Function, expression, and characterization of the serotonin transporter in the native human intestine

The enteric serotonin transporter (SERT) plays a critical role in modulating serotonin availability and thus has been implicated in the pathogenesis of various intestinal disorders. To date, SERT expression and function in the human intestine have not been investigated. Current studies were designed to characterize the function, expression, distribution, and membrane localization of SERT in the native human intestine. Real-time PCR studies showed relatively higher SERT mRNA expression in the human small intestine compared with colon (ileum > duodenum > jejunum). Northern blot analysis revealed three mRNA hybridizing species encoding SERT (3.0, 4.9, and 6.8 kb) in the human ileum. Consistent with SERT mRNA expression, SERT immunostaining was mainly detected in the epithelial cells of human duodenal and ileal resected tissues. Notably, SERT expression was localized predominantly to the apical and intracellular compartments and was distributed throughout the crypt-villus axis. Immunoblotting studies detected a prominent protein band ( approximately 70 kDa) in the ileal apical plasma membrane vesicles (AMVs) isolated from mucosa obtained from organ-donor intestine. Functional studies showed that uptake of [(3)H]serotonin (150 nM) in human ileal AMVs was 1) significantly increased in the presence of both Na(+) and Cl(-); 2) inhibited ( approximately 50%) by the neuronal SERT inhibitor, fluoxetine (10 microM) and by unlabeled 5-HT; and 3) exhibited saturation kinetics indicating the presence of a carrier-mediated process. Our studies demonstrated differential expression of SERT across various regions of the human intestine and provide evidence for the existence of a functional SERT capable of removing intraluminal serotonin in human ileal epithelial cells.     

Functional variation in the 3' untranslated region of the serotonin transporter in human and rhesus macaque

The serotonin system underlies a wide variety of behavioral traits and its dysregulation is the cause of numerous neuropsychiatric disorders. Among genes involved in the system, the serotonin transporter (SERT) is integral and has been repeatedly shown to be associated with disease as well as being a primary drug target. In addition to promoter region variation, we identify here variation in a regulatory region in the 3' untranslated region (UTR) of the SERT gene in both humans and rhesus macaques. We comprehensively survey the 3' UTR of SLC6A4 in Indian-origin rhesus macaques to identify three single nucleotide polymorphisms (SNPs) creating two haplotypes, both derived from an ancestral sequence, that represent the vast majority of the alleles in the population. Through the use of a luciferase reporter gene assay, we are able to show that not only do these alleles have differential effects on gene expression, modulated through changes in messenger RNA stability, but that different commonly occurring SNPs in the human 3' UTR also have similar effects. This finding not only offers additional insight into the regulation, and thus dysregulation, of SERT expression, but also suggests the role of natural selection in maintaining both high and low SERT expression levels broadly across populations of multiple primate species.     

Targeting the murine serotonin transporter: insights into human neurobiology

Mutations resulting in reduced or completely abrogated serotonin-transporter (SERT) function in mice have led to the identification of more than 50 different phenotypic changes, ranging from increased anxiety and stress-related behaviours to gut dysfunction, bone weakness and late-onset obesity with metabolic syndrome. These multiple effects, which can be amplified by gene-environment and gene-gene interactions, are primarily attributable to altered intracellular and extracellular serotonin concentrations during development and adulthood. Much of the human data relating to altered expression of the gene that encodes SERT are based on genetic-association findings or correlations and are therefore not as robust as the experimental mouse results. Nevertheless, SERT-function-modifying gene variants in humans apparently produce many phenotypes that are similar to those that manifest themselves in mice.     

Molecular characterization and intracellular regulation of the human serotonin transporter in Caco-2 cells.

The serotonin transporter (SERT) has shown itself to be an effective pharmacological target in the treatment of mood disorders and some kinds of gastrointestinal syndromes. Most of the molecular studies of SERT in humans have been carried out using heterologous models. In this work, we have investigated the human enterocyte-like Caco-2 cell line as a potential "in vitro" model to study the human SERT. The results show that these cells express a SERT mRNA identical to the human brain SERT, and a 70 kDa protein immunodetected using a specific antibody. The SERT activity levels in Caco-2 cells increased in correlation with the onset and maintenance of the morphological and functional differentiation of the cells. Caco-2 SERT was also shown to be a high affinity (Kt=0.216 microM) saturable, Na(+) -dependent transporter that was inhibited by fluoxetine (IC(50)=17.6 nM). In addition, SERT activity was inhibited by the intracellular modulators protein kinase C and cAMP, either after short or long-term treatment. In short, the expression and molecular characteristics of the human SERT in Caco-2 cells indicate that this cell line may be an ideal tool to study in vitro the physiology and pharmacology of human SERT.     

Characterization of an allosteric citalopram-binding site at the serotonin transporter

The serotonin transporter (SERT), which belongs to a family of sodium/chloride-dependent transporters, is the major pharmacological target in the treatment of several clinical disorders, including depression and anxiety. In the present study we show that the dissociation rate, of [3H]S-citalopram from human SERT, is retarded by the presence of serotonin, as well as by several antidepressants, when present in the dissociation buffer. Dissociation of [3H]S-citalopram from SERT is most potently inhibited by S-citalopram followed by R-citalopram, sertraline, serotonin and paroxetine. EC50 values for S- and R-citalopram are 3.6 +/- 0.4 microm and 19.4 +/- 2.3 microm, respectively. Fluoxetine, venlafaxine and duloxetine have no significant effect on the dissociation of [3H]S-citalopram. Allosteric modulation of dissociation is independent of temperature, or the presence of Na+ in the dissociation buffer. Dissociation of [3H]S-citalopram from a complex with the SERT double-mutant, N208Q/N217Q, which has been suggested to be unable to self-assemble into oligomeric complexes, is retarded to an extent similar to that found with the wild-type, raising the possibility that the allosteric mechanism is mediated within a single subunit. A species-scanning mutagenesis study comparing human and bovine SERT revealed that Met180, Tyr495 and Ser513 are important residues in mediating the allosteric effect, as well as contributing to high-affinity binding at the primary site.     

Serotonin transporters (SERTs) within the rat nucleus of the solitary tract: Subcellular distribution and relation to 5HT2A receptors

Serotonergic transmission is terminated by serotonin transporter (SERT)-mediated uptake following activation of serotonin receptors, several subtypes of which are present in the medial nucleus of the solitary tract (mNTS) at the area postrema level. In this region, serotonin (5HT) is a major modulator of the baroreceptor reflex and also affects gastric motility. This serotonin is derived from multiple sources including local neurons and inputs from raphe and visceral vagal afferents. To determine the relevant functional sites for serotonin uptake in the mNTS, we examined the electron microscopic localization of SERTs using both immunoperoxidase and immunogold labeling in rat brain. In addition, we combined these methods for dual labeling of SERTs and 5HT2A receptors to detect whether the SERT in this region was located near or at a distance from the sites of activation of these G-protein coupled receptors. Intensive SERT immunolabeling was seen on plasma membranes of axons and morphologically heterogeneous axon terminals that formed symmetric or asymmetric synapses on dendrites without detectable 5HT2A immunoreactivity (IR). 5HT2A-IR was, however, located in other nearby neuronal and glial profiles, some of which apposed intensively SERT-labeled terminals or terminals containing lower intensity of SERT immunolabeling. In somatodendritic profiles, co-expression of SERT and 5HT2A receptor immunolabeling was seen near synapses and Golgi lamellae. Our results suggest that in the mNTS 5HT activates 5HT2A receptors at a distance from SERT-mediated uptake sites in diverse cell types including some that express both 5HT2A receptors and SERTs.     

Human Serotonin Transporter Expression During Megakaryocytic Differentiation of MEG-01 Cells

The serotonin (5-HT) transporter (SERT) has been found altered in platelets of patients with genetically complex disorders, including mood-anxiety, pain and eating disorders. In this study, we used cell cultures of platelet precursors as models of investigation on mechanisms of SERT regulation: SERT expression was appraised during megakaryocytic differentiation of human megakaryoblastic MEG-01 cells. Cells were cultured for 8 days with 10^?7M 4-β-12-tetradecanoylphorbol-13-acetate (β-TPA) in the presence of 10% fetal bovine serum (FBS) and SERT was assessed by real time PCR, immunofluorescence microscopy, Western blot and [³H]5-HT re-uptake. Results revealed that SERT is present in control-untreated MEG-01 cells. β-TPA-differentiating MEG-01 cells showed a redistribution of SERT fluorescence, diffuse to cell bodies and blebs along with a 3-fold SERT mRNA increase and a moderate raise in SERT protein (1.5/1.4-fold) by immunoblot and re-uptake assays. In summary, we have shown herein that control megakaryoblasts express the SERT protein. SERT is modulated by differentiation events, implying that SERT density in platelets is under the control of megakaryocytopoiesis stages. Differentiation of MEG-01 cells can provide considerable insight into interactions between SERT genetics, transmitter-hormonal/homeostatic mechanisms and signaling pathways.     

Analysis of Serotonin Transporter in Human Platelets by Immunoblotting Using Site-Specific Antibodies

We have produced a panel of site-specific antibodies recognizing different regions of the human serotonin transporter (SERT). This panel included: 1) monoclonal antibodies 23C5 (mAbs 23C5) to the C-terminal region (amino acid residues 597-630); 2) polyclonal antibodies (pAbs) to the N-terminal region (amino acid residues 69-83); 3) pAbs to the region (amino acid residues 86-100) in the beginning of the first transmembrane domain (TMD). The antibodies were produced using recombinant proteins and synthetic peptides (containing certain sequences of SERT) as antigens. These antibodies were purified by affinity chromatography, conjugated to horseradish peroxidase (HRP), and used for immunoblotting analysis of SERT in extracts of human platelets. Sodium dodecyl sulfate extracts were prepared under conditions preventing non-specific proteolytic degradation of the proteins. In platelet extracts, all antibodies were able to detect the 67 kD protein, apparently corresponding to full-length SERT molecule (its theoretical mass is about 70 kD). These antibodies also detected several polypeptides of smaller size (56, 37, 35, 32, 22, and 14 kD), apparently corresponding to N-terminal, C-terminal, and non-terminal SERT fragments. Specificity of immunostaining was confirmed by preincubation of HRP-labeled anti-SERT antibodies with excess of corresponding antigen, which resulted in disappearance of protein band staining. It is suggested that SERT undergoes a programmed proteolytic cleavage (processing) resulting in formation of several SERT-derived polypeptides of smaller size. It is possible that one of the cleaved SERT species is required for serotonin transport activity. Possible sites for specific proteolysis may be located in the region near TMD1 and in the intracellular loop between TMD4 and TMD5.     

IFN-gamma and TNF-alpha decrease serotonin transporter function and expression in Caco2 cells

Recent studies have shown that mucosal serotonin (5-HT) transporter (SERT) expression is decreased in animal models of colitis, as well as in the colonic mucosa of humans with ulcerative colitis and irritable bowel syndrome. Altered SERT function or expression may underlie the altered motility, secretion, and sensation seen in these inflammatory gut disorders. In an effort to elucidate possible mediators of SERT downregulation, we treated cultured colonic epithelial cells (Caco2) with conditioned medium from activated human lymphocytes. Application of the conditioned medium caused a decrease in fluoxetine-sensitive [(3)H]5-HT uptake. Individual proinflammatory agents were then tested for their ability to affect uptake. Cells were treated for 48 or 72 h with PGE(2) (10 microM), IFN-gamma (500 ng/ml), TNF-alpha (50 ng/ml), IL-12 (50 ng/ml), or the nitric oxide-releasing agent S-nitrosoglutathione (GSNO; 100 microM). [(3)H]5-HT uptake was then measured. Neither PGE nor IL-12 had any effect on [(3)H]5-HT uptake, and GSNO increased uptake. However, after 3-day incubation, both TNF-alpha and IFN-gamma elicited significant decreases in SERT function. Neither TNF-alpha nor IFN-gamma were cytotoxic when used for this period of time and at these concentrations. These two cytokines also induced decreases in SERT mRNA and protein levels. By altering SERT expression, TNF-alpha and IFN-gamma could contribute to the altered motility and expression seen in vivo in ulcerative colitis or irritable bowel syndrome.     

Functional expression of milligram quantities of the synthetic human serotonin transporter gene in a tetracycline-inducible HEK293 cell line

The serotonin transporter (SERT), a member of the solute carrier 6 family, is responsible for reuptake of the monoamine neurotransmitter serotonin (5-hydroxytryptamine) from the synaptic cleft on the neural cells, and a vital target for several antidepressants. To investigate biophysical studies of this pharmacologically relevant transporter, we developed a mammalian expression system with tetracycline-inducible HEK293 cells using synthetic human SERT genes produced by PCR-based self-assembly method. Codon-optimization of this de novo constructed genes and construction of stable cell lines improved expression 3.5-fold and single-step immunoaffinity purification with FLAG-epitope tag yielded around one milligram functional SERT per liter culture medium assessed by [(3)H] imipramine ligand binding. Some characterizations including electrospray ionization MS/MS analysis, subcellular localization and cellular-uptake assay demonstrated that expressed human SERT was properly expressed, folded and fully functional. The long cytosolic N-terminal of SERT was predicted as containing 'intrinsically disordered region (IDR)' (～85 residues) by DISOPRED2 program. We engineered this salient region by step-wise truncation and ligand binding assay determined that dissociation constant for a series of de novo designed truncation constructs was close to the one for full-length wild type SERT. Our expression platform using synthetic codon-optimized gene and mammalian stable cell lines is feasible to produce milligram-scale functional membrane transporter for further biophysical and biochemical studies.     

Cellular and molecular alterations in mice with deficient and reduced serotonin transporters

The function of serotonin transporters (SERTs) is related to mood regulation. Mice with deficient or reduced SERT function (SERT knockout mice) show several behavioral changes, including increased anxiety-like behavior, increased sensitivity to stress, and decreases in aggressive behavior. Some of these behavioral alterations are similar to phenotypes found in humans with short alleles of polymorphism in the 5-hydroxytryptamine (5-HT) transporter-linked promoter region (5-HTTLPR). Therefore, SERT knockout mice can be used as a tool to study 5-HTTLPR-related variations in personality and may be the etiology of affective disorders. This article focuses on the cellular and molecular alterations in SERT knockout mice, including changes in 5-HT concentrations and its metabolism, alterations in 5-HT receptors, impaired hypothalamic-pituitary-adrenal gland axis, developmental changes in the neurons and brain, and influence on other neurotransmitter transporters and receptors. It also discusses the possible relationships between these alterations and the behavioral changes in these mice. The knowledge provides the foundation for understanding the cellular and molecular mechanisms that mediate the SERT-related mood regulation, which may have significant impact on understanding the etiology of affective disorders and developing better therapeutic approaches for affective disorders.     

Cysteine-scanning mutagenesis of serotonin transporter intracellular loop 2 suggests an alpha-helical conformation

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT) in intact cells expressing these mutants, but it inactivated beta-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an alpha-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.     

Interaction of Antidepressants with the Serotonin and Norepinephrine Transporters: MUTATIONAL STUDIES OF THE S1 SUBSTRATE BINDING POCKET*

The serotonin transporter (SERT) and the norepinephrine transporter (NET) are sodium-dependent neurotransmitter transporters responsible for reuptake of released serotonin and norepinephrine, respectively, into nerve terminals in the brain. A wide range of inhibitors of SERT and NET are used as treatment of depression and anxiety disorders or as psychostimulant drugs of abuse. Despite their clinical importance, the molecular mechanisms by which various types of antidepressant drugs bind and inhibit SERT and NET are still elusive for the majority of the inhibitors, including the molecular basis for SERT/NET selectivity. Mutational analyses have suggested that a central substrate binding site (denoted the S1 pocket) also harbors an inhibitor binding site. In this study, we determine the effect of mutating six key S1 residues in human SERT (hSERT) and NET (hNET) on the potency of 15 prototypical SERT/NET inhibitors belonging to different drug classes. Analysis of the resulting drug sensitivity profiles provides novel information on drug binding modes in hSERT and hNET and identifies specific S1 residues as important molecular determinants for inhibitor potency and hSERT/hNET selectivity.     

A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin

Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT.     

Partitioning of the serotonin transporter into lipid microdomains modulates transport of serotonin

The serotonin transporter (SERT) is an integral membrane protein responsible for the clearance of serotonin from the synaptic cleft following the release of the neurotransmitter. SERT plays a prominent role in the regulation of serotoninergic neurotransmission and is a molecular target for multiple antidepressants as well as substances of abuse. Here we show that SERT associates with lipid rafts in both heterologous expression systems and rat brain and that the inclusion of the transporter into lipid microdomains is critical for serotonin uptake activity. SERT is present in a subpopulation of lipid rafts, which is soluble in Triton X-100 but insoluble in other non-ionic detergents such as Brij 58. Disaggregation of lipid rafts upon depletion of cellular cholesterol results in a decrease of serotonin transport capacity (V(max)), due to the reduction of turnover number of serotonin transport. Our data suggest that the association of SERT with lipid rafts may represent a mechanism for regulating the transporter activity and, consequently, serotoninergic signaling in the central nervous system, through the modulation of the cholesterol content in the cell membrane. Furthermore, SERT-containing rafts are detected in both intracellular and cell surface fractions, suggesting that raft association may be important for trafficking and targeting of SERT.     

Serotonin transporter deficiency in rats contributes to impaired object memory

Serotonin is well known for its role in affection, but less known for its role in cognition. The serotonin transporter (SERT) has an essential role in serotonergic neurotransmission as it determines the magnitude and duration of the serotonin signal in the synaptic cleft. There is evidence to suggest that homozygous SERT knockout rats (SERT^−/−), as well as humans with the short SERT allele, show stronger cognitive effects than wild-type control rats (SERT^+/+) and humans with the long SERT allele after acute tryptophan depletion. In rats, SERT genotype is known to affect brain serotonin levels, with SERT^−/− rats having lower intracellular basal serotonin levels than wild-type rats in several brain areas. In the present study, it was investigated whether SERT genotype affects memory performance in an object recognition task with different inter-trial intervals. SERT^−/−, heterozygous SERT knockout (SERT^+/−) and SERT^+/+ rats were tested in an object recognition test applying an inter-trial interval of 2, 4 and 8 h. SERT^−/− and SERT^+/− rats showed impaired object memory with an 8 h inter-trial interval, whereas SERT^+/+ rats showed intact object memory with this inter-trial interval. Although brain serotonin levels cannot fully explain the SERT genotype effect on object memory in rats, these results do indicate that serotonin is an important player in object memory in rats, and that lower intracellular serotonin levels lead to enhanced memory loss. Given its resemblance with the human SERT-linked polymorphic region and propensity to develop depression-like symptoms, our findings may contribute to further understanding of mechanisms underlying cognitive deficits in depression.