The Pro-inflammatory Cytokine TNF-α Regulates the Activity and Expression of the Serotonin Transporter (SERT) in Astrocytes

Pro-inflammatory cytokines have been implicated in the precipitation of depression and related disorders, and the antidepressant sensitive serotonin transporter (SERT) may be a major target for immune regulation in these disorders. Here, we focus on astrocytes, a major class of immune competent cells in the brain, to examine the effects of pro-longed treatment with tumor necrosis factor-alpha (TNF-α) on SERT activity. We first established that high-affinity serotonin uptake into C6 glioma cells occurs through a SERT-dependent mechanism. Functional SERT expression is also confirmed for primary astrocytes. In both cell types, exposure to TNF-α resulted in a dose- and time-dependent increase in SERT-mediated 5-HT uptake, which was sustained for at least 48 h post-stimulation. Further analysis in primary astrocytes revealed that TNF-α enhanced the transport capacity (V_max) of SERT-specific 5-HT uptake, suggesting enhanced transporter expression, consistent with our observation of an increase in SERT mRNA levels. We confirmed that in both, primary astrocytes and C6 glioma cells, treatment with TNF-α activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Pre-treatment with the p38 MAPK inhibitor SB203580 attenuated the TNF-α mediated stimulation of 5-HT transport in both, C6 glioma and primary astrocytes. In summary, we show that SERT gene expression and activity in astrocytes is subject to regulation by TNF-α, an effect that is at least in part dependent on p38 MAPK activation.     

Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter

The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best system for the expression, purification and structural analysis of serotonin transporter (SERT), we expressed SERT in Escherichia coli, Pichia pastoris, the baculovirus expression system and in four different stable mammalian cell lines. Two stable cell lines that constitutively expressed SERT (Imi270 and Coca270) were constructed using episomal plasmids in HEK293 cells expressing the EBNA-1 antigen. SERT expression in the three different inducible stable mammalian cell lines was induced either by a decrease in temperature (cell line pCytTS-SERT), the addition of tetracycline to the growth medium (cell line T-REx-SERT) or by adding DMSO which caused the cells to differentiate (cell line MEL-SERT). All the mammalian cell lines expressed functional SERT, but SERT expressed in E. coli or P. pastoris was nonfunctional as assessed by 5-hydroxytryptamine uptake and inhibitor binding assays. Expression of functional SERT in the mammalian cell lines was assessed by an inhibitor binding assay; the cell lines pCytTS-SERT, Imi270 and Coca270 contained levels of functional SERT similar to that of the standard baculovirus expression system (250,000 copies per cell). The expression of SERT in induced T-REx-SERT cells was 400,000 copies per cell, but in MEL-SERT it was only 80,000 copies per cell. All the mammalian stable cell lines expressed SERT at the plasma membrane as assessed by [³H]-5-hydroxytryptamine uptake into whole cells, but the V_max for the T-Rex-SERT cell line was 10-fold higher than any of the other cell lines. It was noticeable that the cell lines that constitutively expressed SERT grew extremely poorly, compared to the inducible cell lines whose growth rates were similar to the parental cell lines when not induced. In addition, the cell lines MEL-SERT, Imi270 and T-REx-SERT all expressed fully N-glycosylated SERT and no unglycosylated inactive protein, in contrast to the baculovirus expression system where the vast majority of expressed SERT was unglycosylated and nonfunctional.     

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.     

Role of C-terminal region in the functional regulation of rat serotonin transporter (SERT)

Previously, we revealed that the state of the actin cytoskeleton affects the uptake activity of the serotonin transporter (SERT). Recently, it was reported that the C-terminus of SERT interacts with MacMARCKS, a substrate of PKC that can bind to the actin cytoskeleton. To elucidate the importance of the C-terminal region in the regulation of SERT activity and the interaction with the actin cytoskeleton, we examined whether the overexpression of the C-terminus affects the transport activity of SERT. To this end, we overexpressed a GFP-fused 30-amino acid construct of the SERT C-terminus (GFP-SERT-CT) in HEK293 cells stably expressing FLAG-tagged SERT (FL-SERT-HEK293 cells). The SERT uptake activity and transporter current were attenuated in GFP-SERT-CT-expressing FL-SERT-HEK293 cells, as compared with GFP-expressing FL-SERT-HEK293 cells. Eadie-Hofstee analysis revealed that GFP-SERT-CT overexpression attenuated the SERT uptake activity by reducing the Vmax, but not changing the Km, which was consistent with the results of experiments on the cell-surface expression of SET using biotinylation/immunoblot analysis. Immunocytochemical analysis demonstrated that GFP-SERT-CT was co-localized with FLAG-SERT and cortical actin at the plasma membrane. In addition, the SERT C-terminus did not affect dopamine transporter activity. These findings showed the significance of the C-terminal region to the functional regulation of SERT, suggesting that GFP-SERT-CT acts as a molecular decoy to disrupt the interaction between SERT and the actin cytoskeleton.     

Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter

The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best system for the expression, purification and structural analysis of serotonin transporter (SERT), we expressed SERT in Escherichia coli, Pichia pastoris, the baculovirus expression system and in four different stable mammalian cell lines. Two stable cell lines that constitutively expressed SERT (Imi270 and Coca270) were constructed using episomal plasmids in HEK293 cells expressing the EBNA-1 antigen. SERT expression in the three different inducible stable mammalian cell lines was induced either by a decrease in temperature (cell line pCytTS-SERT), the addition of tetracycline to the growth medium (cell line T-REx-SERT) or by adding DMSO which caused the cells to differentiate (cell line MEL-SERT). All the mammalian cell lines expressed functional SERT, but SERT expressed in E. coli or P. pastoris was nonfunctional as assessed by 5-hydroxytryptamine uptake and inhibitor binding assays. Expression of functional SERT in the mammalian cell lines was assessed by an inhibitor binding assay; the cell lines pCytTS-SERT, Imi270 and Coca270 contained levels of functional SERT similar to that of the standard baculovirus expression system (250,000 copies per cell). The expression of SERT in induced T-REx-SERT cells was 400,000 copies per cell, but in MEL-SERT it was only 80,000 copies per cell. All the mammalian stable cell lines expressed SERT at the plasma membrane as assessed by [3H]-5-hydroxytryptamine uptake into whole cells, but the V(max) for the T-Rex-SERT cell line was 10-fold higher than any of the other cell lines. It was noticeable that the cell lines that constitutively expressed SERT grew extremely poorly, compared to the inducible cell lines whose growth rates were similar to the parental cell lines when not induced. In addition, the cell lines MEL-SERT, Imi270 and T-REx-SERT all expressed fully N-glycosylated SERT and no unglycosylated inactive protein, in contrast to the baculovirus expression system where the vast majority of expressed SERT was unglycosylated and nonfunctional.     

Phosphorylation of threonine residue 276 is required for acute regulation of serotonin transporter by cyclic GMP

Cellular protein kinases, phosphatases, and other serotonin transporter (SERT) interacting proteins participate in several signaling mechanisms regulating SERT activity. The molecular mechanisms of protein kinase G (PKG)-mediated SERT regulation and the site of transporter phosphorylation were investigated. Treatment of rat midbrain synaptosomes with 8-bromo-cGMP increased SERT activity, and the increase was selectively blocked by PKG inhibitors. The V(max) value for serotonin (5-HT) transport increased following cGMP treatment. However, surface biotinylation studies showed no change in SERT surface abundance following PKG activation. (32)P metabolic labeling experiments showed increased SERT phosphorylation in the presence of cGMP that was abolished by selectively inhibiting PKG. Phosphoamino acid analysis revealed that cGMP-stimulated native SERT phosphorylation occurred only on threonine residues. When added to CHO-1 cells expressing SERT, 8-bromo-cGMP stimulated 5-HT transport and SERT phosphorylation. Mutation of SERT threonine 276 to alanine completely abolished cGMP-mediated stimulation of 5-HT transport and SERT phosphorylation. Although the T276A mutation had no significant effect on 5-HT transport or SERT protein expression, mutation to aspartate (T276D) increased the level of 5-HT uptake to that of cGMP-stimulated 5-HT uptake in wild-type SERT-expressing cells and was no longer sensitive to cGMP. These findings provide the first identification of a phosphorylation site in SERT and demonstrate that phosphorylation of Thr-276 is required for cGMP-mediated SERT regulation. They also constitute the first evidence that in the central nervous system PKG activation stimulates endogenous SERT activity by a trafficking-independent mechanism.     

The Cellular Distribution of Serotonin Transporter Is Impeded on Serotonin-Altered Vimentin Network

Background

The C-terminus of the serotonin transporter (SERT) contains binding domains for different proteins and is critical for its functional expression. In endogenous and heterologous expression systems, our proteomic and biochemical analysis demonstrated that an intermediate filament, vimentin, binds to the C-terminus of SERT. It has been reported that 5HT-stimulation of cells leads to disassembly and spatial reorientation of vimentin filaments.

Methodology/Principal Findings

We tested the impact of 5HT-stimulation on vimentin-SERT association and found that 5HT-stimulation accelerates the translocation of SERT from the plasma membrane via enhancing the level of association between phosphovimentin and SERT. Furthermore a progressive truncation of the C-terminus of SERT was performed to map the vimentin-SERT association domain. Deletion of up to 20, but not 14 amino acids arrested the transporters at intracellular locations. Although, truncation of the last 14 amino acids, did not alter 5HT uptake rates of transporter but abolished its association with vimentin.To understand the involvement of 5HT in phosphovimentin-SERT association from the plasma membrane, we further investigated the six amino acids between Δ14 and Δ20, i.e., the SITPET sequence of SERT. While the triple mutation on the possible kinase action sites, S₆₁₁, T₆₁₃, and T₆₁₆ arrested the transporter at intracellular locations, replacing the residues with aspartic acid one at a time altered neither the 5HT uptake rates nor the vimentin association of these mutants. However, replacing the three target sites with alanine, either simultaneously or one at a time, had no significant effect on 5HT uptake rates or the vimentin association with transporter.

Conclusions/Significance

Based on our findings, we propose that phosphate modification of the SITPET sequence differentially, one at a time exposes the vimentin binding domain on the C-terminus of SERT. Conversely, following 5HT stimulation, the association between vimentin-SERT is enhanced which changes the cellular distribution of SERT on an altered vimentin network.     

Modulation of monoamine transporter expression and function by repetitive transcranial magnetic stimulation

Repetitive transcranial magnetic stimulation (rTMS) is a new tool for the treatment of neuropsychiatric disorders. However, the mechanisms underlying the effects of rTMS are still unclear. In this study, we analyzed mRNA expression changes of monoamine transporter (MAT) genes, which are targets for antidepressants and psychostimulants. Following a 20-day rTMS treatment, these genes were found to be differentially expressed in the mouse brain. Down-regulation of serotonin transporter (SERT) mRNA levels and the subsequent decrease in serotonin uptake and binding were observed after chronic rTMS. In contrast to the SERT changes, increased mRNA levels of dopamine transporter (DAT) and norepinephrine transporter (NET) were observed. For NET, but not DAT, there were accompanying changes in uptake and binding. Similar effect on NET was observed in PC12 cells stimulated by rTMS for 15 days. These results indicate that modulation of MATs by chronic rTMS may be one therapeutic mechanism for the treatment of neuropsychiatric disorders.     

The mechanistic basis for noncompetitive ibogaine inhibition of serotonin and dopamine transporters

Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study.     

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.     

Discovery of SMP-304, a novel benzylpiperidine derivative with serotonin transporter inhibitory activity and 5-HT1A weak partial agonistic activity showing the antidepressant-like effect

We report the discovery of a novel benzylpiperidine derivative with serotonin transporter (SERT) inhibitory activity and 5-HT_1A receptor weak partial agonistic activity showing the antidepressant-like effect. The 3-methoxyphenyl group and the phenethyl group of compound 1, which has weak SERT binding activity, but potent 5-HT_1A binding activity, were optimized, leading to compound 35 with potent and balanced dual SERT and 5-HT_1A binding activity, but also potent CYP2D6 inhibitory activity. Replacement of the methoxy group in the left part of compound 35 with a larger alkoxy group, such as ethoxy, isopropoxy or methoxy-ethoxy group ameliorated CYP2D6 inhibition, giving SMP-304 as a candidate. SMP-304 with serotonin uptake inhibitory activity and 5-HT_1A weak partial agonistic activity, which could work as a 5-HT_1A antagonist, displayed faster onset of antidepressant-like effect than a representative SSRI paroxetine in an animal model.     

Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat

It has been proposed that serotonin (5-HT) facilitates the chemosensory activity of the carotid body (CB). In the present study, we investigated mRNA expression and immunohistochemical localization of the 5-HT synthetic enzyme isoforms, tryptophan hydroxylase 1 (TPH1) and TPH2, and the 5-HT plasma membrane transport protein, 5-HT transporter (SERT), in the CB of the rat. RT-PCR analysis detected the expression of mRNA for TPH1 and SERT in extracts of the CB. Using immunohistochemistry, 5-HT immunoreactivity was observed in a few glomus cells. TPH1 and SERT immunoreactivities were observed in almost all glomus cells. SERT immunoreactivity was seen on nerve fibers with TPH1 immunoreactivity. SERT immunoreactivity was also observed in varicose nerve fibers immunoreactive for dopamine beta-hydroxylase, but not in nerve fibers immunoreactive for vesicular acetylcholine transporters or nerve terminals immunoreactive for P2X₃ purinoreceptors. These results suggest that 5-HT is synthesized and released from glomus cells and sympathetic nerve fibers in the CB of the rat, and that the chemosensory activity of the CB is regulated by 5-HT from glomus cells and sympathetic nerve fibers.     

Thermostabilization of the Human Serotonin Transporter in an Antidepressant-Bound Conformation

Serotonin is a ubiquitous chemical transmitter with particularly important roles in the gastrointestinal, cardiovascular and central nervous systems. Modulation of serotonergic signaling occurs, in part, by uptake of the transmitter by the serotonin transporter (SERT). In the brain, SERT is the target for numerous antidepressants including tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs). Despite the importance of SERT in human physiology, biochemical, biophysical and high-resolution structural studies have been hampered due to the instability of SERT in detergent micelles. To identify a human SERT (hSERT) construct suitable for detailed biochemical and structural studies, we developed an efficient thermostability screening protocol and rapidly screened 219 mutations for thermostabilization of hSERT in complex with the SSRI paroxetine. We discovered three mutations—Y110A, I291A and T439S –that, when combined into a single construct, deemed TS3, yielded a hSERT variant with an apparent melting temperature (Tm) 19°C greater than that of the wild-type transporter, albeit with a loss of transport activity. Further investigation yielded a double mutant—I291A and T439S—defined as TS2, with a 12°C increase in Tm and retention of robust transport activity. Both TS2 and TS3 were more stable in short-chain detergents in comparison to the wild-type transporter. This thermostability screening protocol, as well as the specific hSERT variants, will prove useful in studies of other integral membrane receptors and transporters and in the investigation of structure and function relationships in hSERT.     

Distinct Effects of Imipramine on 5-Hydroxytryptamine Uptake Mediated by the Recombinant Rat Serotonin Transporter SERT1

Abstract: Tricyclic and nontricyclic serotonin [5-hydroxytryptamine (5-HT)] uptake inhibitors are widely used for the treatment of depression. Here, we show that both the tricyclic antidepressant imipramine and the nontricyclic antidepressant citalopram competitively inhibit 5-HT transport mediated by the recombinant rat 5-HT transporter SERT1. For citalopram, the concentration producing half-maximal transport inhibition was in the same order of magnitude as its K _D value determined by equilibrium binding. In contrast, the inhibitory potency of imipramine was more than one order of magnitude lower than its K _D value. Our data are consistent with low-affinity imipramine binding occurring at or close to the substrate recognition site, which also binds citalopram. Occupation of the high-affinity imipramine binding site on SERT1 did not affect 5-HT transport but allosterically displaced citalopram from the substrate recognition site. Consequently, low concentrations of imipramine partially protected 5-HT transport from citalopram inhibition. This protection was only observed in the presence of Na⁺ because high-affinity imipramine binding is strictly sodium-dependent. Thus, depending on which of its binding sites on SERT1 is occupied, imipramine may exert distinct effects on 5-HT uptake mediated by the recombinant rat 5-HT transporter.     

Glycosyl modification facilitates homo- and hetero-oligomerization of the serotonin transporter. A specific role for sialic acid residues

The serotonin transporter (SERT) is an oligomeric glycoprotein with two sialic acid residues on each of two complex oligosaccharide molecules. In this study, we investigated the contribution of N-glycosyl modification to the structure and function of SERT in two model systems: wild-type SERT expressed in sialic acid-defective Lec4 Chinese hamster ovary (CHO) cells and a mutant form (after site-directed mutagenesis of Asn-208 and Asn-217 to Gln) of SERT, QQ, expressed in parental CHO cells. In both systems, SERT monomers required modification with both complex oligosaccharide residues to associate with each other and to function in homo-oligomeric forms. However, defects in sialylated N-glycans did not alter surface expression of the SERT protein. Furthermore, in heterologous (CHO and Lec4 cells) and endogenous (placental choriocarcinoma JAR cells) expression systems, we tested whether glycosyl modification also manipulates the hetero-oligomeric interactions of SERT, specifically with myosin IIA. SERT is phosphorylated by cGMP-dependent protein kinase G through interactions with anchoring proteins, and myosin is a protein kinase G-anchoring protein. A physical interaction between myosin and SERT was apparent; however, defects in sialylated N-glycans impaired association of SERT with myosin as well as the stimulation of the serotonin uptake function in the cGMP-dependent pathway. We propose that sialylated N-glycans provide a favorable conformation to SERT that allows the transporter to function most efficiently via its protein-protein interactions.     

Serotonin transporter phosphorylation modulated by tetanus toxin

Tetanus toxin (TeTx) modifies Na(+)-dependent, high-affinity 5-hydroxytryptamine (5-HT, serotonin) uptake in a synaptosomal-enriched P(2) fraction from rat brain. The effect corresponds to a rapid and non-competitive uptake inhibition, and it is preceded by induction of phospholipase C (PLC) activity and translocation and down-regulation of the classical protein kinase C (PKC-alpha, -beta and -gamma) isoforms. The effects on serotonin transport and on cPKC activation were similar to the effects exhibited by phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Moreover, after treatment with TeTx, an increase in Ser- and Tyr-specific phosphorylation was found. Activation of PKC by both TeTx and TPA results in a loss of transport capacity and serotonin transporter (SERT) phosphorylation, which are abolished by coapplication of the specific PKC inhibitor bisindolylmaleimide-1. Since a specific PLCgamma1 phosphorylation prior to TeTx's inducing SERT phosphorylation was found, the studies suggest that part of the action of TeTx consists of modifying the signal cascade initiated in tyrosine kinase receptors on nerve tissue previous to its cellular internalization, resulting in transporter phosphorylation.     

Substrate binding and translocation of the serotonin transporter studied by docking and molecular dynamics simulations

The serotonin (5-HT) transporter (SERT) plays an important role in the termination of 5-HT-mediated neurotransmission by transporting 5-HT away from the synaptic cleft and into the presynaptic neuron. In addition, SERT is the main target for antidepressant drugs, including the selective serotonin reuptake inhibitors (SSRIs). The three-dimensional (3D) structure of SERT has not yet been determined, and little is known about the molecular mechanisms of substrate binding and transport, though such information is very important for the development of new antidepressant drugs. In this study, a homology model of SERT was constructed based on the 3D structure of a prokaryotic homologous leucine transporter (LeuT) (PDB id: 2A65). Eleven tryptamine derivates (including 5-HT) and the SSRI (S)-citalopram were docked into the putative substrate binding site, and two possible binding modes of the ligands were found. To study the conformational effect that ligand binding may have on SERT, two SERT–5-HT and two SERT–(S)-citalopram complexes, as well as the SERT apo structure, were embedded in POPC lipid bilayers and comparative molecular dynamics (MD) simulations were performed. Our results show that 5-HT in the SERT–5-HT^B complex induced larger conformational changes in the cytoplasmic parts of the transmembrane helices of SERT than any of the other ligands. Based on these results, we suggest that the formation and breakage of ionic interactions with amino acids in transmembrane helices 6 and 8 and intracellular loop 1 may be of importance for substrate translocation.     

Effect of a new potential psychotropic drug, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride,on the expression of serotonin-related genes in the mouse brain

Investigation of molecular mechanisms underlying psychotropic drug action is the main aim of molecular psychopharmacology. Previously, a new synthetic varacin analog, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine (TC-2153) was shown to produce anxiolytic and anticonvulsant effects in mice. This study investigated the effects of chronic TC-2153 administration on the expression of some serotonin-related genes in the mouse brain. The drug was administered (10 mg/kg, per os, 16 days) to adult male mice of the ASC (Antidepressant Sensitive Catalepsy) strain characterized by altered behavior and hereditary impairment of the brain serotonin system. Expression of genes encoding tryptophan hydroxylase 2 (TPH2), the key enzyme of serotonin synthesis, monoamine oxydase A (MAOA), the major serotonin-degrading enzyme, 5-HT transporter (SERT), and 5-HT_1A receptor was studied using quantitative RT-PCR. TC-2153 significantly reduced the 5-HT_1A receptor and MAOA mRNA levels in the midbrain, but did not have any effect on the expression of these genes in the frontal cortex and the hippocampus. The drug did not affect the expression of TPH2 and SERT in the midbrain. The results indicate that the brain 5-HT system is involved in the molecular basis of TC-2153 action.     

The C-terminus is critical for the functional expression of the human serotonin transporter

The plasma membrane serotonin transporter (SERT) has an important role in terminating serotonergic neurotransmission by re-uptake of 5-HT from the synaptic cleft. The expression of SERT on the cell surface is therefore a critical factor. In this study, we examined the role of the carboxyl terminus of SERT in trafficking to the plasma membrane. 5-HT uptake activity was used to measure the effects of systematic deletions or alanine substitutions in the C-terminus. We found that deletion of 16 amino acids in the distal C-terminus had no effect on uptake activity, whereas further deletion was detrimental for the function of SERT. Cell surface biotinylation was used to determine the role of the C-terminus in localization and trafficking. We showed that the C-terminus is crucial for the delivery of SERT to the plasma membrane and that the deletion of this part of the transporter results in a lack of mature glycosylation and impaired trafficking to the plasma membrane. Furthermore, the C-terminally truncated mutants were shown to have a dominant negative effect on wild-type SERT uptake activity.