SLC6 neurotransmitter transporters: structure, function, and regulation

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.     

Impact of genetic polymorphisms in transmembrane carrier-systems on drug and xenobiotic distribution

Active transport across biological membranes has become a noticeable factor in the absorption, distribution, and excretion of an increasing number of drugs. Different transmembrane transport systems including organic anion transporters (OATP, solute carrier family SLC21A), organic cation transporters (OCT, SLC22A), dipeptide transporters (PEPT, SLC15A), nucleoside transporters (CNT, SLC28A), monocarboxylate carriers (MCT, SLC2A), and members of the large ATP-binding cassette family (ABC, SLC3A) are involved in drug disposition. Genetic polymorphisms in transport proteins frequently occur and contribute to interindividual differences in the efficacy and safety of pharmatherapy. Currently, the most advanced research has been done on P-glycoprotein (ABCB1, SLC3A1.201.1). Knowledge of this transporter indicates that haplotype analysis rather than association with single nucleotide polymorphisms (SNPs) provides the most appropriate interpretation of pharmacogenetic data from drug transporters. This review gives an overview and update on the pharmacological impact of genetic variants in transmembrane transporters.An erratum to this article can be found at     

Systematic identification and characterization of cynomolgus macaque solute carrier transporters

Cynomolgus macaques are used in preclinical studies in part because of their evolutionary closeness to humans. However, drug transporters [including solute carrier (SLC) transporters] essential for the absorption and excretion of drugs have not been fully investigated at the molecular level in cynomolgus macaques. We identified and characterized cynomolgus macaque SLC15A1, SLC15A2, SLC22A1, SLC22A2, SLC22A6, SLC22A8, SLC47A1, and SLC47A2, along with SLCO (formerly SLC21A) transporters SLCO1A2, SLCO1B1, SLCO1B3, and SLCO2B1. These cynomolgus SLC transporters had high amino acid sequence identities (92–97%) with their human orthologs and contained sequence motifs characteristic of SLC transporters. Phylogenetic analysis showed that these cynomolgus SLC transporters were more closely clustered with their human orthologs than with those of dogs, rats, or mice. Gene structure and genomic organization were similar in macaques and humans. Cynomolgus SLC transporter mRNAs showed distinct tissue expression patterns, being most abundantly expressed in jejunum (SLC15A1), liver (SLC22A1, SLCO1B1, and SLCO2B1), and kidney (SLC15A2, SLC22A2, SLC22A6, SLC22A8, SLC47A1, SLC47A2, and SLCO1A2). In contrast, cynomolgus SLCO2B1 mRNA was more ubiquitously expressed. Among these SLC mRNAs, the most abundant in liver was SLCO1B1, in jejunum SLC15A1, and in kidney SLC22A2. These results suggest similar characteristics of SLC transporters in cynomolgus macaques and humans.     

Organic cation transporters (OCTs, MATEs), in vitro and in vivo evidence for the importance in drug therapy

Organic cation transporters (OCTs) of the solute carrier family (SLC) 22 and multidrug and toxin extrusion (MATE) transporters of the SLC47 family have been identified as uptake and efflux transporters, respectively, for xenobiotics including several clinically used drugs such as the antidiabetic agent metformin, the antiviral agent lamivudine, and the anticancer drug oxaliplatin. Expression of human OCT1 (SLC22A1) and OCT2 (SLC22A2) is highly restricted to the liver and kidney, respectively. By contrast, OCT3 (SLC22A3) is more widely distributed. MATEs (SLC47A1, SLC47A2) are predominantly expressed in human kidney. Data on in vitro studies reporting a large number of substrates and inhibitors of OCTs and MATEs are systematically summarized. Several genetic variants of human OCTs and in part of MATE1 have been reported, and some of them result in reduced in vitro transport activity corroborating data from studies with knockout mice. A comprehensive overview is given on currently known genotype-phenotype correlations for variants in OCTs and MATE1 related to protein expression, pharmacokinetics/-dynamics of transporter substrates, treatment outcome, and disease susceptibility.     

Polyspecific Organic Cation Transporters: Structure,Function, Physiological Roles,and Biopharmaceutical Implications

The body is equipped with broad-specificity transporters for the excretion and distribution of endogeneous organic cations and for the uptake, elimination and distribution of cationic drugs, toxins and environmental waste products. This group of transporters consists of the electrogenic cation transporters OCT1-3 (SLC22A1-3), the cation and carnitine transporters OCTN1 (SLC22A4), OCTN2 (SLC22A5) and OCT6 (SLC22A16), and the proton/cation antiporters MATE1, MATE2-K and MATE2-B. The transporters show broadly overlapping sites of expression in many tissues such as small intestine, liver, kidney, heart, skeletal muscle, placenta, lung, brain, cells of the immune system, and tumors. In epithelial cells they may be located in the basolateral or luminal membranes. Transcellular cation movement in small intestine, kidney and liver is mediated by the combined action of electrogenic OCT-type uptake systems and MATE-type efflux transporters that operate as cation/proton antiporters. Recent data showed that OCT-type transporters participate in the regulation of extracellular concentrations of neurotransmitters in brain, mediate the release of acetylcholine in non-neuronal cholinergic reactions, and are critically involved in the regulation of histamine release from basophils. The recent identification of polymorphisms in human OCTs and OCTNs allows the identification of patients with an increased risk for adverse drug reactions. Transport studies with expressed OCTs will help to optimize pharmacokinetics during development of new drugs.     

Functional characterization and haplotype analysis of polymorphisms in the human equilibrative nucleoside transporter, ENT2.

The equilibrative nucleoside transporter 2 (ENT2; SLC29A2) is a bidirectional transporter that is involved in the disposition of naturally occurring nucleosides as well as a variety of anticancer and antiviral nucleoside analogs. The goal of the current study was to evaluate the function of genetic variants in ENT2 in cellular assays and to determine the haplotype structure of the coding and flanking intronic region of the gene. As part of a large study focused on genetic variation in membrane transporters (Leabman et al., 2003), DNA samples from ethnically diverse populations (100 African-Americans, 100 European-Americans, 30 Asians, 10 Mexicans, and 7 Pacific Islanders) were screened for variants in membrane transporters, including SLC29A2. Fourteen polymorphic sites in SLC29A2 were found, including 11 in the coding region. Five protein-altering variants were identified: three nonsynonymous variants, and two deletions. Each of the protein-altering variants was found at a very low frequency, occurring only once in the sample population. The nonsynonymous variants and the deletions were constructed via site-directed mutagenesis and were subsequently characterized in Xenopus laevis oocytes. All variants were able to take up inosine with the exception of ENT2-Delta845-846, which resulted in a frameshift mutation that prematurely truncated the protein. ENT2 showed very infrequent variation compared with most other transporter proteins studied, and it was found that five haplotypes were sufficient to describe the entire sample set. The low overall genetic diversity in SLC29A2 makes it unlikely that variation in the coding region contributes significantly to clinically observed differences in drug response.     

Monoamine transporter pharmacology and mutant mice

Monoamine transporters, such as the dopamine transporter, 5-HT transporter and noradrenaline transporter, in the plasma membrane provide effective control over the intensity of monoamine-mediated signaling by recapturing neurotransmitters released by presynaptic neurons. These proteins represent established targets for several psychotropic drugs, including psychostimulants and antidepressants; however, important issues regarding the selectivity and mechanisms of action of these drugs remain unresolved. Although monoamine transporter knockout mice have profound changes in neurotransmission, they provide useful in vivo models to analyze the effects of psychotropic drugs. In this review, we summarize recent insights into the pharmacology of psychotropic drugs using mice in which the genes encoding these transporters have been deleted.     

The Na<Superscript>+</Superscript>/Cl<Superscript>−</Superscript>-Coupled,Broad-Specific,Amino Acid Transporter SLC6A14 (ATB<Superscript>0,+</Superscript>): Emerging Roles in Multiple Diseases and Therapeutic Potential for Treatment and Diagnosis

Amino acids are essential building blocks of all mammalian cells, and amino acid transporters play a vital role in transporting them into cells and their further distribution among the various cellular compartments. There are ~?430 known transporters in the solute-linked carrier (SLC) gene family, divided into 52 distinct families. Eleven of these gene families contain one or more amino acid transporters. These transporters differ significantly from each other in terms of substrate specificity, ion dependence, and energetics. Given the variety of roles they fulfill in human physiology, it is not surprising that a number of diseases are associated with the malfunction of these transporters. In particular, as amino acids are critical for cell growth, survival, and proliferation, the role of amino acid transporters in cancer is gaining increasing attention in recent years. The present review primarily focuses on one particular amino acid transporter, SLC6A14 (also known as ATB^0,+), with regard to its relevance to specific diseases, including cancer, and the molecular mechanisms underlying the disease-related alterations in the expression of the transporter. Furthermore, the review highlights the possible utility of this transporter in drug delivery and also its therapeutic potential for the treatment and diagnosis of cancer.     

Genetic polymorphisms of drug transporters: pharmacokinetic and pharmacodynamic consequences in pharmacotherapy

There has been increasing appreciation of the role of drug transporters in pharmacokinetic and pharmacodynamic consequences in pharmacotherapy. The clinical relevance of drug transporters depends on the localisation in human tissues (i.e., vectorial movement), the therapeutic index of the substrates and inherent interindividual variability. With regard to variability, polymorphisms of drug transporter genes have recently been reported to be associated with alterations in the pharmacokinetics and pharmacodynamics of clinically useful drugs. A growing number of preclinical and clinical studies have demonstrated that the application of genetic information may be useful in individualised pharmacotherapy for numerous diseases. However, the reported effects of variants in certain drug transporter genes have been inconsistent and, in some cases, conflicting among studies. Furthermore, the incidence of almost all known variants in transporter genes tends to be racially dependent. These observations suggest the necessity of considering interethnic variability before extrapolating pharmacokinetic data obtained in one ethic group to another, especially in the early phase of drug development. This review focuses on the impact of genetic variations in the function of drug transporters (ABC, organic anion and cation transporters) and the implications of these variations for pharmacotherapy from pharmacokinetic and pharmacodynamic viewpoints.     

Effects of culture duration on gene expression of p450 isoforms, uptake and efflux transporters in primary hepatocytes cultured in the absence and presence of interleukin- 6: implications for experimental design for the evaluation of downregulatory effects of biotherapeutics

We report here a comprehensive evaluation of the effects of culture duration on the gene expression of P450 isoforms, uptake transporters and efflux transporters in human hepatocyte cultured in the absence and presence of the prototypical proinflammatory cytokine, interleukin-6 (IL-6). Primary collagen-matrigel sandwich cultures of human hepatocytes were cultured in supplemented William's E medium containing 0, 0.1, 0.5 and 5 ng/mL of IL-6 for the time periods of 2, 6, 12, 24 and 48 hrs. Real-time PCR was performed to quantify gene expression of acute phase proteins (suppressor of cytokine signaling 3 (SOCS-3), c-reactive protein (CRP) and lipopolysaccharide (LPS)-binding proteins (LBP)); P450 isoforms (CYPs 1A2, 2B6, 2C8, 2C9, 2D6, 3A4, and 3A5), uptake transporters (SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, SLCO2B1) and efflux transporters (ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2). SOCS-3, CRP, and LBP were extensively induced by IL-6, with maximal induction observed at 2 (SOCS-3) and 12 hrs (CRP; LBP), demonstrating that the cultured human hepatocytes responded to IL-6 treatment. In the untreated group (control), gene expression of P450 isoforms and uptake transporters decreased while efflux transporters remained relatively stable or increased with cultured duration. IL-6 predominantly caused down regulations of the genes studied, with the most significant changes observed at different treatment durations, apparently related to the stability of the basal levels of gene expression. For instance, for genes with unstable expression, which would decrease rapidly in culture (e.g CYP3A4), the most definitive down regulatory effects were observed at a relatively early time point (e.g. 12 hrs). In contrast, a longer treatment duration (e.g. 48 hrs) was required for genes with relatively stable expression levels in culture (e.g. ABCB1). Based on our findings, evaluation of multiple treatment durations rather than single treatment duration is recommended for the evaluation of biotherapeutics in cultured human hepatocytes where down regulation is expected.     

Renal Transport of Adefovir,Cidofovir, and Tenofovir by SLC22A Family Members (hOAT1, hOAT3, and hOCT2)

Purpose The nephrotoxicity of the nucleotide antivirals adefovir, cidofovir and tenofovir is considered to depend on the renal tubular transport of them. Although it is known that the antivirals are substrates of the human renal organic anion transporter hOAT1 (SLC22A6), there is no information available on other organic ion transporters. The aim of the present study was to investigate whether the other renal organic anion transporter hOAT3 (SLC22A8) and organic cation transporter hOCT2 (SLC22A2) transport the antivirals. Materials and Methods Uptake experiments were performed using HEK293 cells transfected with cDNA of the organic ion transporters. Results The uptake of adefovir, cidofovir and tenofovir in monolayers stably expressing hOAT3 increased time-dependently, compared with control. Probenecid, a typical inhibitor of organic anion transporters, completely inhibited their transport. The amounts of the antivirals taken up by hOAT3 were much lower than those by hOAT1. The transient expression of hOCT2 did not increase uptake of the antivirals. Conclusion These results indicate that adefovir, cidofovir and tenofovir are substrates of hOAT3 as well as hOAT1, but that quantitatively hOAT1 is the major renal transporter for these drugs.     

Drug transporter expression and activity in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice with humanized livers

Chimeric mice with humanized liver are thought to represent a sustainable source of isolated human hepatocytes for in vitro studying detoxification of drugs in humans. Because drug transporters are now recognized as key-actors of the hepatic detoxifying process, the present study was designed to characterize mRNA expression and activity of main hepatic drug transporters in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice and termed HepaSH cells. Such cells after thawing were shown to exhibit a profile of hepatic solute carrier (SLC) and ATP-binding cassette (ABC) drug transporter mRNA levels well correlated to those found in cryopreserved primary human hepatocytes or human livers. HepaSH cells used either as suspensions or as 24 h-cultures additionally displayed notable activities of uptake SLCs, including organic anion transporting polypeptides (OATPs), organic anion transporter 2 (OAT2) or sodium-taurocholate co-transporting polypeptide (NTCP). SLC transporter mRNA expression, as well as SLC activities, nevertheless fell in HepaSH cells cultured for 120 h, which may reflect a partial dedifferentiation of these cells with time in culture in the conventional monolayer culture conditions used in the study. These data therefore support the use of cryopreserved HepaSH cells as either suspensions or short-term cultures for drug transport studies.     

Opioid transport by ATP-binding cassette transporters at the blood-brain barrier: implications for neuropsychopharmacology

Some of the ATP-binding cassette (ABC) transporters like P-glycoprotein (P-gp; ABCB1, MDR1), BCRP (ABCG2) and MRPs (ABCCs) that are present at the blood-brain barrier (BBB) influence the brain pharmacokinetics (PK) of their substrates by restricting their uptake or enhancing their clearance from the brain into the blood, which has consequences for their CNS pharmacodynamics (PD). Opioid drugs have been invaluable tools for understanding the PK-PD relationships of these ABC-transporters. The effects of morphine, methadone and loperamide on the CNS are modulated by P-gp. This review examines the ways in which other opioid drugs and some of their active metabolites interact with ABC transporters and suggests new mechanisms that may be involved in the variability of the response of the CNS to these drugs like carrier-mediated system belonging to the solute carrier (SLC) superfamily. Exposure to opioids may also alter the expression of ABC transporters. P-gp can be overproduced during morphine treatment, suggesting that the drug has a direct or, more likely, an indirect action. Variations in cerebral neurotransmitters during exposure to opioids and the release of cytokines during pain could be new endogenous stimuli affecting transporter synthesis. This review concludes with an analysis of the pharmacotherapeutic and clinical impacts of the interactions between ABC transporters and opioids.     

Interaction of the human plasma membrane monoamine transporter (hPMAT) with antidepressants and antipsychotics

Monoamine neurotransmission is efficiently terminated through synaptic reuptake of released neurotransmitters by high-affinity Na⁺- and Cl⁻-dependent neuronal monoamine transporters of the SLC6A family located in the plasma membrane of presynaptic nerve terminals. Recently, a low-affinity, high-capacity Na⁺- and Cl⁻-independent plasma membrane monoamine transporter (PMAT) belonging to the SLC29 solute carrier family has been cloned. PMAT was shown to transport monoamine neurotransmitters as well as organic cations such as 1-phenyl-4-methyl-pyridinium (MPP⁺). Thus, the PMAT which is highly expressed in the human brain may be involved in the modulation of central monoaminergic neurotransmission and it may be a target for drugs used to treat depression and schizophrenia, i.e., dysregulations of the monoamine homeostasis in the central nervous system (CNS). Therefore, we examined in transfected cells the influence on [³H]-MPP⁺ transport by the human PMAT (hPMAT) of nine monoamine transport inhibiting antidepressants (ADs) belonging to pharmacologically diverse classes (imipramine, desipramine, amitriptyline, bupropion, fluoxetine, sertraline, paroxetine, reboxetine, and venlafaxine), of the atypical ADs tianeptine and trimipramine and of five antipsychotics (levomepromazine, haloperidol, clozapine, olanzapine, and risperidone). All examined drugs inhibited the hPMAT; however, half-maximum inhibition (IC₅₀) was observed at concentrations which were much higher than reported clinical plasma concentrations of these drugs. Thus, inhibition of the hPMAT by these CNS drugs may not (or only marginally) contribute to their therapeutic effects.     

Pharmacogenetic insights to monoaminergic dysfunction in alcohol dependence

Rationale Alcohol dependence is characterized by the development of tolerance, withdrawal symptoms, and craving for alcohol. Chronic alcohol consumption causes neuroadaptive changes in the central dopaminergic and serotonergic system, which are partially reversible after detoxification. The severity and time-course of recovery of these neuroadaptive changes may depend on the genetic constitution of monoamine transporters and receptors and contribute to the relapse risk of alcoholics.Objectives To assess the interaction between the genetic constitution and the in vivo availability of dopamine and serotonin transporters and receptors, chronic alcohol intake, alcohol craving and withdrawal.Methods Review of brain imaging studies that assess the genotype and availability of dopamine and serotonin transporters in detoxified alcoholics and healthy control subjects.Results Chronic alcohol intake induced neuroadaptive reductions in striatal dopamine transporter (DAT) availability, which were reversible during early abstinence. A polymorphism of the DAT gene (SLC6A3) was associated with the in vivo transporter availability and with the severity of alcohol withdrawal. Neurotoxic reductions in 5-HTT protein expression were limited to homozygous carriers of the long allele in the 5-HTT gene (SCL6A4) regulatory region and correlated with negative mood states.Conclusion Genetic constitution interacts with the in vivo availability of central dopamine and serotonin transporters during alcohol detoxification and may affect the severity of alcohol withdrawal and clinical depression.     

Neurotransmitter transporters: molecular function of important drug targets

The concentration of neurotransmitters in the extracellular space is tightly controlled by distinct classes of membrane transport proteins. This review focuses on the molecular function of two major classes of neurotransmitter transporter that are present in the cell membrane of neurons and/or glial cells: the solute carrier (SLC)1 transporter family, which includes the transporters that mediate the Na(+)-dependent uptake of glutamate, and the SLC6 transporter family, which includes the transporters that mediate the Na(+)-dependent uptake of dopamine, 5-HT, norepinephrine, glycine and GABA. Recent research has provided substantial insight into the structure and function of these transporters. In particular, the recent crystallizations of bacterial homologs are of the utmost importance, enabling the first reliable structural models of the mammalian neurotransmitter transporters to be generated. These models should be an important tool for developing specific drugs that, through selective interaction with transporters, could improve the treatment of serious neurological and psychiatric disorders.