Identification and characterization of antidepressant-sensitive serotonin transporter proteins using site-specific antibodies (original) (raw)
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Going with the Flow: Trafficking-Dependent and -Independent Regulation of Serotonin Transport
Traffic, 2008
Antidepressant-, cocaine-and 3,4-methylenedioxymethamphetamine-sensitive serotonin (5hydroxytryptamine, 5-HT) transporters (SERTs) are expressed on presynaptic membranes of 5-HTsecreting 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 mitogenactivated 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 traffickingindependent modes of SERT regulation and also the suspected roles played in regulation by SERTassociated 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.
Regulated phosphorylation and trafficking of antidepressant-sensitive serotonin transporter proteins
Biological Psychiatry, 1998
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 transportermediated 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.
The serotonin transporter from human brain: Purification and partial characterization
Neurochemistry International, 1996
The serotonin (5-HT) transporter from hhman striatum was solubilized by digitonin and purified by affinity chromatography. The native protein-detergent complex had a molecular mass of 205 kDa, as estimated by gel-exclusion chromatography of the eluates obtained from affinity chromatography. The purified 5-HT transporter migrated as a single band of 67 kDa in SDS-PAGE. To clarify the spatial relationships between the binding sites of the tricyclic antidepressants, as [3H]-imipramine, and of the selective serotonin reuptake inhibitors, as [3H]-paroxetine, on the 5-HT transporter, both radioligands were used to label it in the purification steps.
Gene structure and 5′-flanking regulatory region of the murine serotonin transporter
Molecular Brain Research, 1997
By modulating the magnitude and duration of postsynaptic responses, carrier-facilitated serotonin (5-HT) transport into and release from the presynaptic neuron is central to the fine tuning of serotonergic neurotransmission. The 5-HT transporter (5-HTT) is the prime target for widely used antidepressants, psychostimulants, drugs of abuse and neurotoxins. We have isolated the gene encoding the murine 5-HTT and determined the sequence of all exons including adjacent intronic regions and approximately 3.6 kb of the 5'-flanking regulatory region. The murine 5-HTT gene is composed of 14 exons spanning approximately 34 kb. The single gene transcript after splicing is 2744 bp in length and it contains 186 bp of 5' untranslated region (5'-UTR) and 668 bp of 3'-UTR. A TATA-like motif and several potential binding sites for transcription factors including AP1, AP2, AP4, SP1 as well as CRE- and GRE-like motifs are present in the GC-rich 5'-flanking region. The characterization of murine 5-HTT cDNA and genomic organization will facilitate studies of 5-HT uptake function with molecular pharmacologic and transgenic strategies as well as investigations of its role in quantitative traits and psychiatric disorders.
Journal of Biological Chemistry, 2006
In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [ 125 I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/ I172M had a synergistic impact on (RS)-CIT recognition (ϳ10,000fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/ I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.
Characterization of a genetically reconstituted high-affinity system for serotonin transport
Proceedings of the National Academy of Sciences, 1989
By transfecting mouse fibroblast L-M cells with human genomic DNA, we have established and identified several clonal cell lines that stably express a high-affinity serotonin (5-HT)-uptake mechanism absent in untransfected host cells. One such cell line, L-S1, possesses features of 5-[3H]HT uptake similar to those previously characterized in the central nervous system and blood platelets: (i) specificity for 5-HT; (ii) antagonism by imipramine, a known inhibitor of high-affinity 5-HT uptake; (iii) both Na+ and temperature dependences; (iv) kinetic saturability; and (v) high affinity for 5-HT (Km = 0.39 +/- 0.10 microM; Vmax = 2.14 +/- 0.55 pmol/min per mg of protein). This cell line can be used to compare the relative efficacies of known blockers of 5-HT uptake and thereby offers a rapid and reliable assay system for testing novel inhibitors of this system. Since L-S1 contains stably integrated human DNA in its genome, we postulate that the observed 5-HT-uptake system resulted from t...
Antidepressant-induced internalization of the serotonin transporter in serotonergic neurons
The FASEB Journal, 2008
A deficiency of serotonergic signaling is thought to be involved in the etiology of depression. Thus, drugs blocking the reuptake of serotonin back into the neurons are widely used in treatment of this disease; however, their delayed effect in remission of patients suggests that the clinical response does not rely on simple serotonin uptake inhibition but may include further regulatory mechanisms. We have analyzed cellular serotonin transporter (SERT) expression on exposure to the selective serotonin reuptake inhibitor citalopram in serotonergic neurons expressing the native SERT allele in its natural surroundings. Biotinylation of SERT-expressing HEK293 cells, as well as confocal microscopy analysis in these cells and in serotonergic neurons, revealed that exposure to citalopram time dependently reduces the amount of cell surface-expressed SERT. Furthermore, in serotonergic neurons, longer exposure to citalopram not only caused an internalization of SERT proteins from the cell surface but also induced a redistribution of SERT from neurite extensions into the soma. This process was reversible on drug removal. Microarray analysis performed on citalopram-treated serotonergic neurons revealed that antidepressant treatment does not alter SERT mRNA expression, suggesting that SERT trafficking from and to the cell membrane is regulated on the posttranscriptional level.-Lau, T., Horschitz, S., Berger, S., Bartsch, D., Schloss, P. Antidepressantinduced internalization of the serotonin transporter in serotonergic neurons. FASEB J. 22, 1702-1714 (2008)
Neuroscience, 2007
Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by N-ethyl-N-nitrosurea (ENU)-driven target-selected mutagenesis. Biochemical characterization revealed that SERT mRNA and functional protein are completely absent in homozygous knockout (SERT ؊/؊ ) rats, and that there is a gene dose-dependent reduction in the expression and function of the SERT in heterozygous knockout rats. As a result, 5-HT homeostasis was found to be severely affected in SERT ؊/؊ rats: 5-HT tissue levels and depolarization-induced 5-HT release were significantly reduced, and basal extracellular 5-HT levels in the hippocampus were ninefold increased. Interestingly, we found no compensatory changes in in vitro activity of tryptophan hydroxylase and monoamine oxidase, the primary enzymes involved in 5-HT synthesis and degradation, respectively. Similarly, no major adaptations in nonserotonergic systems were found, as determined by dopamine and noradrenaline transporter binding, monoamine tissue levels, and depolarization-induced release of dopamine, noradrenaline, glutamate and GABA. In conclusion, neurochemical changes in the SERT knockout rat are primarily limited to the serotonergic system, making this novel rat model potentially very useful for studying the behavioral and neurobiological consequences of disturbed 5-HT homeostasis. transporter; SERT Ϫ/Ϫ , homozygous serotonin transporter knockout rat; SERT ϩ/Ϫ , heterozygous serotonin transporter knockout rat; SERT ϩ/ϩ , wildtype littermate of heterozygous serotonin transporter knockout and homozygous serotonin transporter knockout rats; SSRI, selective serotonin reuptake inhibitor; TPH, tryptophan hydroxylase; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HTP, 5-hydroxytryptophan.
Research report Cellular localization and expression of the serotonin transporter in mouse brain
The high-affinity serotonin 5-HT transporter 5-HTT plays an important role in the removal of extracellular serotonin, thereby modulating and terminating the action of this neurotransmitter at various pre- and post-synaptic serotonergic receptors and heterorecep- tors. In order to characterize the anatomical distribution of the 5-HTT in mouse brain, in situ hybridization histochemistry using 35 w125 x S-labeled riboprobes was performed. These results were compared with 5-HTT binding site distribution as evaluated by I RTI-55 autoradiography. High levels of 5-HTT mRNA were detected in all brain stem raphe nuclei, with variations in labeling among the various subnuclei. Those brain areas known to possess serotonergic cell bodies stained intensely for both 5-HTT mRNA and 5-HTT binding sites. In contrast to previous findings in rat brain, the highest densities of 5-HTT sites were found in areas outside the raphe complex, particularly in the substantia nigra, globus pallidus, and superior...