Sitte, Harald H., Hiptmair, Birgit, Zwach, Julia, Pifl, Christian, Singer, Ernst A., Scholze, Petra
Quantitative Analysis of Inward and Outward Transport Rates in Cells Stably Expressing the Cloned Human Serotonin Transporter: Inconsistencies with the Hypothesis of Facilitated Exchange Diffusion
Mol Pharmacol 2001 59: 1129-1137
"Quantitative aspects of inward and outward transport of substrates by the human plasmalemmal serotonin transporter (hSERT) were investigated. Uptake and superfusion experiments were performed on human embryonic kidney 293 cells permanently expressing the hSERT using [(3)H]serotonin (5-HT) and [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) as substrates. Saturation analyses rendered K(m) values of 0.60 and 17.0 microM for the uptake of [(3)H]5-HT and [(3)H]MPP(+), respectively. Kinetic analysis of outward transport was performed by prelabeling the cells with increasing concentrations of the two substrates and exposing them to a saturating concentration of p-chloroamphetamine (PCA; 10 microM). Apparent K(m) values for PCA induced transport were 564 microM and about 7 mM intracellular [(3)H]5-HT and [(3)H]MPP(+), respectively. Lowering the extracellular Na(+) concentrations in uptake and superfusion experiments revealed differential effects on substrate transport: at 10 mM Na(+) the K(m) value for [(3)H]5-HT uptake increased approximately 5-fold and the V(max) value remained unchanged. The K(m) value for [(3)H]MPP(+) uptake also increased, but the V(max) value was reduced by 50%. When efflux was studied at saturating prelabeling conditions of both substrates, PCA as well as unlabeled 5-HT and MPP(+) (all substances at saturating concentrations) induced the same efflux at 10 mM and 120 mM Na(+). Thus, notwithstanding a 50% reduction in the V(max) value of transport into the cell, MPP(+) was still able to induce maximal outward transport of either substrate. Thus, hSERT-mediated inward and outward transport seems to be independently modulated and may indicate inconsistencies with the classical model of facilitated exchange diffusion." [Full Text]

Adams, Scott V., DeFelice, Louis J.
Ionic Currents in the Human Serotonin Transporter Reveal Inconsistencies in the Alternating Access Hypothesis
Biophys. J. 2003 85: 1548-1559
"We have investigated the conduction states of human serotonin transporter (hSERT) using the voltage clamp, cut-open frog oocyte method under different internal and external ionic conditions. Our data indicate discrepancies in the alternating access model of cotransport, which cannot consistently explain substrate transport and electrophysiological data. We are able simultaneously to isolate distinct external and internal binding sites for substrate, which exert different effects upon currents conducted by hSERT, in contradiction to the alternating access model. External binding sites of coupled Na ions are likewise simultaneously accessible from the internal and external face. Although Na and Cl are putatively cotransported, they have opposite effects on the internal face of the transporter. Finally, the internal K ion does not compete with internal 5-hydroxytryptamine for empty transporters. These data can be explained more readily in the language of ion channels, rather than carrier models distinguished by alternating access mechanisms: in a channel model of coupled transport, the currents represent different states of the same permeation path through hSERT and coupling occurs in a common pore." [Abstract]

Kocabas AM, Rudnick G, Kilic F.
Functional consequences of homo- but not hetero-oligomerization between transporters for the biogenic amine neurotransmitters.
J Neurochem. 2003 Jun;85(6):1513-20.
"Before this study, the human norepinephrine transporter (hNET) was the only member of the biogenic amine neurotransmitter transporter family that had not been demonstrated to be a functional homo-oligomer. Here, using two forms of the transporter, I155C and hNET-myc, with distinct antigenicity and inhibitor sensitivity, we demonstrated that hNET exists as a homo-oligomer. hNET I155C is a functional mutant and is sensitive to inactivation by the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate, while hNET-myc is resistant to inactivation by this reagent. Coimmunoprecipitation of these two forms demonstrated that a physical interaction exists between norepinephrine transporter monomers. Further characterization of this physical interaction has revealed that the activity of norepinephrine transporters depends on interactions between monomers. Because norepinephrine transporters and serotonin transporters are the only two members of the neurotransmitter transporter family endogenously expressed in the cell membrane of the same cells, placental syncytiotrophoblasts, we tested the ability of norepinephrine transporters and serotonin transporters to associate and function in a hetero-oligomeric form. Similarly, coexpression of hNET-myc with serotonin transporter-FLAG showed a physical interaction in coimmunoprecipitation assays. However, coexpression of serotonin and norepinephrine transporters did not sensitize norepinephrine transporter activity to inhibition by citalopram, a selective serotonin transport inhibitor. Thus, the norepinephrine transporter-serotonin transporter physical association did not produce functional consequences. Based on this, we propose that the transporters for biogenic amine neurotransmitters interact functionally in homo- but not hetero-oligomeric forms." [Abstract]

Deniz Ozaslan, Sophie Wang, Billow A. Ahmed, Arif M. Kocabas, John C. McCastlain, Anca Bene, and Fusun Kilic
Glycosyl Modification Facilitates Homo- and Hetero-oligomerization of the Serotonin Transporter: A SPECIFIC ROLE FOR SIALIC ACID RESIDUES
J. Biol. Chem. 278: 43991-44000.
"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." [Full Text]

Sammanda Ramamoorthy, Elena Giovanetti, Yan Qian, and Randy D. Blakely
Phosphorylation and Regulation of Antidepressant-sensitive Serotonin Transporters
J. Biol. Chem. 273: 2458-2466, January 1998. [Full Text]

Masson, J., Sagne, C., Hamon, M., Mestikawy, S. El
Neurotransmitter Transporters in the Central Nervous System
Pharmacol Rev 1999 51: 439-464 [Full Text]

Pineyro, Graciela, Blier, Pierre
Autoregulation of Serotonin Neurons: Role in Antidepressant Drug Action
Pharmacol Rev 1999 51: 533-591 [Full Text]

Qian, Yan, Galli, Aurelio, Ramamoorthy, Sammanda, Risso, Stefania, DeFelice, Louis J., Blakely, Randy D.
Protein Kinase C Activation Regulates Human Serotonin Transporters in HEK-293 Cells via Altered Cell Surface Expression
J. Neurosci. 1997 17: 45-57 [Full Text]

Yoel Smicun, Scott D. Campbell, Marisa A. Chen, Howard Gu, and Gary Rudnick
The Role of External Loop Regions in Serotonin Transport. LOOP SCANNING MUTAGENESIS OF THE SEROTONIN TRANSPORTER EXTERNAL DOMAIN
J. Biol. Chem. 274: 36058-36064, December 1999. [Full Text]

Andreas Androutsellis-Theotokis, Farshid Ghassemi, and Gary Rudnick
A Conformationally Sensitive Residue on the Cytoplasmic Surface of Serotonin Transporter
J. Biol. Chem. 276: 45933-45938, December 2001. [Full Text]

Christopher G. Tate, Erik Whiteley, and Michael J. Betenbaugh
Molecular Chaperones Stimulate the Functional Expression of the Cocaine-sensitive Serotonin Transporter
J. Biol. Chem. 274: 17551-17558.
"The serotonin transporter (SERT) is an N-glycosylated integral membrane protein that is predicted to contain 12 transmembrane regions. SERT is the major binding site in the brain for antidepressant drugs, and it also binds amphetamines and cocaine. The ability of various molecular chaperones to interact with a tagged version of SERT (Myc-SERT) was investigated using the baculovirus expression system. Overexpression of Myc-SERT using the baculovirus system led to substantial quantities of inactive transporter, together with small amounts of fully active and, therefore, correctly folded molecules. The high levels of inactive Myc-SERT probably arose because folding was rate-limiting due, perhaps, to insufficient molecular chaperones. Therefore, Myc-SERT was co-expressed with the endoplasmic reticulum (ER) molecular chaperones calnexin, calreticulin and immunoglobulin heavy chain binding protein (BiP), and the foldase, ERp57. The expression of functional Myc-SERT, as determined by an inhibitor binding assay, was enhanced nearly 3-fold by co-expressing calnexin, and to a lesser degree on co-expression of calreticulin and BiP. Co-expression of ERp57 did not increase the functional expression of Myc-SERT. A physical interaction between Myc-SERT-calnexin and Myc-SERT-calreticulin was demonstrated by co-immunoprecipitation. These associations were inhibited in vivo by deoxynojirimycin, an inhibitor of N-glycan precusor trimming that is known to prevent the calnexin/calreticulin-N-glycan interaction. Functional expression of the unglycosylated SERT mutant, SERT-QQ, was also increased on co-expression of calnexin, suggesting that the interaction between calnexin and SERT is not entirely dictated by the N-glycan. SERT is the first member of the neurotransmitter transporter family whose folding has been shown to be assisted by the molecular chaperones calnexin, calreticulin, and BiP." [Full Text]

Murphy, Dennis L., Lerner, Alicja, Rudnick, Gary, Lesch, Klaus-Peter
Serotonin Transporter: Gene, Genetic Disorders, and Pharmacogenetics
Mol. Interv. 2004 4: 109-123
"The highly evolutionarily conserved serotonin transporter (SERT) regulates the entire serotoninergic system and its receptors via mod-ulation of extracellular fluid serotonin concentrations. Differences in SERT expression and function produced by three SERT genes and their variants show associations with multiple human disorders. Screens of DNA from patients with autism, ADHD, bipolar disorder, and Tourette’s syndrome have detected signals in the chromosome 17q region where SERT is locat-ed. Parallel investigations of SERT knockout mice have uncovered multiple phenotypes that identify SERT as a candidate gene for additional human disorders ranging from irritable bowel syndrome to obesity. Replicated studies have demonstrated that the SERT 5'-flanking region polymorphism SS genotype is associated with poorer therapeutic responses and more frequent serious side effects during treatment with antidepressant SERT antagonists, namely, the serotonin reuptake inhibitors (SRIs)." [Abstract]

Tafet GE, Idoyaga-Vargas VP, Abulafia DP, Calandria JM, Roffman SS, Chiovetta A, Shinitzky M.
Correlation between cortisol level and serotonin uptake in patients with chronic stress and depression.
Cogn Affect Behav Neurosci 2001 Dec;1(4):388-93
"In a recent study (Tafet, Toister-Achituv, & Shinitzky, 2001), we demonstrated that cortisol induces an increase in the expression of the gene coding for the serotonin transporter, associated with a subsequent elevation in the uptake of serotonin. This stimulatory effect, produced upon incubation with cortisol in vitro, was observed in peripheral blood lymphocytes from normal subjects. In the present work we investigated the cortisol-induced increase in serotonin uptake in lymphocytes from hypercortisolemic patients, including subjects with major depressive disorder (n = 8), and subjects with generalized anxiety disorder (n = 12), in comparison with a control group of normal healthy subjects (n = 8). A significant increase in serotonin uptake (+37% + 14, M + SD) was observed in the control group, whereas neither the generalized anxiety disorder nor the major depression group exhibited changes in serotonin uptake upon incubation with cortisol. It is likely that under chronic stress or depression, the capacity for increase in serotonin transporter has reached its limit due to the chronically elevated blood cortisol level. The physiological and diagnostic implications of this observation are discussed." [Abstract]

Jayanthi D. Ramamoorthy, Sammanda Ramamoorthy, Andreas Papapetropoulos, John D. Catravas, Frederick H. Leibach, and Vadivel Ganapathy
Cyclic AMP-independent Up-regulation of the Human Serotonin Transporter by Staurosporine in Choriocarcinoma Cells
J. Biol. Chem. 270: 17189-17195, 1995.
"Treatment of confluent cultures of JAR human placental choriocarcinoma cells with staurosporine caused a marked stimulation of serotonin transport activity in these cells. The stimulatory effect was noticeable at nanomolar concentrations of staurosporine, and a treatment time of > 4 h was required for staurosporine to elicit the effect. At 40 nM and with a treatment time of 16 h, the stimulation of the transport activity was 3.5-6.0-fold. None of the several other protein kinase inhibitors tested had similar effect except KT 5720, a protein kinase A inhibitor, which showed a small but significant (approximately 1.4-fold) stimulatory effect at a concentration of 5 microM. Blockade of RNA synthesis and protein synthesis in the cells prevented completely the stimulation of the transport activity induced by staurosporine. The stimulation was observed not only in intact cells but also in plasma membrane vesicles prepared from staurosporine-treated cells. The stimulation was accompanied by a 5-7-fold increase in the steady state levels of the transporter-specific mRNAs, by a 7-fold increase in the maximal velocity of the transport process, and by a 6-fold increase in the transporter density in the plasma membrane. Even though both staurosporine and cholera toxin had similar effects on the serotonin transport activity in these cells, the effect was not additive when the cells were treated with both reagents together. While treatment of the cells with cholera toxin markedly elevated intracellular levels of cAMP, staurosporine did not have any effect on the cellular levels of this cyclic nucleotide. It is concluded that staurosporine up-regulates the serotonin transport activity in JAR cells by increasing the steady state levels of the serotonin transporter mRNA and by the consequent increase in the transporter density in the plasma membrane and that the process involves a cAMP-independent signaling pathway." [Full Text]

Kekuda, Ramesh, Leibach, Frederick H., Furesz, Todd C., Smith, Carl H., Ganapathy, Vadivel
Polarized Distribution of Interleukin-1 Receptors and Their Role in Regulation of Serotonin Transporter in Placenta
J Pharmacol Exp Ther 2000 292: 1032-1041
"We investigated the expression of interleukin-1 (IL-1) receptors and their involvement in the regulation of the serotonin transporter gene expression in human placenta. IL-1beta is an activator of the serotonin transporter gene expression in JAR human placental choriocarcinoma cells as demonstrated by an increase in the steady-state levels of the transporter mRNA and in serotonin transport activity. This activation is blocked by IL-1 receptor antagonist. Genistein also blocks the effect of IL-1beta, indicating involvement of tyrosine phosphorylation in the process. Treatment of JAR cells with IL-1beta activates mitogen-activated protein kinases and nuclear factor-kappaB. The nuclear factor-kappaB that is responsive to IL-1beta in these cells is the p65 homodimer. Northern blot analysis and reverse transcription-polymerase chain reaction revealed that JAR cells and human placenta express type I and type II IL-1 receptors. The binding sites for [125]I-IL-1beta are localized predominantly in the maternal-facing brush border membrane of the syncytiotrophoblast. These results show that IL-1 in the maternal circulation is likely to play a critical role in the regulation of the serotonin transporter gene expression in the placenta." [Full Text]

Persico, Antonio M., Mengual, Elisa, Moessner, Rainald, Hall, Scott F., Revay, Randal S., Sora, Ichiro, Arellano, Jon, DeFelipe, Javier, Gimenez-Amaya, Jose Manuel, Conciatori, Monica, Marino, Ramona, Baldi, Alfonso, Cabib, Simona, Pascucci, Tiziana, Uhl, George R., Murphy, Dennis L., Lesch, K. Peter, Keller, Flavio
Barrel Pattern Formation Requires Serotonin Uptake by Thalamocortical Afferents, and Not Vesicular Monoamine Release
J. Neurosci. 2001 21: 6862-6873 [Full Text]

Upton, A. L., Salichon, N., Lebrand, C., Ravary, A., Blakely, R., Seif, I., Gaspar, P.
Excess of Serotonin (5-HT) Alters the Segregation of Ispilateral and Contralateral Retinal Projections in Monoamine Oxidase A Knock-Out Mice: Possible Role of 5-HT Uptake in Retinal Ganglion Cells During Development
J. Neurosci. 1999 19: 7007-7024
"In this paper we show that in MAOA-KO mice, elevated levels of brain 5-HT during the first 2 weeks of postnatal development prevent the ipsilateral and contralateral retinal projections from segregating into eye-specific areas in their target structures. Furthermore, we show that in normal and MAOA-KO mice SERT, VMAT2, and 5-HT1B are jointly expressed by a subpopulation of developing RGCs during the period of axonal remodeling. We propose that 5-HT could, via these molecules, influence retinofugal pathways and thereby help in sculpting their adult pattern of connections." [Full Text]

Salichon, Nathalie, Gaspar, Patricia, Upton, A. Louise, Picaud, Sandrine, Hanoun, Naima, Hamon, Michel, De Maeyer, Edward, Murphy, Dennis L., Mossner, Rainald, Lesch, Klaus Peter, Hen, Rene, Seif, Isabelle
Excessive Activation of Serotonin (5-HT) 1B Receptors Disrupts the Formation of Sensory Maps in Monoamine Oxidase A and 5-HT Transporter Knock-Out Mice
J. Neurosci. 2001 21: 884-896 [Full Text]

Yura A, Kiuchi Y, Uchikawa T, Uchida J, Yamazaki K, Oguchi K.
Possible involvement of calmodulin-dependent kinases in Ca(2+)-dependent enhancement of [3H]5-hydroxytryptamine uptake in rat cortex.
Brain Res 1996 Oct 28;738(1):96-102
"Effects of Ca2+ on [3H]5-hydroxytryptamine (5-HT) uptake into rat cortical synaptosomes were studied. The uptake was enhanced in the presence of Ca2+ in Krebs-Ringer medium and the uptake at 0.3-5 mM Ca2+ was 2.4-2.7 times greater than that observed in the absence of Ca2+. The maximal increase at the concentration of 1 mM Ca2+ was achieved after 2 min preincubation. Ca(2+)-dependent enhancement of the [3H]5-HT uptake reflected an increase in Vmax of the uptake process. However, Kd and Bmax values for [3H]paroxetine were not significantly changed in the presence of 1 mM Ca2+ compared with Ca(2+)-free condition. On the other hand, uptake was still enhanced after synaptosomes were washed with Ca(2+)-free after preincubation with 1 mM Ca2+. Staurosporine (a protein kinase C inhibitor) and wortmannin (a myosin light chain kinase inhibitor) did not affect Ca(2+)-dependent enhancement of the uptake, whereas 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazin e (KN-62, an inhibitor of Ca2+ /calmodulin-dependent kinase II) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a calmodulin antagonist) significantly reduced it. Moreover, L-type, but not P- or N-type, voltage-dependent Ca(2+)-channel blockers suppressed enhancement of the uptake. These results indicate that Ca(2+)-dependent enhancement of [3H]5-HT uptake is mediated by activation of calmodulin-dependent protein kinases, suggesting a possibility of calmodulin-dependent regulation of in vivo 5-HT uptake." [Abstract]

Ase, Ariel R., Reader, Tomas A., Hen, Rene, Riad, Mustapha, Descarries, Laurent
Regional changes in density of serotonin transporter in the brain of 5-HT1A and 5-HT1B knockout mice, and of serotonin innervation in the 5-HT1B knockout.
J Neurochem 2001 78: 619-630 [Abstract]

S Ramamoorthy, DR Cool, VB Mahesh, FH Leibach, HE Melikian, RD Blakely, and V Ganapathy
Regulation of the human serotonin transporter. Cholera toxin-induced stimulation of serotonin uptake in human placental choriocarcinoma cells is accompanied by increased serotonin transporter mRNA levels and serotonin transporter-specific ligand binding
J. Biol. Chem. 268: 21626-21631, October 1993.
"Treatment of confluent cultures of JAR human placental choriocarcinoma cells with cholera toxin or forskolin for 16 h markedly stimulated (2.4- fold) serotonin transport activity in these cells. Cycloheximide, an inhibitor of protein synthesis or actinomycin D, an inhibitor of mRNA synthesis effectively blocked this stimulation. Northern blot analysis revealed that treatment with cholera toxin resulted in severalfold increase in the concentrations of the three mRNA species (6.8, 4.9 and 3.0 kilobases in size) which hybridized to the human placental serotonin transporter cDNA. Under similar conditions, the concentrations of the mRNA species which hybridized to the human placental taurine transporter cDNA or to the human beta-actin cDNA were not affected. Analysis of paroxetine-sensitive binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane to the membranes prepared from control and cholera toxin-treated cells indicated that the maximal binding capacity was increased 2.5-fold by cholera toxin, with no significant change in the binding affinity. Thus, stimulation of serotonin transporter activity in the placental choriocarcinoma cells following cholera toxin treatment is likely a result of an increase in cell surface density of the serotonin transporter protein as a consequence of increased steady state serotonin transporter mRNA levels." [Abstract/Full Text]

Roxanne A. Vaughan, Robin A. Huff, George R. Uhl, and Michael J. Kuhar
Protein Kinase C-mediated Phosphorylation and Functional Regulation of Dopamine Transporters in Striatal Synaptosomes
J. Biol. Chem. 272: 15541-15546, 1997.
"Dopamine transporters (DATs) are members of a family of Na+- and Cl-dependent neurotransmitter transporters responsible for the rapid clearance of dopamine from synaptic clefts. The predicted primary sequence of DAT contains numerous consensus phosphorylation sites. In this report we demonstrate that DATs undergo endogenous phosphorylation in striatal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [32P]orthophosphate, and solubilized homogenates were subjected to immunoprecipitation with an antiserum specific for DAT. Basal phosphorylation occurred in the absence of exogenous treatments, and the phosphorylation level was rapidly increased when synaptosomes were treated with the phosphatase inhibitors okadaic acid or calyculin. Treatment of synaptosomes with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also increased the level of phosphate incorporation. This occurred within 10 min and was dosedependent between 0.1 and 1 µM PMA. DAT phosphorylation was also significantly increased by two other protein kinase C activators, ()-indolactam V and 1-oleoyl-2-acetyl-sn-glycerol. The inactive phorbol ester 4-phorbol 12,13-didecanoate at 10 µM was without effect, and PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors staurosporine and bisindoylmaleimide. These results indicate that DATs undergo rapid in vivo phosphorylation in response to protein kinase C activation and that a robust mechanism exists in synaptosomes for DAT dephosphorylation. Dopamine transport activity in synaptosomes was reduced by all treatments that promoted DAT phosphorylation, with comparable dose, time, and inhibitor characteristics. The change in transport activity was produced by a reduction in Vmax with no significant effect on the Km for dopamine. These results suggest that synaptosomal dopamine transport activity is regulated by phosphorylation of DAT and present a potential mechanism for local neuronal control of synaptic neurotransmitter levels and consequent downstream neural activity." [Full Text]

Carvelli, Lucia, Moron, Jose A, Kahlig, Kristopher M, Ferrer, Jasmine V, Sen, Namita, Lechleiter, James D, Leeb-Lundberg, L. M. Fredrik, Merrill, Gerald, Lafer, Eileen M, Ballou, Lisa M, Shippenberg, Toni S, Javitch, Jonathan A, Lin, Richard Z, Galli, Aurelio
PI 3-kinase regulation of dopamine uptake
J Neurochem 2002 81: 859-869
"The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3-kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3 H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3-kinase, increased [3 H]DA uptake and blocked the amphetamine-induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3-kinase. The LY294002-induced reduction in [3 H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin-dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs." [Abstract]

Miller, Dennis K., Sumithran, Sangeetha P., Dwoskin, Linda P.
Bupropion Inhibits Nicotine-Evoked [3H]Overflow from Rat Striatal Slices Preloaded with [3H]Dopamine and from Rat Hippocampal Slices Preloaded with [3H]Norepinephrine
J Pharmacol Exp Ther 2002 302: 1113-1122
"Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters (DAT and NET, respectively). Recently, bupropion has been reported to noncompetitively inhibit alpha3beta2, alpha3beta4, and alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes or established cell lines. The present study evaluated bupropion-induced inhibition of native alpha3beta2* and alpha3beta4* nAChRs using functional neurotransmitter release assays, nicotine-evoked [(3)H]overflow from superfused rat striatal slices preloaded with [(3)H]dopamine ([(3)H]DA), and nicotine-evoked [(3)H]overflow from hippocampal slices preloaded with [(3)H]norepinephrine ([(3)H]NE). The mechanism of inhibition was evaluated using Schild analysis. To eliminate the interaction of bupropion with DAT or NET, nomifensine or desipramine, respectively, was included in the superfusion buffer. A high bupropion concentration (100 microM) elicited intrinsic activity in the [(3)H]DA release assay. However, none of the concentrations (1 nM-100 microM) examined evoked [(3)H]NE overflow and, thus, were without intrinsic activity in this assay. Moreover, bupropion inhibited both nicotine-evoked [(3)H]DA overflow (IC(50) = 1.27 microM) and nicotine-evoked [(3)H]NE overflow (IC(50) = 323 nM) at bupropion concentrations well below those eliciting intrinsic activity. Results from Schild analyses suggest that bupropion competitively inhibits nicotine-evoked [(3)H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of bupropion inhibition of nicotine-evoked [(3)H]NE overflow. Thus, bupropion acts as an antagonist at alpha3beta2* and alpha3beta4* nAChRs in rat striatum and hippocampus, respectively, across the same concentration range that inhibits DAT and NET function. The combination of nAChR and transporter inhibition produced by bupropion may contribute to its clinical efficacy as a smoking cessation agent." [Abstract]

Miller, Dennis K., Wong, Erik H. F., Chesnut, M. Dathan, Dwoskin, Linda P.
Reboxetine: Functional Inhibition of Monoamine Transporters and Nicotinic Acetylcholine Receptors
J Pharmacol Exp Ther 2002 302: 687-695
"The present study determined whether repeated administration of the antidepressant and selective norepinephrine (NE) uptake inhibitor reboxetine resulted in an adaptive modification of the function of the NE transporters (NETs), serotonin (5-HT) transporters, or dopamine (DA) transporters. Because antidepressants may be effective tobacco smoking cessation agents and because antidepressants have recently been shown to interact with nicotinic acetylcholine receptors (nAChRs), the interaction of reboxetine with nAChRs was also evaluated. Repeated administration of reboxetine (10 mg/kg i.p., twice daily for 14 days) did not alter the potency or selectivity of reboxetine inhibition of [3H]NE, [3H]DA, or [3H]5-HT uptake into striatal or hippocampal synaptosomes (IC50 values = 8.5 nM, 89 µM, and 6.9 µM, respectively). In a separate series of experiments, reboxetine did not inhibit (Ki > 1 µM) [3H]methyllycaconitine, [3H]cytisine, or [3H]epibatidine binding to rat whole brain membranes. However, at concentrations that did not exhibit intrinsic activity, reboxetine potently inhibited (IC50 value = 7.29 nM) nicotine-evoked [3H]NE overflow from superfused hippocampal slices via a noncompetitive mechanism. In the latter experiments, the involvement of NET was eliminated by inclusion of desipramine (10 µM) in the superfusion buffer. Reboxetine also inhibited (IC50 value = 650 nM) nicotine-evoked 86Rb+ efflux at reboxetine concentrations that did not exhibit intrinsic activity in this assay. Thus, in addition to inhibition of NET function, reboxetine inhibits nAChR function, suggesting that it may have potential as a smoking cessation agent." [Abstract]

Bauman, Andrea L., Apparsundaram, Subbu, Ramamoorthy, Sammanda, Wadzinski, Brian E., Vaughan, Roxanne A., Blakely, Randy D.
Cocaine and Antidepressant-Sensitive Biogenic Amine Transporters Exist in Regulated Complexes with Protein Phosphatase 2A
J. Neurosci. 2000 20: 7571-7578
"Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration." [Full Text]

Francis, Michael M., Vazquez, Raymond W., Papke, Roger L., Oswald, Robert E.
Subtype-Selective Inhibition of Neuronal Nicotinic Acetylcholine Receptors by Cocaine Is Determined by the alpha 4 and beta 4 Subunits
Mol Pharmacol 2000 58: 109-119 [Full Text]

Prasad PD, Leibach FH, Mahesh VB, Ganapathy V.
Human placenta as a target organ for cocaine action: interaction of cocaine with the placental serotonin transporter.
Placenta 1994 Apr;15(3):267-78
"When equilibrium interaction was allowed, cocaine inhibited the function of the transporter with a Ki of 0.09 microM. It is concluded that cocaine and its analog RTI-55 are potent inhibitors of the function of the serotonin transporter that is expressed in the normal human placenta and in cultured human placental choriocarcinoma cells. Since the reported values for cocaine concentration in the blood of cocaine users are several-fold higher than the inhibition constant for cocaine, the present study strongly suggests that the function of the placental serotonin transporter may be severely impaired by maternal use of cocaine during pregnancy. These findings may be relevant to fetal and placental complications of cocaine abuse during pregnancy." [Abstract]

Ramamoorthy JD, Ramamoorthy S, Leibach FH, Ganapathy V.
Human placental monoamine transporters as targets for amphetamines.
Am J Obstet Gynecol 1995 Dec;173(6):1782-7
"The results show that the norepinephrine transporter and, to a lesser extent, the serotonin transporter are cellular targets in the human placenta for the abusable drugs amphetamine and methamphetamine." [Abstract]

Haughey, Heather M., Fleckenstein, Annette E., Metzger, Ryan R., Hanson, Glen R.
The Effects of Methamphetamine on Serotonin Transporter Activity: Role of Dopamine and Hyperthermia
J Neurochem 2000 75: 1608-1617 [Abstract]

Brown, Pierre, Molliver, Mark E.
Dual Serotonin (5-HT) Projections to the Nucleus Accumbens Core and Shell: Relation of the 5-HT Transporter to Amphetamine-Induced Neurotoxicity
J. Neurosci. 2000 20: 1952-1963 [Full Text]

Gobbi, M., Moia, M., Pirona, L., Ceglia, I., Reyes-Parada, M., Scorza, C., Mennini, T.
p -Methylthioamphetamine and 1-(m -chlorophenyl)piperazine, two non-neurotoxic 5-HT releasers in vivo, differ from neurotoxic amphetamine derivatives in their mode of action at 5-HT nerve endings in vitro.
J Neurochem 2002 82: 1435-1443 [Abstract]

Quick MW.
Regulating the conducting states of a mammalian serotonin transporter.
Neuron. 2003 Oct 30; 40(3): 537-49.
"Serotonin transporters (SERTs), sites of psychostimulant action, display multiple conducting states in expression systems. These include a substrate-independent transient conductance, two separate substrate-independent leak conductances associated with Na(+) and H(+), and a substrate-dependent conductance of variable stoichiometry, which exceeds that predicted from electroneutral substrate transport. The present data show that the SNARE protein syntaxin 1A binds the N-terminal tail of SERT, and this interaction regulates two SERT-conducting states. First, substrate-induced currents are absent because Na(+) flux becomes strictly coupled to 5HT transport. Second, Na(+)-mediated leak currents are eliminated. These two SERT-conducting states are present endogenously in thalamocortical neurons, act to depolarize the membrane potential, and are modulated by molecules that disrupt SERT and syntaxin 1A interactions. These data show that protein interactions govern SERT activity and suggest that both cell excitability and psychostimulant-mediated effects will be dependent upon the state of association among SERT and its interacting partners." [Abstract]

Joel W. Schwartz, Randy D. Blakely, and Louis J. DeFelice
Binding and Transport in Norepinephrine Transporters. REAL-TIME, SPATIALLY RESOLVED ANALYSIS IN SINGLE CELLS USING A FLUORESCENT SUBSTRATE
J. Biol. Chem. 278: 9768-9777.
"Monoamine transporters, the molecular targets for drugs of abuse and antidepressants, clear norepinephrine, dopamine, or serotonin from the synaptic cleft. Neurotransmitters, amphetamines, and neurotoxins bind before being transported, whereas cocaine and antidepressants bind to block transport. Although binding is crucial to transport, few assays separate binding from transport, nor do they provide adequate temporal or spatial resolution to describe real-time kinetics or localize sites of active uptake. Here, we report a new method that distinguishes substrate binding from substrate transport using single-cell, space-resolved, real-time fluorescence microscopy. For these studies we use a fluorescent analogue of 1-methyl-4-phenylpyridinium, a neurotoxic metabolite and known substrate of monoamine transporters, to assess binding and transport with 50-ms, sub-micron resolution. We show that ASP+ (4-(4-(dimethylamino)styrl)-N-methylpyridinium) has micromolar potency for the human norepinephrine transporter, that ASP+ accumulation is Na+-, Cl-, cocaine-, and desipramine-sensitive and temperature-dependent, and that ASP+ competes with norepinephrine uptake. Using this method we demonstrate that norepinephrine transporters are efficient buffers for substrate, with binding rates exceeding transport rates by 100-fold. Furthermore, substrates bind deep within the transporter, isolated from both the bath and the lipid bilayer. Although transport per se depends on Na+ and Cl, binding is independent of Na+ and actually increases in low Cl. We further demonstrate that ASP+ interacts with transporters not only in transfected cells but in cultured neurons. ASP+ is also a substrate for dopamine and serotonin transporters and therefore represents a powerful new technique for studying the biophysical properties of monoamine transporters, an approach also amenable to high throughput assays for drug discovery." [Abstract]

Androutsellis-Theotokis, Andreas, Rudnick, Gary
Accessibility and Conformational Coupling in Serotonin Transporter Predicted Internal Domains
J. Neurosci. 2002 22: 8370-8378
"The intracellular topology of serotonin transporter (SERT) was examined using mutants containing single cysteine residues in the predicted cytoplasmic domain of the protein. Cysteine residues in each predicted cytoplasmic domain, including the NH2 and COOH termini and the five predicted internal loops, reacted with methanethiosulfonate (MTS) reagents only when the plasma membrane was permeabilized with digitonin or in membrane preparations but not in intact cells. The reaction was monitored by inactivation of high-affinity binding activity and by incorporation of biotin groups into the protein. Of the seven endogenous cysteine residues predicted to lie in the cytoplasmic domain, modification of only Cys-357 in the third internal loop (IL3) led to loss of activity. Cys-15 in the NH2 terminus and Cys-622 in the COOH terminus also reacted with MTS reagents. Modification of cysteine residues inserted at positions 137 in IL1, 277 in IL2, and 441 in IL4 also led to inactivation, and at positions 157 in IL1 and 532 in IL5, cysteine was modified without an effect on binding activity. These results are in agreement with the originally proposed topology for SERT and argue against an alternative topology proposed for the closely related GABA and glycine transporters. The reactivity of many of the cytoplasmic cysteine residues studied was influenced by ion and ligand binding, suggesting that the internal domains of SERT participate in conformational changes during neurotransmitter transport." [Abstract]

Fusun Kilic, and Gary Rudnick
Oligomerization of serotonin transporter and its functional consequences
PNAS 97: 3106-3111; published online before print as 10.1073/pnas.060408997
"Two forms of serotonin transporter (SERT) were prepared with different epitope tags. When co-expressed in HeLa cells, the form containing a FLAG tag (Res-FLAG) was associated with the form containing a c-myc tag (Sens-myc). Antibody against c-myc precipitated Res-FLAG from detergent extracts of cells expressing both forms, but not when Res-FLAG was expressed alone. The specificity of the interaction was demonstrated by the observation that anti-myc antibodies did not precipitate the unrelated vesicular stomatitis virus coat glycoprotein when it was co-expressed with Sens-myc. Sens-myc contained a reactive cysteine at position 172, which reacted with both (2-aminoethyl)methanethiosulfonate and N-biotinylaminoethyl methanethiosulfonate on the surface of intact cells. Sens-myc, but not Res-FLAG, was inactivated by these reagents. When co-expressed with Sens-myc, functionally active Res-FLAG was precipitated by immobilized streptavidin from digitonin-solubilized cells that had been treated with N-biotinylaminoethyl methanethiosulfonate. In cells co-expressing mixtures of Sens-myc and Res-FLAG, the amount of inactivation by (2-aminoethyl)methanethiosulfonate was less than expected if the two forms were independent. The results are consistent with a dimeric form of SERT with functional interactions between subunits, and with association of dimers into a higher order complex, possibly a tetramer." [Full Text]

Kilic, Fusun, Murphy, Dennis L., Rudnick, Gary
A Human Serotonin Transporter Mutation Causes Constitutive Activation of Transport Activity
Mol Pharmacol 2003 64: 440-446
"A rarely occurring variant of human serotonin transporter (hSERT) was tested for its functional consequences in HeLa and COS-7 cells. The variant, in which Ile-425 is converted to Val, was significantly different from wild type with respect to its catalytic properties. In both cell types, rates of serotonin (5-HT) transport were higher for the I425V variant. Both an increase in Vmax and a decrease in KM caused this increase in rate. The increase in Vmax was not accounted for by increases in transporter expression or in the distribution of transporter between the cell surface and intracellular pools. The decrease in KM was accompanied by a decrease in the KD for binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane. In both HeLa and COS-7 cells, the nitric oxide donor S-nitroso-N-acetylpenicillamine increased the activity of wild-type hSERT to that of the variant but did not change the activity of the I425V variant. This stimulation was prevented by the presence of oxyhemoglobin, which quenches nitric oxide, and by an inhibitor of guanylyl cyclase." [Abstract]

KJ Miller, and BJ Hoffman
Adenosine A3 receptors regulate serotonin transport via nitric oxide and cGMP
J. Biol. Chem. 269: 27351-27356, November 1994.
"Many antidepressants inhibit 5-hydroxytryptamine (5HT) transport resulting in increased 5HT levels in the synapse. However, physiological regulation of neurotransmitter uptake has not been demonstrated. We have examined the effect of receptor-activated second messengers on the 5HT transporter in rat basophilic leukemia cells (RBL 2H3). Here, we show that activation of an A3 adenosine receptor results in an increase of 5HT uptake in RBL cells, due to an increase in maximum velocity (Vmax). The A3 adenosine receptor-stimulated increase in transport is blocked by inhibitors of nitric oxide synthase and by a cGMP-dependent kinase inhibitor. In fact, compounds that generate nitric oxide (NO) and the cGMP analog 8-bromo-cGMP mimicked the effect of A3 receptor stimulation, suggesting that the elevation in transport occurs through the generation of the gaseous second messenger NO and a subsequent elevation in cGMP. Additionally, the 5HT transporter is differentially regulated by second messengers since direct activation of protein kinase C by phorbol esters decreases 5HT uptake by decreasing Vmax. Our results suggest that the changes in transport are due to a direct modification of the 5HT transporter, possibly by phosphorylation, which appears to alter the rate at which transport occurs. As the 5HT transporter in RBL cells is identical to that in neurons, our results suggest that analogous mechanisms may operate in the brain." [Abstract/Full Text]

I. Scott Ramsey, and Louis J. DeFelice
Serotonin Transporter Function and Pharmacology Are Sensitive to Expression Level. EVIDENCE FOR AN ENDOGENOUS REGULATORY FACTOR
J. Biol. Chem. 277: 14475-14482, April 2002.
"We express mammalian serotonin transporters (SERTs) in Xenopus oocytes by cRNA injection and measure 5-hydroxytryptamine (5-HT) transport and 5-HT-induced current at varying expression levels. Transport and current both increase sigmoidally with the amount of cRNA injected, but current requires ~5-fold more cRNA to elicit a half-maximal response. Western blots of SERT protein demonstrate that current, but not transport, correlates linearly with the amount of SERT on the plasma membrane. In oocytes co-injected with wild-type SERT and an inactive SERT mutant, transport is similar to SERT alone, but current is attenuated. The charge/transport ratio reports the differential sensitivity of transport and current to increasing SERT cRNA injection and mutant co-expression. Manipulations that alter the charge/transport ratio also perturb substrate and inhibitor recognition. 5-HT, d-amphetamine, cocaine, and paroxetine inhibit transport more potently at lower expression levels; however, 5-HT potency for induction of current is similar at high and low expression. Moreover, the apparent potency of cRNA for transport depends on 5-HT concentration. We postulate that SERT interacts allosterically with an endogenous factor of limited abundance to alter substrate and inhibitor potency and the balance of 5-HT transport and channel-like activity." [Abstract]

Ramamoorthy, Sammanda, Blakely, Randy D.
Phosphorylation and Sequestration of Serotonin Transporters Differentially Modulated by Psychostimulants
Science 1999 285: 763-766
"Many psychotropic drugs interfere with the reuptake of dopamine, norepinephrine, and serotonin. Transport capacity is regulated by kinase-linked pathways, particularly those involving protein kinase C (PKC), resulting in transporter phosphorylation and sequestration. Phosphorylation and sequestration of the serotonin transporter (SERT) were substantially impacted by ligand occupancy. Ligands that can permeate the transporter, such as serotonin or the amphetamines, prevented PKC-dependent SERT phosphorylation. Nontransported SERT antagonists such as cocaine and antidepressants were permissive for SERT phosphorylation but blocked serotonin effects. PKC-dependent SERT sequestration was also blocked by serotonin. These findings reveal activity-dependent modulation of neurotransmitter reuptake and identify previously unknown consequences of amphetamine, cocaine, and antidepressant action." [Full Text]

Whitworth, Terri L., Herndon, Laura C., Quick, Michael W.
Psychostimulants Differentially Regulate Serotonin Transporter Expression in Thalamocortical Neurons
J. Neurosci. 2002 22: 192-
"5-HT transporters (SERTs) are transiently expressed in thalamocortical neurons during development, permitting these glutamatergic neurons to co-release 5-HT as a "borrowed" transmitter. The high level of SERT expression in these neurons is likely important in the serotonergic modulation of neocortical circuits and provides a system for examining endogenous SERT regulation. We tested the hypothesis that developmental expression of SERT in thalamocortical neurons is regulated by psychostimulants that are agonists and antagonists of SERT. Cultured thalamocortical neurons from embryonic day 18 rats were examined for SERT expression until P15. In untreated cultures, SERT protein levels peaked at postnatal day 3 (P3) and were absent by P10. Chronic treatment with SERT substrates (5-HT, 3,4-methylenedioxymethamphetamine) increased both peak SERT protein levels (fourfold) and the time course of SERT expression. SERT substrates also shifted the relative functional expression of SERT by redistributing intracellular SERT protein to the plasma membrane. The subcellular redistribution was prevented by PKC activators. SERT antagonists (e.g., fluoxetine, cocaine) reduced total SERT expression levels and the time course of SERT expression. These data (1) show that endogenous SERT is differentially regulated by 5-HT and psychostimulants, (2) indicate that SERT modulation occurs via changes in both total SERT protein levels and subcellular redistribution of the transporter, and (3) suggest that some of the actions of drugs of abuse in neocortical development may be attributable to alterations in SERT expression and concomitant changes in 5-HT signaling." [Full Text]

G Rudnick, and SC Wall
The Molecular Mechanism of "Ecstasy" [3,4-Methylenedioxy-Methamphetamine (MDMA)]: Serotonin Transporters are Targets for MDMA-Induced Serotonin Release
PNAS 89: 1817-1821, 1992.
"MDMA ("ecstasy") has been widely reported as a drug of abuse and as a neurotoxin. This report describes the mechanism of MDMA action at serotonin transporters from plasma membranes and secretory vesicles. MDMA stimulates serotonin efflux from both types of membrane vesicle. In plasma membrane vesicles isolated from human platelets, MDMA inhibits serotonin transport and [3H]imipramine binding by direct interaction with the Na+-dependent serotonin transporter. MDMA stimulates radiolabel efflux from plasma membrane vesicles preloaded with [3H]serotonin in a stereospecific, Na+-dependent, and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, which contain the vesicular biogenic amine transporter, MDMA inhibits ATP-dependent [3H]serotonin accumulation and stimulates efflux of previously accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due to dissipation of the transmembrane pH difference generated by ATP hydrolysis and to direct interaction with the vesicular amine transporter." [Abstract/Full Text]

Fletcher PJ, Korth KM, Robinson SR, Baker GB.
Multiple 5-HT receptors are involved in the effects of acute MDMA treatment: studies on locomotor activity and responding for conditioned reinforcement.
Psychopharmacology (Berl) 2002 Jul;162(3):282-91
"AbstractRATIONALE. Responding for conditioned reinforcement is increased by the dopamine releasing agent amphetamine, but reduced by drugs that enhance serotonin (5-HT) function. The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) releases both monoamines.OBJECTIVES. The primary purpose of this study was to examine the effects of MDMA on responding for conditioned reinforcement as well as on locomotor activity. The roles of several 5-HT receptor sub-types in mediating these behavioural effects of MDMA were also examined.METHODS. Locomotion was measured in photocell activity monitors. For conditioned reinforcement experiments thirsty rats learned to associate a conditioned stimulus (CS) with water in operant chambers. Subsequently, two response levers were available; responding on one lever delivered the CS, while responding on the second lever had no consequences. Drug effects on this operant response were measured.RESULTS. MDMA dose-dependently increased locomotion but reduced responding for conditioned reinforcement. This latter effect differs from that induced by amphetamine, which potentiates conditioned reinforcement responding. The stimulant effect of MDMA was attenuated by GR127935 and ketanserin, indicating facilitatory roles of 5-HT(1B) and 5-HT(2A) receptors in mediating this effect. The 5-HT(2C) antagonist SB242084 enhanced the stimulant effect of MDMA. Only SB242084 attenuated the suppressant effect of MDMA on responding for conditioned reinforcement.CONCLUSIONS. The results show that 5-HT(2A) and 5-HT(1B/1D) receptors play a facilitatory role in mediating the stimulant effect of MDMA, whereas 5-HT(2C) receptors are inhibitory. Activation of 5-HT(2C) receptors also contributes to the deficit in operant responding. Multiple 5-HT receptor sub-types appear to contribute to the behavioural effects of MDMA." [Abstract]

Bengel, Dietmar, Murphy, Dennis L., Andrews, Anne M., Wichems, Christine H., Feltner, Douglas, Heils, Armin, Mossner, Rainald, Westphal, Heiner, Lesch, Klaus-Peter
Altered Brain Serotonin Homeostasis and Locomotor Insensitivity to 3,4-Methylenedioxymethamphetamine ("Ecstasy") in Serotonin Transporter-Deficient Mice
Mol Pharmacol 1998 53: 649-655 [Full Text]

Byrne SE, Rothschild AJ.
Loss of antidepressant efficacy during maintenance therapy: possible mechanisms and treatments.
J Clin Psychiatry 1998 Jun;59(6):279-88
"BACKGROUND: Many patients with unipolar depression experience a return of depressive symptoms while taking a constant maintenance dose of an antidepressant. METHOD: All cited studies were found using computerized literature searches of the MEDLINE database since 1966. RESULTS: The return of depressive symptoms during maintenance antidepressant treatment has occurred in 9% to 57% of patients in published trials. Possible explanations include loss of placebo effect, pharmacologic tolerance, increase in disease severity, change in disease pathogenesis, the accumulation of a detrimental metabolite, unrecognized rapid cycling, and prophylactic inefficacy. CONCLUSION: Although several strategies have been proposed to overcome the loss of antidepressant efficacy, double-blind controlled studies are needed to ascertain the optimal strategy for this perplexing clinical problem." [Abstract]

Fryer, John D, Lukas, Ronald J
Antidepressants Noncompetitively Inhibit Nicotinic Acetylcholine Receptor Function
J Neurochem 1999 72: 1117-1124
"Nicotinic acetylcholine receptors (nAChRs) are diverse members of the neurotransmitter-gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes for the first time the acute functional effects of sertraline (Zoloft), paroxetine (Paxil), nefazodone (Serzone), and venlafaxine (Effexor) on two human and one chick nAChR subtype. This study also confirms previous findings of nAChR functional block by fluoxetine (Prozac). Function of human muscle-type nAChR (alpha1/beta gammadelta) in TE671/RD cells, human autonomic nAChR (alpha3/beta4alpha5 +/- beta2) in SH-SY5Y neuroblastoma cells, or chick V274T mutant alpha7-nAChR heterologously expressed in native nAChR-null SH-EP1 epithelial cells was measured using 86Rb+ efflux assays. Functional blockade of human muscle-type and autonomic nAChRs is produced by each of the drugs in the low to intermediate micromolar range, and functional blockade of chick V274T-alpha7-nAChR is produced in the intermediate to high micromolar range. Functional blockade is insurmountable by increasing agonist concentrations at each nAChR subtype tested for each of these drugs, suggesting noncompetitive inhibition of nAChR function. These studies open the possibilities that nAChR subtypes in the brain could be targets for therapeutic antidepressants and could play roles in clinical depression." [Abstract]

Hennings, Esteban C. P., Kiss, Janos P., Oliveira, Karine De, Toth, Peter T., Vizi, E. Sylvester
Nicotinic Acetylcholine Receptor Antagonistic Activity of Monoamine Uptake Blockers in Rat Hippocampal Slices
J Neurochem 1999 73: 1043-1050
"The aim of our study was to investigate the effect of different monoamine uptake blockers on the nicotine-evoked release of [3H]noradrenaline ([3H]NA) from rat hippocampal slices. We found that desipramine (DMI), nisoxetine, cocaine, citalopram, and nomifensine inhibit the nicotine-evoked release of [3H]NA with an IC50 of 0.36, 0.59, 0.81, 0.93, and 1.84 µM, respectively. These IC50 values showed no correlation with the inhibitory effect (Ki) of monoamine uptake blockers on the neuronal NA transporter (r = 0.17, slope = 0.02), indicating that the NA uptake system is not involved in the process. In whole-cell patch clamp experiments neither drug blocked Na+ currents at 1 µM in sympathetic neurons from rat superior cervical ganglia, and only DMI produced a pronounced inhibition (52% decrease) at 10 µM. Comparison of the effect of DMI and tetrodotoxin (TTX) on the electrical stimulation- and nicotine-evoked release of [3H]NA showed that DMI, in contrast to TTX, inhibits only the nicotine-induced response, indicating that the target of DMI is not the Na+ channel. Our data suggest that monoamine uptake blockers with different chemical structure and selectivity are able to inhibit the nicotinic acetylcholine receptors in the CNS. Because these compounds are widely used in the therapy of depressed patients, our findings may have great importance in the evaluation of their clinical effects." [Abstract]

XM Guan and WJ McBride
Fluoxetine increases the extracellular levels of serotonin in the nucleus accumbens.
Brain Res Bull, Jul 1988; 21(1): 43-6.
"The effects of an IP injection of the monoamine uptake inhibitor fluoxetine on the extracellular concentration of serotonin (5-HT), dopamine (DA), 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens of awake and freely moving rats were examined using a push-pull perfusion technique. Baseline values of 5-HT, 5-HIAA, DA, DOPAC and HVA in the perfusates were approximately 0.07, 13, 0.8, 49 and 12 pmol/hr, respectively. The IP administration of 5 and 10 mg/kg fluoxetine dose-dependently elevated the amounts of 5-HT 3- and 13-fold, respectively, in the push-pull perfusate, with the maximum reached within one hour after drug administration. Moreover, 10 mg/kg fluoxetine also significantly decreased the levels of 5-HIAA in the perfusate as much as 50% within 2-3 hours. On the other hand, no significant effect of 5 or 10 mg/kg fluoxetine was observed on the contents of DA, DOPAC and HVA in the push-pull perfusates. The data indicate that fluoxetine, in accord with its role as a 5-HT uptake inhibitor, increases the physiologically active pool of 5-HT in the nucleus accumbens under in vivo conditions." [Abstract]

Cabrera-Vera, Theresa M., Garcia, Francisca, Pinto, Wilfred, Battaglia, George
Effect of Prenatal Fluoxetine (Prozac) Exposure on Brain Serotonin Neurons in Prepubescent and Adult Male Rat Offspring
J Pharmacol Exp Ther 1997 280: 138-145
"The present study examines the consequences of prenatal fluoxetine exposure on brain serotonin [5-hydroxytryptamine (5-HT)] neurons in male offspring. Pregnant rats were administered either saline or fluoxetine (10 mg/kg s.c.) daily from gestational day 13 through gestational day 20. The biochemical status of brain 5-HT neurons was assessed in prepubescent and adult offspring by measuring 1) the 5-HT and 5-hydroxyindoleacetic acid content, 2) the density of [3H]paroxetine-labeled 5-HT uptake sites and 3) the ability of the 5-HT-releasing drug p-chloroamphetamine to reduce 5-HT content. Biochemical parameters were assessed in the frontal cortex, hypothalamus, hippocampus, striatum and midbrain. Comparative effects on dopamine and norepinephrine content in selected regions were also determined. Prenatal exposure to fluoxetine significantly reduced (28%) 5-HT content in the frontal cortex of prepubescent but not adult male offspring. In contrast, in adult progeny prenatal fluoxetine exposure produced a significant decrease only in midbrain 5-HT content (28%). In addition, p-chloroamphetamine markedly reduced 5-HT content in all brain regions examined, but the ability of p-chloroamphetamine to reduce 5-HT content was significantly attenuated only in the midbrain of adult progeny prenatally exposed to fluoxetine. No significant differences were observed between control and fluoxetine-exposed progeny with respect to brain 5-hydroxyindoleacetic acid content, the 5-hydroxyindoleacetic acid/5-HT ratio or the density of 5-HT uptake sites, regardless of the brain region examined or the age of the offspring. These data provide additional evidence that prenatal exposure to fluoxetine can produce limited, rather than global, changes in brain 5-HT neurons in male rat offspring and that the effects observed are region-specific and age-dependent. The potential functional consequences and clinical implications of these alterations in brain 5-HT systems remain to be elucidated." [Full Text]

Thomas, Dierk, Gut, Bernd, Wendt-Nordahl, Gunnar, Kiehn, Johann
The Antidepressant Drug Fluoxetine Is an Inhibitor of Human Ether-A-Go-Go-Related Gene (HERG) Potassium Channels
J Pharmacol Exp Ther 2002 300: 543-548
"Fluoxetine is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Although this group of antidepressant drugs is generally believed to cause fewer proarrhythmic side effects compared with tricyclic antidepressants, serious concerns have been raised by case reports of tachycardia and syncopes associated with fluoxetine treatment. To determine the electrophysiological basis for the arrhythmogenic potential of fluoxetine, we investigated the effects of this drug on cloned human ether-a-go-go-related gene (HERG) potassium channels heterologously expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. We found that fluoxetine blocked HERG channels with an IC50 value of 3.1 µM. Inhibition occurred fast to open channels with very slow unbinding kinetics. Analysis of the voltage dependence of block revealed loss of inhibition at membrane potentials greater than 40 mV, indicating that channel inactivation prevented block by fluoxetine. No pronounced changes in electrophysiological parameters such as voltage dependence of activation or inactivation, or inactivation time constant could be observed, and block was not frequency-dependent. This is the first study demonstrating that HERG potassium channels are blocked by the selective serotonin reuptake inhibitor fluoxetine. We conclude that HERG current inhibition might be an explanation for the arrhythmogenic side effects of this drug." [Abstract]

J. García-Colunga, J. N. Awad, and R. Miledi
Blockage of muscle and neuronal nicotinic acetylcholine receptors by fluoxetine (Prozac)
PNAS 94: 2041-2044, March 1997.
"Fluoxetine (Prozac), a widely used antidepressant, is said to exert its medicinal effects almost exclusively by blocking the serotonin uptake systems. The present study shows that both muscle and neuronal nicotinic acetylcholine receptors are blocked, in a noncompetitive and voltage-dependent way, by fluoxetine, which also increases the rate of desensitization of the nicotinic receptors. Because these receptors are very widely distributed in the both central and peripheral nervous systems, the blocking action of fluoxetine on nicotinic receptors may play an important role in its antidepressant and other therapeutical effects. Our findings will help to understand the mode of action of fluoxetine, and they may also help to develop more specific medicinal drugs." [Full Text]

Blakely, Randy D.
Physiological Genomics of Antidepressant Targets: Keeping the Periphery in Mind
J. Neurosci. 2001 21: 8319-8323
"The plasma membrane transporters that clear extracellular serotonin (5-HT) and norepinephrine (NE), serotonin transporters (SERTs) and NE transporters (NETs), have received considerable attention over the past four decades because of their roles in amine neurotransmitter inactivation. In addition, they interact with many centrally active drugs, including multiple classes of antidepressants such as the serotonin-selective reuptake inhibitors, typified by fluoxetine (Prozac), and the more recently developed norepinephrine-selective transporter antagonists, such as reboxetine. The therapeutic utility of these agents supports biogenic amine theories of affective disorders and raises the question as to whether SERT and NET exhibit a functional genetic variation that could influence risk for behavioral disorders. Although evidence exists that a promoter polymorphism in SERT may influence behavioral states, this contention is not without complexity and its mechanism of action remains poorly understood. The identification of coding variants of NETs and SERTs would offer important opportunities to connect genotype to phenotype. However, given the limited frequency of transporter coding variations evident to date in general population surveys or in psychiatric genetic studies, the identification of informative functional variants of transporters will likely require refined phenotypes. In this regard, NET and SERT play critical roles in cardiovascular and gastrointestinal physiology, respectively. This perspective reviews recent human and mouse studies that suggest how peripheral autonomic phenotypes, linked to genetic disruption of NET and SERT function, can aid in the phenotypic segregation needed for advanced theories of biogenic amine dysfunction and pharmacogenetics." [Abstract]