Serotonin (5-hydroxytryptamine, 5-HT) Transporter Research Articles

Overfeeding during development induces temporally-dependent changes in areas controlling food intake in the brains of male Wistar rats

AimsWe sought to evaluate the effects of overfeeding during lactation on the feeding behavior and expression of specific regulatory genes in brain areas associated with food intake in 22- and 60-day old male rats. MethodsWe evaluated body weight, food intake of standard and palatable diet, and mRNA expression of dopamine receptor D1 (DDR1), dopamine receptor (DDR2), melanocortin 4 receptor (MC4R), the μ-opioid receptor (MOR), neuropeptide Y (NPY), agouti-related protein (AGRP), proopiomelanocortin (POMC), cocaine-and amphetamine-regulated transcript (CART), serotonin (5-hydroxytryptamine; 5-HT) transporter (SERT), 5-hydroxytryptamine receptor 1B (5-HT1B), 5-hydroxytryptamine receptor 2C receptor (5-HT2C), Clock (CLOK), cryptochrome protein 1 (Cry1) and period circadian protein homolog 2 (Per2) in the striatum, hypothalamus and brainstem of male rats at post-natal days (PND) 22 and 60. Key findingsOverfeeding resulted in significantly increased body weight through PND60, and a 2-fold increase in palatable food intake at PND22, but not at PND60. We observed significant increases in DDR1, DDR2, and MC4R gene expression in the striatum and brainstem and POMC/CART in the hypothalamus of the OF group at PND22 that were reversed by PND60. Hypothalamic levels of 5-HT1B, 5-HT2C and NPY/AGRP on the other hand were decreased at PND22 and increased at PND60 in OF animals. Clock genes were unaffected by OF at PND22, but were significantly elevated at PND60. SignificanceOverfeeding during early development of the rat brain results in obesity and altered feeding behavior in early adulthood. The altered behavior might be the consequence of the changes in food intake and reward gene expression.

Sexually dimorphic responses of rats to fluoxetine in the forced swimming test are unrelated to the function of the serotonin transporter in the brain

Due to the prevalence of depression in women, female rats may be a better models for antidepressant research than males. In male rats, fluoxetine inhibited the serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) which is reducing the immobility time in the repeated forced swimming test (rFST). The performance of female rats in this test is unknown. In this study, responses of male and female rats in the rFST under chronic treatment with fluoxetine and the function of SERT in their brains were examined. Wistar rats received oral fluoxetine (females: 0, 1, 2.5, or 5mgkg-1 day-1 ; males: 0 or 2.5mgkg-1 day-1 ; in sucrose 10%, 1.5ml/rat) 1 hr before the test daily for 12days over the course of the rFST. rFST consisted of a 15 min pretest followed by 5 min sessions of swimming at 1 (test), 7 (retest 1), and 14 (retest 2) days later. SERT functioning was assessed by ex vivo assays of the frontal cortex and hippocampus of rats. Fluoxetine reduced immobility time of males in the rFST while it failed to do so in females. In vitro treatment with fluoxetine inhibited the uptake of 5-HT of both sexes similarly, while in vivo chronic administration of fluoxetine failed to do so. In summary, rats responded to the chronic treatment with fluoxetine in a sexually dimorphic fashion during the rFST despite the functioning of SERT in their brains remaining equally unchanged. Hence, our data suggest that sexually dimorphic responses to fluoxetine in rFST may be unrelated to the function of SERT in rat brains.

Identification of novel serotonin reuptake inhibitors targeting central and allosteric binding sites: A virtual screening and molecular dynamics simulations study

The serotonin (5-hydroxytryptamine, 5HT) transporter (SERT) is a member of neurotransmitter sodium symporter (NSS) family, which maintains neurotransmitter by reuptaking 5HT into synapses. Decrease in serotonin concentrations in synaptic clefts have been reported to cause psychological and neurological disorders. Therefore, inhibition of SERT is a potent strategy for the treatment of related diseases such as depression. In this study, approximately 260,000 small molecules from an available chemical database have been virtually screened both at central and allosteric binding sites of SERT to identify potent novel candidate SERT inhibitors. A set of docking algorithms were used to predict binding modes and energies of compounds. Screening analyses led three top-ranked hit compounds (160234, Otava ID: 7118020138; 159166, Otava ID: 7117171303; and 69419, Otava ID: 118671819) for central binding site (S1) and one compound (93507, Otava ID: 6248262) for allosteric binding site (S2). These promising compounds are then subjected to long multiple molecular dynamics (MD) simulations to elucidate their structural and dynamical profiles at the binding cavities of SERT. Higher predicted binding affinities of identified compounds were also confirmed with binding free energy calculations (MM/GBSA) in comparison with the reference central and allosteric binding site inhibitors, paroxetine (8PR) and escitalopram (68P), respectively. To the best of our knowledge, the present work is the first structure-based high throughput virtual screening study reported using recently revealed crystal structure of SERT for screening inhibitors from chemical databases on S1 and S2 binding sites. Small molecule library screening study yielded candidate compounds both at central and allosteric binding site of SERT, and further experimentation may pave the way for developing novel strong inhibitors.

Methamphetamine self-administration attenuates hippocampal serotonergic deficits: role of brain-derived neurotrophic factor

Preclinical studies suggest that prior treatment with escalating doses of methamphetamine (METH) attenuates the persistent deficits in hippocampal serotonin (5-hydroxytryptamine; 5HT) transporter (SERT) function resulting from a subsequent 'binge' METH exposure. Previous work also demonstrates that brain-derived neurotrophic factor (BDNF) exposure increases SERT function. The current study investigated changes in hippocampal BDNF protein and SERT function in rats exposed to saline or METH self-administration prior to a binge exposure to METH or saline. Results revealed that METH self-administration increased hippocampal mature BDNF (mBDNF) immunoreactivity compared to saline-treated rats as assessed 24h after the start of the last session. Further, mBDNF immunoreactivity was increased and SERT function was not altered in rats that self-administered METH prior to the binge METH exposure as assessed 24h after the binge exposure. These results suggest that prior exposure to contingent METH increases hippocampal mBDNF, and this may contribute to attenuated deficits in SERT function.

Identification of Novel Serotonin Transporter Compounds by Virtual Screening

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) plays an essential role in the termination of serotonergic neurotransmission by removing 5-HT from the synaptic cleft into the presynaptic neuron. It is also of pharmacological importance being targeted by antidepressants and psychostimulant drugs. Here, five commercial databases containing approximately 3.24 million drug-like compounds have been screened using a combination of two-dimensional (2D) fingerprint-based and three-dimensional (3D) pharmacophore-based screening and flexible docking into multiple conformations of the binding pocket detected in an outward-open SERT homology model. Following virtual screening (VS), selected compounds were evaluated using in vitro screening and full binding assays and an in silico hit-to-lead (H2L) screening was performed to obtain analogues of the identified compounds. Using this multistep VS/H2L approach, 74 active compounds, 46 of which had Ki values of ≤1000 nM, belonging to 16 structural classes, have been identified, and multiple compounds share no structural resemblance with known SERT binders.

Quantitative trait loci mapping and gene network analysis implicate protocadherin‐15 as a determinant of brain serotonin transporter expression

Presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporters (SERT) regulate 5-HT signaling via antidepressant-sensitive clearance of released neurotransmitter. Polymorphisms in the human SERT gene (SLC6A4) have been linked to risk for multiple neuropsychiatric disorders, including depression, obsessive-compulsive disorder and autism. Using BXD recombinant inbred mice, a genetic reference population that can support the discovery of novel determinants of complex traits, merging collective trait assessments with bioinformatics approaches, we examine phenotypic and molecular networks associated with SERT gene and protein expression. Correlational analyses revealed a network of genes that significantly associated with SERT mRNA levels. We quantified SERT protein expression levels and identified region- and gender-specific quantitative trait loci (QTLs), one of which associated with male midbrain SERT protein expression, centered on the protocadherin-15 gene (Pcdh15), overlapped with a QTL for midbrain 5-HT levels. Pcdh15 was also the only QTL-associated gene whose midbrain mRNA expression significantly associated with both SERT protein and 5-HT traits, suggesting an unrecognized role of the cell adhesion protein in the development or function of 5-HT neurons. To test this hypothesis, we assessed SERT protein and 5-HT traits in the Pcdh15 functional null line (Pcdh15(av-) (3J) ), studies that revealed a strong, negative influence of Pcdh15 on these phenotypes. Together, our findings illustrate the power of multidimensional profiling of recombinant inbred lines in the analysis of molecular networks that support synaptic signaling, and that, as in the case of Pcdh15, can reveal novel relationships that may underlie risk for mental illness.

Platelet SERT as a Peripheral Biomarker of Serotonergic Neurotransmission in the Central Nervous System

Alterations in serotonergic activity have been observed in many pathological conditions, including neuro psychiatric diseases, irritable bowel syndrome, and hypertension. The serotonin (5-hydroxytryptamine; 5-HT) transporter(SERT) in the brain clears 5-HT from extracellular spaces, modulating the strength and duration of serotonergic signaling.Outside the central nervous system, it is also present in platelets, where it takes up 5-HT from plasma, keeping levels very low (i.e., ~1 nM). Importantly, it is generally accepted that SERT protein expressed in platelets is identical to the one found in neurons, displaying similar structural and functional properties in both tissues. At the present time, it is technically difficult to measure SERT binding and function in vivo since imaging methods are limited by a number of factors,especially the cost and the selectivity of the available radioligands. One of the most frequently used molecular imaging techniques to study SERT is positron emission tomography (PET). Although an impressive number of PET radio ligands have been synthesized and validated, there is still a lack of suitable ligands for a large part of the 5-HT system. Interest in determining both the molecular characteristics and the regulation of SERT has been enormous over the last decade, but the difficulty in obtaining human tissues and the ethical limitations in human experiments have turned researchers to look for alternative models. This review summarizes recent clinical and preclinical data relevant to the use of blood platelets asa peripheral marker for the central 5-HT system, and outlines future directions in this field.

A Fluorescence Displacement Assay for Antidepressant Drug Discovery Based on Ligand-Conjugated Quantum Dots

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) protein plays a central role in terminating 5-HT neurotransmission and is the most important therapeutic target for the treatment of major depression and anxiety disorders. We report an innovative, versatile, and target-selective quantum dot (QD) labeling approach for SERT in single Xenopus oocytes that can be adopted as a drug-screening platform. Our labeling approach employs a custom-made, QD-tagged indoleamine derivative ligand, IDT318, that is structurally similar to 5-HT and accesses the primary binding site with enhanced human SERT selectivity. Incubating QD-labeled oocytes with paroxetine (Paxil), a high-affinity SERT-specific inhibitor, showed a concentration- and time-dependent decrease in QD fluorescence, demonstrating the utility of our approach for the identification of SERT modulators. Furthermore, with the development of ligands aimed at other pharmacologically relevant targets, our approach may potentially form the basis for a multitarget drug discovery platform.

Monoamines suppressed phosphorylation of SNAP-25 in mouse brain

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) clear the amine from extracellular spaces in the CNS and periphery as a mechanism for transmitter inactivation and recycling. Although it is known that SERTs are preferentially expressed on basolateral domains in transfected epithelial cells, details of the transporter's membrane localizationin vivoare lacking. 5HT and 5HT receptors have been identified in the rodent adrenal gland. Using SERT antagonist autoradiography, we establish the presence of antidepressant-sensitive transport sites in the rat adrenal medulla. Immunofluorescence experiments using antibodies specific for the SERT COOH and NH2termini, for 5HT, or for catecholamine biosynthetic enzymes suggest that SERT mediates intracellular 5HT accumulation by epinephrine-secreting chromaffin cells. Using confocal microscopy, we establish that SERT expression is nonuniformly distributed along the plasma membrane of chromaffin cells. Notably, SERT immunoreactivity is largely absent from plasma membranes bordering smooth muscle that surrounds vascular sinusoids. Rather, SERT is highly expressed in membranes adjoining other chromaffin cells, consistent with a role for 5HT and SERT in autocrine or paracrine control of chromaffin cell physiology. SNAP-25, a t-SNARE protein implicated in neurotransmitter release, was found to colocalize with SERT. In contrast, Na,K ATPase and NCAM are uniformly distributed along the entire perimeter of chromaffin cell membranes. These findings underscore a role for 5HT and SERT in adrenal physiology, reveal unrecognized polarity of chromaffin cell plasma membranes, and warrant a consideration of common targeting mechanisms localizing amine transporters near release sites.

CGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

BackgroundThe Na+/Cl--dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear.ResultsIn this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIα specifically associates with hSERT.ConclusionOur findings provide evidence of a physical and compartmentalized association between SERT and PKGIα that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIα supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.

Going with the Flow: Trafficking‐Dependent and ‐Independent Regulation of Serotonin Transport

Antidepressant-, cocaine- and 3,4-methylenedioxymethamphetamine-sensitive serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) are expressed on presynaptic membranes of 5-HT-secreting neurons to provide efficient uptake of the biogenic amine after release. SERTs also support 5-HT transport across platelet, placental, gastrointestinal and pulmonary membranes and thus play a critical role in central nervous system and peripheral nervous system 5-HT signaling. SERTs are subject to multiple levels of posttranslational regulation that can rapidly alter 5-HT uptake and clearance rates. Specific cell surface receptors are now known to regulate SERT trafficking and/or catalytic function, with pathways activating protein kinase C, protein kinase G and p38 mitogen-activated protein kinase receiving the greatest attention. Remarkably, disease-associated mutations in SERT not only impact basal SERT activity but also selectively impact one or more SERT regulatory pathway(s). In this review, we describe both trafficking-dependent and trafficking-independent modes of SERT regulation and also the suspected roles played in regulation by SERT-associated proteins. Elucidation of the SERT 'regulome' provides important depth to our understanding of the likely origins of 5-HT-associated disorders and may help orient research to develop novel therapeutics.

P38 MAPK Activation Elevates Serotonin Transport Activity via a Trafficking-independent, Protein Phosphatase 2A-dependent Process

Presynaptic, plasma membrane serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) clear 5-HT following vesicular release and are regulated through trafficking-dependent pathways. Recently, we provided evidence for a trafficking-independent mode of SERT regulation downstream of adenosine receptor (AR) activation that is sensitive to p38 MAPK inhibitors. Here, we probe this pathway in greater detail, demonstrating elevation of 5-HT transport by multiple p38 MAPK activators (anisomycin, H(2)O(2), and UV radiation), in parallel with p38 MAPK phosphorylation, as well as suppression of anisomycin stimulation by p38 MAPK siRNA treatments. Studies with transporter-transfected Chinese hamster ovary cells reveal that SERT stimulation is shared with the human norepinephrine transporter but not the human dopamine transporter. Saturation kinetic analyses of anisomycin-SERT activity reveal a selective reduction in 5-HT K(m) supported by a commensurate increase in 5-HT potency (K(i)) for displacing surface antagonist binding. Anisomycin treatments that stimulate SERT activity do not elevate surface SERT surface density whereas stimulation is lost with preexposure of cells to the surface-SERT inactivating reagent, 2-(trimethylammonium)ethyl methane thiosulfonate. Guanylyl cyclase (1H-(1,2,4)-oxadiazolo[4,3-a]-quinoxalin-1-one) and protein kinase G inhibitors (H8, DT-2) block AR stimulation of SERT yet fail to antagonize SERT stimulation by anisomycin. We thus place p38 MAPK activation downstream of protein kinase G in a SERT-catalytic regulatory pathway, distinct from events controlling SERT surface density. In contrast, the activity of protein phosphatase 2A inhibitors (fostriecin and calyculin A) to attenuate anisomycin stimulation of 5-HT transport suggests that protein phosphatase 2A is a critical component of the pathway responsible for p38 MAPK up-regulation of SERT catalytic activity.

Adenosine receptor, protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation of serotonin transporters involves both transporter trafficking and activation.

Serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) are critical determinants of synaptic 5-HT inactivation and the targets for multiple drugs used to treat psychiatric disorders. In support of prior studies, we found that short-term (5-30 min) application of the adenosine receptor (AR) agonist 5'-N-ethylcarboxamidoadenosine (NECA) induces an increase in 5-HT uptake Vmax in rat basophilic leukemia 2H3 cells that is enhanced by pretreatment with the cGMP phosphodiesterase inhibitor sildenafil. NECA stimulation is blocked by the A3 AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(+/-)dihydropyridine-3,5-dicarboxylate (MRS1191), by the phospholipase C inhibitor 1-(6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl)-1H-pyrrole-2,5-dione (U73122), by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, and by the guanyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Hydroxylamine, a nitric-oxide donor, and 8-bromo-cGMP, a membrane-permeant analog of cGMP, mimic the effects of NECA on 5-HT uptake, whereas the protein kinase G (PKG) inhibitor N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) blocks NECA, hydroxylamine, and 8-bromo-cGMP effects. NECA stimulation activates p38 mitogen-activated protein kinase (MAPK), whereas p38 MAPK inhibitors block NECA stimulation of SERT activity, as does the protein phosphatase 2A (PP2A) inhibitor calyculin A. 5-HT-displaceable [125I]3beta-(4-iodophenyl)-tropane-2beta-carboxylic acid methylester tartrate (RTI-55) whole-cell binding is increased by NECA or sildenafil, and both surface binding and cell surface SERT protein are elevated after NECA or sildenafil stimulation of AR/SERT-cotransfected Chinese hamster ovary cells. Whereas p38 MAPK inhibition blocks NECA stimulation of 5-HT activity, it fails to blunt stimulation of SERT surface density. Moreover, inactivation of existing surface SERTs fails to eliminate NECA stimulation of SERT. Together, these results reveal two PKG-dependent pathways supporting rapid SERT regulation by A3 ARs, one leading to enhanced SERT surface trafficking, and a separate, p38 MAPK-dependent process augmenting SERT intrinsic activity.

Flux Coupling in the Human Serotonin Transporter

The serotonin (5-hydroxytryptamine; 5HT) transporter (SERT) catalyzes the movement of 5HT across cellular membranes. In the brain, SERT clears 5HT from extracellular spaces, modulating the strength and duration of serotonergic signaling. SERT is also an important pharmacological target for antidepressants and drugs of abuse. We have studied the flux of radio-labeled 5HT through the transporter stably expressed in HEK-293 cells. Analysis of the time course of net transport, the equilibrium 5HT gradient sustained, and the ratio of the unidirectional influx to efflux of 5HT indicate that mechanistically, human SERT functions as a 5HT channel rather than a classical carrier. This is especially apparent at relatively high [5HT] out (≥10 μM), but is not restricted to this regime of external 5HT.

Interactions of tryptamine derivatives with serotonin transporter species variants implicate transmembrane domain I in substrate recognition.

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is responsible for the inactivation of synaptic 5-HT and is also a target for multiple psychostimulants. Despite the critical role of SERT in 5-HT inactivation and psychostimulant response, many aspects of the transporter's recognition of ligands are poorly defined. We took advantage of sequence divergence of SERT species variants to identify structural determinants of substrate recognition. Tryptamine derivatives with substitutions at the 4 and 7 positions on the phenyl ring, the indole nitrogen, and the beta position show up to 40-fold potency differences for inhibiting [(3)H]5-HT transport in cells transfected with either human or Drosophila melanogaster SERT cDNAs. Species selectivities of these derivatives were largely recapitulated in antagonist binding. Human/D. melanogaster SERT chimera studies implicated the first two SERT transmembrane domains (TMDs) in the potency of the indole nitrogen-substituted compounds N-isopropyltryptamine (NIT), 5-methoxy-N-isopropyltryptamine (5-MNIT), and the 7-substituted compound 7-benzyloxytryptamine (7BT). Potency differences of analogs with substitutions at the 4 and beta positions are influenced by sequences distal to this region. Within TMD I-II, species-scanning mutagenesis implicated a single residue (Y95 in human SERT, F90 in D. melanogaster SERT) in the recognition of NIT, 5-MNIT, and 7BT. Remarkably, this is the same site we established previously in species-specific recognition of the antagonists citalopram and mazindol. These findings support a critical role for TMD I residues in defining shared aspects of SERT substrate and antagonist recognition.

Regulated phosphorylation and trafficking of antidepressant-sensitive serotonin transporter proteins

Presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) mediate antidepressant-sensitive clearance of 5-HT following release. Although we have been aware for decades that SERT-mediated 5-HT clearance can be modulated by exogenous agents including serotonin-selective reuptake inhibitors, amphetamines, and cocaine, we have had little reason to speculate that SERT activity was actively controlled through endogenous pathways. Recent studies indicate that SERTs are likely to be trafficked to specific plasma membrane subdomains to achieve localized clearance of 5-HT, and that the number of SERTs resident in the plasma membrane is controlled through kinase- and phosphatase-linked pathways. In particular, roles for protein kinase C and phosphatase 2A become apparent through studies with enzyme activators and inhibitors in SERT-transfected cells, where SERT proteins are rapidly phosphorylated in parallel with transporter redistribution and loss of functional uptake capacity. Based on our findings, and the studies of others in native tissues and transfected cells, we propose a model whereby SERTs are organized in a macromolecular complex in the plasma membrane that may serve to locate reuptake activity near release sites. Although many elements of this model remain hypothetical, our findings suggest a much more dynamic picture of transporter-mediated 5-HT reuptake than typically described and suggest opportunities both for the development of new SERT regulatory agents and for the identification of regulatory pathways that may be compromised in mental illness.

Phosphorylation and Regulation of Antidepressant-sensitive Serotonin Transporters

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) are responsible for efficient synaptic clearance of extracellular 5HT. Previously (Qian, Y., Galli, A., Ramamoorthy, S., Risso, S., DeFelice, L. J., and Blakely, R. D. (1997) J. Neurosci. 17, 45-47), we demonstrated that protein kinase (PKC)-linked pathways in transfected HEK-293 cells lead to the internalization of cell-surface human (h) SERT protein and a reduction in 5HT uptake capacity. In the present study, we report that PKC activators rapidly, and in a concentration-dependent manner, elevate the basal level of hSERT phosphorylation 5-6-fold. Similarly, protein phosphatase (PP1/PP2A) inhibitors down-regulate 5HT transport and significantly elevate hSERT 32P incorporation, effects that are additive with those of PKC activators. Moreover, hSERT phosphorylation induced by beta-phorbol 12-myristate 13-acetate is abolished selectively by the PKC inhibitors staurosporine and bisindolylmaleimide I, whereas hSERT phosphorylation induced by phosphatase inhibitors is insensitive to these agents at comparable concentrations. Protein kinase A and protein kinase G activators fail to acutely down-regulate 5HT uptake but significantly enhance hSERT phosphorylation. Basal hSERT and okadaic acid-induced phosphorylation were insensitive to chelation of intracellular calcium and Ca2+/calmodulin-dependent protein kinase inhibitors. Together these results reveal hSERT to be a phosphoprotein whose phosphorylation state is likely to be tightly controlled by multiple kinase and phosphatase pathways that may also influence the transporter's regulated trafficking.

Polarized Expression of the Antidepressant-Sensitive Serotonin Transporter in Epinephrine-Synthesizing Chromaffin Cells of the Rat Adrenal Gland

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) clear the amine from extracellular spaces in the CNS and periphery as a mechanism for transmitter inactivation and recycling. Although it is known that SERTs are preferentially expressed on basolateral domains in transfected epithelial cells, details of the transporter's membrane localizationin vivoare lacking. 5HT and 5HT receptors have been identified in the rodent adrenal gland. Using SERT antagonist autoradiography, we establish the presence of antidepressant-sensitive transport sites in the rat adrenal medulla. Immunofluorescence experiments using antibodies specific for the SERT COOH and NH2termini, for 5HT, or for catecholamine biosynthetic enzymes suggest that SERT mediates intracellular 5HT accumulation by epinephrine-secreting chromaffin cells. Using confocal microscopy, we establish that SERT expression is nonuniformly distributed along the plasma membrane of chromaffin cells. Notably, SERT immunoreactivity is largely absent from plasma membranes bordering smooth muscle that surrounds vascular sinusoids. Rather, SERT is highly expressed in membranes adjoining other chromaffin cells, consistent with a role for 5HT and SERT in autocrine or paracrine control of chromaffin cell physiology. SNAP-25, a t-SNARE protein implicated in neurotransmitter release, was found to colocalize with SERT. In contrast, Na,K ATPase and NCAM are uniformly distributed along the entire perimeter of chromaffin cell membranes. These findings underscore a role for 5HT and SERT in adrenal physiology, reveal unrecognized polarity of chromaffin cell plasma membranes, and warrant a consideration of common targeting mechanisms localizing amine transporters near release sites.

Protein Kinase C Activation Regulates Human Serotonin Transporters in HEK-293 Cells via Altered Cell Surface Expression

Antidepressant- and cocaine-sensitive serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) dictate clearance of extracellular 5-HT after release. To explore protein kinase C-mediated SERT regulation, we generated a stable human SERT (hSERT)-expressing cell line (293-hSERT) and evaluated modulation of 5-HT activity via studies of 5-HT flux, hSERT-mediated currents under voltage clamp, and surface distribution of SERT protein. 293-hSERT cells exhibit saturable, high-affinity, and antidepressant-sensitive 5-HT uptake as well as hSERT-dependent whole-cell currents. In these cells, the protein kinase C activator beta-PMA caused a time-dependent reduction in 5-HT uptake capacity (Vmax) after acute application and a reduction in SERT-mediated currents. Effects of beta-PMA were mimicked by the phorbol ester beta-PDBu, were not observed with the inactive alpha-isomers, and could be blocked by treatment of cells with the protein kinase C inhibitor staurosporine. Biotinylation/immunoblot analyses showed that activity reductions are paralleled by a staurosporine-sensitive loss of surface SERT protein. These data indicate that altered surface abundance, rather than reduced catalytic transport efficiency, mediates acute PKC-dependent modulation of 5-HT uptake.