The serotonin transporter (SERT) is an integral membrane protein responsible for the clearance of serotonin from the synaptic cleft following the release of the neurotransmitter. SERT plays a prominent role in the regulation of serotoninergic neurotransmission and is a molecular target for multiple antidepressants as well as substances of abuse. Here we show that SERT associates with lipid rafts in both heterologous expression systems and rat brain and that the inclusion of the transporter into lipid microdomains is critical for serotonin uptake activity. SERT is present in a subpopulation of lipid rafts, which is soluble in Triton X-100 but insoluble in other non-ionic detergents such as Brij 58. Disaggregation of lipid rafts upon depletion of cellular cholesterol results in a decrease of serotonin transport capacity (V max ), due to the reduction of turnover number of serotonin transport. Our data suggest that the association of SERT with lipid rafts may represent a mechanism for regulating the transporter activity and, consequently, serotoninergic signaling in the central nervous system, through the modulation of the cholesterol content in the cell membrane. Furthermore, SERT-containing rafts are detected in both intracellular and cell surface fractions, suggesting that raft association may be important for trafficking and targeting of SERT. The serotonin transporter (SERT)1 is a member of a Na ϩ / Cl Ϫ -dependent family of integral membrane proteins that includes carriers for neurotransmitters, osmolytes, and nutrients (1-3). SERT, the norepinephrine transporter, and the dopamine transporter are most closely related, defining a subgroup characterized by a high degree of similarity both in sequence and pharmacological properties (4). SERT is responsible for the clearance of serotonin (5-hydroxytryptamine, 5HT) from the synaptic cleft following the release of the neurotransmitter (2).5HT is believed to accumulate inside the cell through cotransport with Na ϩ and Cl Ϫ and countertransport with K ϩ (4). SERT is an important pharmacological target for substances of abuse, such as cocaine, 3,4-methylenedioxymethamphetamine (Ecstasy), and p-chloramphetamine, and a variety of therapeutic antidepressants (1, 5, 6).Acute changes in SERT endogenous activity are likely to originate from local variations in 5HT concentration, cell surface distribution of SERT, interaction with regulatory proteins, or reversible post-translational modifications. To date SERT expression on the plasma membrane has been shown to be down-regulated by protein kinase C activation, following elevation of intracellular levels of Ca 2ϩ (7,8), or by phorbol 12-myristate 13-acetate treatment (9 -12). The SNARE protein syntaxin 1A (Syn 1A) is one of the few proteins known to modulate SERT function via protein-protein interaction by regulating the number of SERT molecules on the plasma membrane (13,14).To carry out clearance of extracellular 5HT, SERT must be inserted into the plasma membrane in an active form. This may require targeting the transport...

The serotonin transporter (SERT) plays a critical role in the maintenance of normal neurotransmission by serotonin [5-hydroxytryptamine (5-HT)]. Recent evidence suggests that SERT and other neurotransmitter transporters are tightly regulated. Activation of protein kinase C results in a decrease in SERT-mediated 5-HT uptake, which is due to an internalization of the transporter. However, to date little is known about the mechanism and proteins involved in the down-regulation of the transporter. One candidate SERT-regulatory protein is the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) protein, syntaxin 1A (Syn1A), which has recently been implicated in the regulation of ion channels as well as the SERT-related gamma-aminobutyric acid- and glycine-transporters. Using 5-HT uptake assays, confocal microscopy and glutathione S-transferase (GST) pull-down assays we showed that Syn1A also interacts with SERT and alters the subcellular localization of the transporter, resulting in a reduction of 5-HT transport. In addition, we have used the yeast two-hybrid system to search for novel regulatory proteins that interact with the cytoplasmic N-terminal domain of SERT. By screening rat brain cDNA library we have identified six potential SERT-binding proteins. Here we also present progress towards the elucidation of the biological relevance of these proteins and their potential role for the regulation of the serotonin transporter.

ConclusionWe have demonstrated that synaptosomal Lcystine uptake occurs primarily by a sodiumdependent mechanism that involves the X,,(;high-affinity glutamate transporters. However, it is apparent that the sites to which glutamate and cystine bind on these proteins are not the same, and the full implication of this, in terms of the physiological role of this transport system, and how it would be affected by pathological conditions remains to be determined. W e are grateful to Enterprise Ireland and the lnsh Motor Neurone Disease Association for financial support.

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The serotonin transporter (SERT) plays a critical role for the maintenance of normal neurotransmission by serotonin (5-hydroxytryptamine, 5-HT). SERT itself is thought to be tightly regulated. For example, it was previously shown that activation of PKC results in a down-regulation of SERT-mediated 5-HT uptake due to an internalisation of the transporter. However, to-date little is known about the mechanisms and proteins involved in this process. One candidate SERT-regulatory protein is t-SNARE protein syntaxin 1A, which has recently been implicated in the regulation of ion channels, as well as the SERT-related GABA and glycine transporters. Using 5-HT uptake assays, confocal microscopy, and GST-pulldown assays we could show, that syntaxin 1A does indeed interact with SERT and functionally regulates the transporter. In addition, we have used the yeast two-hybrid system to search for novel regulatory proteins that interact with the N-terminal domain of SERT. Screening a rat brain cDNA library we have identified six potential SERT-binding proteins. Here we present progress towards the elucidation of the biological relevance of these proteins and their potential role for the regulation of the serotonin transporter B6 O n the structure, function and regulation of the serotonin transporter S. Horschitz, R. Hummerich, P. SchlossThe serotonin transporter (SERT) terminates serotonergic neurotransmission by rapid reuptake of 5-hydroxytryptamine (5-HT) back into the nerve terminal or axonal varicosities. SERT represents the target of various antidepressants which inhibit 5-HT transport and are widely used for the pharmacotherapy of depression. In order to gain insight into the molecular mechanism of substrate transport potential interactions of SERT with the norepinephrine transporter (NET), which is also a target for antidepressants, have been investigated in a heterologous cell culture system. In addition, the regulatory effects of antidepressants on the expression of the transport proteins was studied. Following exposure to antidepressants substrate transport and antidepressant binding parameters were determined. Long-term exposure of SERT to the selective serotonin reuptake inhibitor citalopram resulted in down-regulation of the expression level, as was also observed with NET exposed to desipramine. Analysis of substrate transport activity and determination of protein expression (estimated by radioligand binding experiments) revealed that these regulatory effects are dependent on drug concentration and exposure time. The observed long-term effects upon chronic exposure to antidepressants may provide clues to the delayed effects that these drugs have in an antidepressant therapy.Recent biochemical evidence suggests the formation of oligomeric structures in transporter proteins such as the transporters for serotonin, dopamine and glucose. Furthermore, a glutamate transporter has been shown to exist as a homopentamer. We investigated whether the human serotonin transporter (hSERT) exists as an oligomer in living cells and ...