram) of exogenously given m1A was decreased to near the glomerular filtration rates by Oct1/2 double knockout or Mate1 inhibition by pyrimethamine (16.6 ± 2.6 and 24.3 ± 0.6 ml/min per kilogram, respectively), accompanied by significantly higher plasma concentrations. In vivo inhibition of OCT2/MATE2-K by a single dose of 7-[(3R)-3-(1-aminocyclopropyl)pyrrolidin-1-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxoquinoline-3-carboxylic acid in cynomolgus monkeys resulted in the elevation of the area under the curve of m1A (1.72-fold) as well as metformin (2.18-fold). The plasma m1A concentration profile showed low diurnal and interindividual variation in healthy volunteers. The renal clearance of m1A in younger (21-45 year old) and older (65-79 year old) volunteers (244 ± 58 and 169 ± 22 ml/min per kilogram, respectively) was about 2-fold higher than the creatinine clearance. The renal clearances of m1A and creatinine were 31% and 17% smaller in older than in younger volunteers. Thus, m1A could be a surrogate probe for the evaluation of DDIs involving OCT2/MATE2-K. SIGNIFICANCE STATEMENT: Endogenous substrates can serve as surrogate probes for clinical drug-drug interaction studies involving drug transporters or enzymes. In this study, m1A was found to be a novel substrate of renal cationic drug transporters OCT2 and MATE2-K. N 1-methyladenosine was revealed to have some advantages compared to other OCT2/MATE substrates (creatinine and N 1-methylnicotinamide). The genetic or chemical impairment of OCT2 or MATE2-K caused a significant increase in the plasma m1A concentration in mice and cynomolgus monkeys due to the high contribution of tubular secretion to the net elimination of m1A. The plasma m1A concentration profile showed low diurnal and interindividual variation in healthy volunteers. Thus, m1A could be a better biomarker of variations in OCT2/MATE2-K activity caused by inhibitory drugs.

[Possible Treatment of Neuropsychiatric Disorders by Promotion of Neuronal Differentiation through Organic Cation Transporters].

Neurons differentiated from neural stem cells mature to form a neuronal network. Neuronal maturation enables neurotransmission that regulates brain function. Therefore, abnormal neuronal differentiation causes dysfunction in neurotransmission, and is involved in the onset of various neuropsychiatric disorders. Most of the drugs currently available for the treatment of neuropsychiatric disorders act on membrane receptors and reuptake transporters of neurotransmitters, and control neurotransmission. These membrane proteins have a high affinity for a specific neurotransmitter, and are highly expressed in synapses. By contrast, xenobiotic transporters have a relatively lower affinity for neurotransmitters, but widely recognize various organic compounds, and are also expressed in brain neural cells. It has remained largely unknown why such xenobiotic transporters are expressed in neural cells that play a key role in neurotransmission. We have therefore attempted to clarify the physiological roles of organic cation transporters (OCTs) in neural stem cells in order to obtain new insight into the treatment of neuropsychiatric disorders. The carnitine/organic cation transporter OCTN1/SLC22A4 is expressed at much higher levels in neural stem cells compared with other OCTs, and promotes their differentiation into neurons through the uptake of the food-derived hydrophilic antioxidant ergothioneine after oral administration. In this review, we introduce current topics on the physiological/pathophysiological roles of OCTs in neural stem cells, and discuss their possible application to the treatment of neuropsychiatric disorders.

Choline transport for phospholipid synthesis: An emerging role of choline transporter-like protein 1.

IMPACT STATEMENT: This review will provide a summary of recent advances in choline transport research and highlight important novel areas of focus in the field.

Phylogenetic analysis of upland cotton MATE gene family reveals a conserved subfamily involved in transport of proanthocyanidins.

The multidrug and toxic compound extrusion (MATE) protein belongs to a secondary transporter family, which plays a role in transporting different kinds of substrates like phytohormones and secondary metabolites. In plant, MATE transporters related to the endogenous and exogenous mechanisms of detoxification for secondary metabolites such as alkaloids, flavonoids, anthocyanins and other secondary metabolites have been studied. However, a genome-wide analysis of the MATE family is rarely reported in upland cotton (Gossypium hirsutum L.). In the study, a total of 72 GhMATEs were identified from the genome of upland cotton, which were classified into four subfamilies with possible diverse functions such as transport of proanthocyanidins (PAs), accumulation of alkaloids, extrusion of xenobiotic compounds, regulation of disease resistance and response to abiotic stresses. Meanwhile, the gene structure, evolutionary relationship, physical location, conservative motifs, subcellular localization and gene expression pattern of GhMATEs have been further analysed. Three of these MATE genes (GhMATE12, GhMATE16 and GhMATE38) were identified as candidate genes due to their functions in transport of PA similar to GhTT12. These results provide a new perspective on upland cotton MATE gene family for their potential roles in transport of PA and a theoretical basis for further analyzing the function of MATE genes and improving the fiber quality of brown cotton.

Genome-wide analysis of the MATE gene family in potato.

The multidrug and toxic compound extrusion (MATE) protein family is a newly discovered family of secondary transporters that extrude metabolic waste and a variety of antibiotics out of the cell using an electrochemical gradient of H+ or Na+ across the membrane. The main function of MATE gene family is to participate in the process of plant detoxification and morphogenesis. The genome-wide analysis of the MATE genes in potato genome was conducted. At least 48 genes were initially identified and classified into six subfamilies. The chromosomal localization of MATE gene family showed that they could be distributed on 11 chromosomes except chromosome 9. The number of amino acids is 145-616, the molecular weight of proteins is 15.96-66.13 KD, the isoelectric point is 4.97-9.17, and they were located on the endoplasmic reticulum with having 4-13 transmembrane segments. They contain only two parts of the exons and UTR without introns. Some members of the first subfamily of potato MATE gene family are clustered with At2g04070 and they may be related to the transport of toxic compounds such as alkaloids and heavy metal. The function of the members of the second subfamily may be similar to that of At3g23560, which is related to tetramethylammonium transport. Some members of the third subfamily are clustered with At3g59030 and they may be involved in the transport of flavonoids. The fifth subfamily may be related to the transport of iron ions. The function of the sixth subfamily may be similar to that of At4g39030, which is related to salicylic acid transport. There are three kinds of conserved motifs in potato MATE genes, including the motif 1, motif 2, and motif 3. Each motif has 50 amino acids. The number of each motif is different in the gene sequence, of which 45 MATE genes contain at least a motif, but there is no motif in ST0015301, ST0045283, and ST0082336. These results provide a reference for further research on the function of potato MATE genes.

DKB114, A Mixture of Chrysanthemum Indicum Linne Flower and Cinnamomum Cassia (L.) J. Presl Bark Extracts, Improves Hyperuricemia through Inhibition of Xanthine Oxidase Activity and Increasing Urine Excretion.

Chrysanthemum indicum Linne flower (CF) and Cinnamomum cassia (L.) J. Presl bark (CB) extracts have been used as the main ingredients in several prescriptions to treat the hyperuricemia and gout in traditional medicine. In the present study, we investigated the antihyperuricemic effects of DKB114, a CF, and CB mixture, and the underlying mechanisms in vitro and in vivo. DKB114 markedly reduced serum uric acid levels in normal rats and rats with PO-induced hyperuricemia, while increasing renal uric acid excretion. Furthermore, it inhibited the activity of xanthine oxidase (XOD) in vitro and in the liver in addition to reducing hepatic uric acid production. DKB114 decreased cellular uric acid uptake in oocytes and HEK293 cells expressing human urate transporter (hURAT)1 and decreased the protein expression levels of urate transporters, URAT1, and glucose transporter, GLUT9, associated with the reabsorption of uric acid in the kidney. DKB114 exerts antihyperuricemic effects and uricosuric effects, which are accompanied, partially, by a reduction in the production of uric acid and promotion of uric acid excretion via the inhibition of XOD activity and reabsorption of uric acid. Therefore, it may have potential as a treatment for hyperuricemia and gout.

The permeation mechanism of organic cations through a CNG mimic channel.

Several channels, ranging from TRP receptors to Gap junctions, allow the exchange of small organic solute across cell membrane. However, very little is known about the molecular mechanism of their permeation. Cyclic Nucleotide Gated (CNG) channels, despite their homology with K+ channels and in contrast with them, allow the passage of larger methylated and ethylated ammonium ions like dimethylammonium (DMA) and ethylammonium (EA). We combined electrophysiology and molecular dynamics simulations to examine how DMA interacts with the pore and permeates through it. Due to the presence of hydrophobic groups, DMA enters easily in the channel and, unlike the alkali cations, does not need to cross any barrier. We also show that while the crystal structure is consistent with the presence of a single DMA ion at full occupancy, the channel is able to conduct a sizable current of DMA ions only when two ions are present inside the channel. Moreover, the second DMA ion dramatically changes the free energy landscape, destabilizing the crystallographic binding site and lowering by almost 25 kJ/mol the binding affinity between DMA and the channel. Based on the results of the simulation the experimental electron density maps can be re-interpreted with the presence of a second ion at lower occupancy. In this mechanism the flexibility of the channel plays a key role, extending the classical multi-ion permeation paradigm in which conductance is enhanced by the plain interaction between the ions.

Organic cation transporter 3: A cellular mechanism underlying rapid, non-genomic glucocorticoid regulation of monoaminergic neurotransmission, physiology, and behavior.

Contribution to Special Issue on Fast effects of steroids. Corticosteroid hormones act at intracellular glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) to alter gene expression, leading to diverse physiological and behavioral responses. In addition to these classical genomic effects, corticosteroid hormones also exert rapid actions on physiology and behavior through a variety of non-genomic mechanisms, some of which involve GR or MR, and others of which are independent of these receptors. One such GR-independent mechanism involves corticosteroid-induced inhibition of monoamine transport mediated by "uptake2" transporters, including organic cation transporter 3 (OCT3), a low-affinity, high-capacity transporter for norepinephrine, epinephrine, dopamine, serotonin and histamine. Corticosterone directly and acutely inhibits OCT3-mediated transport. This review describes the studies that initially characterized uptake2 processes in peripheral tissues, and outlines studies that demonstrated OCT3 expression and corticosterone-sensitive monoamine transport in the brain. Evidence is presented supporting the hypothesis that corticosterone can exert rapid, GR-independent actions on neuronal physiology and behavior by inhibiting OCT3-mediated monoamine clearance. Implications of this mechanism for glucocorticoid-monoamine interactions in the context-dependent regulation of behavior are discussed.

[Role of drug transporters in rational use of drugs at high altitude area].

Pharmacokinetics plays a key role in rational use of medicines. Many factors can affect the drug's pharmacokinetics. Previous studies mainly focused on the impact of hypoxia on hepatic drug metabolizing enzyme, but uncommon on drug transporters. Actually, drug transporter is a key factor for activation of the drugs transport across the cell membrane into the inside of cells, such as multidrug resistance protein (MDR), breast cancer resistance protein (BCRP), multidrug resistance associated protein (MRP), organic cation transporter (OCT), organic anion-transporting polypeptide (OATP), organic anion transporter (OAT), qligopeptide transporter (PEPT), etc. They are widely present in the small intestine villus epithelial cells, renal tubular epithelial cells, hepatocytes and biliary epithelial cells. They play a very important role in drug absorption, distribution, metabolism and excretion. The changes in drug transporters under hypoxia in intestinal could affect the bioavailability of drugs; the changes in drug transporters in organs could affect drug's distribution, subsequent drug's indications and adverse reactions; the changes in drug transporters in liver and kidney could affect the metabolism and excretion rate of drugs, thereby the drug's residence time and half-life.

Integrated analysis of gene expression signatures associated with colon cancer from three datasets.

PURPOSE: The present study aimed to elucidate the pathogenesis of colon cancer and identify genes associated with tumor development. METHODS: Three datasets, two (GSE74602 and GSE44861) from the Gene Expression Omnibus database and RNA-Seq colon cancer data from The Cancer Genome Atlas data portal, were downloaded. These three datasets were grouped using a meta-analysis approach, and differentially expressed genes (DEGs) were identified between colon tumor samples and adjacent normal samples. Functional enrichment analysis and regulatory factor predication were performed for significant genes. Additionally, small-molecule drugs associated with colon cancer were predicted, and a prognostic risk model was constructed. RESULTS: There were 251 overlapping DEGs (135 up- and 116 downregulated) between cancer samples and control samples in the three datasets. The DEGs were mainly involved in protein transport and apoptotic and neurotrophin signaling pathways. A total of 70 small-molecule drugs were predicated to be associated with colon cancer. Additionally, in the miRNA-target regulatory network, we found that SLC44A1 can be targeted by hsa-miR-183, hsa-miR-206, and hsa-miR-147, while KLF13 can be regulated by hsa-miR-182, hsa-miR-206, and hsa-miR-153. Moreover, the results of the prognostic risk model showed that four genes (VAMP1, P2RX5, CACNB1, and CRY2) could divide the samples into high and low risk groups. CONCLUSION: SLC44A1 and KLF13 may be involved in tumorigenesis and the metastasis of colon cancer by miRNA regulation. In addition, a four-gene (VAMP1, P2RX5, CACNB1, and CRY2) expression signature may have prognostic and predictive value in colon cancer.

Multiple blood-brain barrier transport mechanisms limit bumetanide accumulation, and therapeutic potential, in the mammalian brain.

There is accumulating evidence that bumetanide, which has been used over decades as a potent loop diuretic, also exerts effects on brain disorders, including autism, neonatal seizures, and epilepsy, which are not related to its effects on the kidney but rather mediated by inhibition of the neuronal Na-K-Cl cotransporter isoform NKCC1. However, following systemic administration, brain levels of bumetanide are typically below those needed to inhibit NKCC1, which critically limits its clinical use for treating brain disorders. Recently, active efflux transport at the blood-brain barrier (BBB) has been suggested as a process involved in the low brain:plasma ratio of bumetanide, but it is presently not clear which transporters are involved. Understanding the processes explaining the poor brain penetration of bumetanide is needed for developing strategies to improve the brain delivery of this drug. In the present study, we administered probenecid and more selective inhibitors of active transport carriers at the BBB directly into the brain of mice to minimize the contribution of peripheral effects on the brain penetration of bumetanide. Furthermore, in vitro experiments with mouse organic anion transporter 3 (Oat3)-overexpressing Chinese hamster ovary cells were performed to study the interaction of bumetanide, bumetanide derivatives, and several known inhibitors of Oats on Oat3-mediated transport. The in vivo experiments demonstrated that the uptake and efflux of bumetanide at the BBB is much more complex than previously thought. It seems that both restricted passive diffusion and active efflux transport, mediated by Oat3 but also organic anion-transporting polypeptide (Oatp) Oatp1a4 and multidrug resistance protein 4 explain the extremely low brain concentrations that are achieved after systemic administration of bumetanide, limiting the use of this drug for targeting abnormal expression of neuronal NKCC1 in brain diseases.

Multidrug and toxin extrusion proteins mediate cellular transport of cadmium.

Cadmium (Cd) is an environmentally prevalent toxicant posing increasing risk to human health worldwide. As compared to the extensive research in Cd tissue accumulation, little was known about the elimination of Cd, particularly its toxic form, Cd ion (Cd2+). In this study, we aimed to examine whether Cd2+ is a substrate of multidrug and toxin extrusion proteins (MATEs) that are important in renal xenobiotic elimination. HEK-293 cells overexpressing the human MATE1 (HEK-hMATE1), human MATE2-K (HEK-hMATE2-K) and mouse Mate1 (HEK-mMate1) were used to study the cellular transport and toxicity of Cd2+. The cells overexpressing MATEs showed a 2-4 fold increase of Cd2+ uptake that could be blocked by the MATE inhibitor cimetidine. A saturable transport profile was observed with the Michaelis-Menten constant (Km) of 130±15.8µM for HEK-hMATE1; 139±21.3µM for HEK-hMATE2-K; and 88.7±13.5µM for HEK-mMate1, respectively. Cd2+ could inhibit the uptake of metformin, a substrate of MATE transporters, with the half maximal inhibitory concentration (IC50) of 97.5±6.0µM, 20.2±2.6µM, and 49.9±6.9µM in HEK-hMATE1, HEK-hMATE2-K, and HEK-mMate1 cells, respectively. In addition, hMATE1 could transport preloaded Cd2+ out of the HEK-hMATE1 cells, thus resulting in a significant decrease of Cd2+-induced cytotoxicity. The present study has provided the first evidence supporting that MATEs transport Cd2+ and may function as cellular elimination machinery in Cd intoxication.

[Recent advances in urate metabolism]. / Nuovi avanzamenti nelle conoscenze del metabolismo dell'urato.

In the last fifteen years, genomics and other -omics sciences have revolutionized our understanding of biological processes at the molecular level. An illustrative example is urate metabolism. Before the publication of the complete human genome, in 2003 it was believed that a single enzyme (urate oxidase) was responsible for uricolysis that is the conversion of urate into the more soluble allantoin. Now we know with great detail that this process requires the consecutive action of three enzymes that have been lost by gene inactivation in our hominoid ancestor. Similarly, a single urate transporter (URAT1) was known at that time. Now we have evidence that urate homeostasis depends on a complex set of transporters located on the epithelial cells of the kidney and the intestine. In this review article, we give an account of the recent discoveries on urate metabolism and how these discoveries can be applied to the development of novel drugs to treat hyperuricemia, tumor lysis syndrome and the Lesch-Nyhan disease.