Plasma serotonin levels and the platelet serotonin transporter

Serotonin (5HT) is a platelet-stored vasoconstrictor. Altered concentrations of circulating 5HT are implicated in several pathologic conditions, including hypertension. The actions of 5HT are mediated by different types of receptors and terminated by a single 5HT transporter (SERT). Therefore, SERT is a major mechanism that regulates plasma 5HT levels to prevent vasoconstriction and thereby secure a stable blood flow. In this study, the response of platelet SERT to the plasma 5HT levels was examined within two models: (i) in subjects with chronic hypertension or normotension; (ii) on platelets isolated from normotensive subjects and pretreated with 5HT at various concentrations. The platelet 5HT uptake rates were lower during hypertension due to a decrease in Vmax with a similar Km; also, the decrease in Vmax was primarily due to a decrease in the density of SERT on the platelet membrane, with no change in whole cell expression. Additionally, while the platelet 5HT content decreased 33%, the plasma 5HT content increased 33%. Furthermore, exogenous 5HT altered the 5HT uptake rates by changing the density of SERT molecules on the plasma membrane in a biphasic manner. Therefore, we hypothesize that in a hypertensive state, the elevated plasma 5HT levels induces a loss in 5HT uptake function in platelets via a decrease in the density of SERT molecules on the plasma membrane. Through the feedback effect of this proposed mechanism, plasma 5HT controls its own concentration levels by modulating the uptake properties of platelet SERT.     

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) (> or =10 microM), but is not restricted to this regime of external 5HT.     

Serotonin-, protein kinase C-, and Hic-5-associated redistribution of the platelet serotonin transporter

Emerging data indicate the existence of multiple regulatory processes supporting serotonin (5HT) transporter (SERT) capacity including regulated trafficking and catalytic activation, influenced by post-translational modifications and transporter-associated proteins. In the present study, using differential extraction and sedimentation procedures optimized for the purification of cytoskeletal and membrane-skeletal associated proteins, we analyze SERT localization in platelets. We find that most of the plasma membrane SERT is associated with the membrane skeleton. This association can be enhanced by both transporter activation and 5HT2A receptor activation. Inactivation of transport activity by phorbol ester treatment of intact platelets relocates SERT to the cytoskeleton fraction, consequently leading to transporter internalization. The translocation of SERT between these compartments is correlated with changes in the interaction with the LIM domain adaptor protein Hic-5. Co-immunoprecipitation and uptake activity studies suggest that Hic-5 is a determinant of transporter inactivation and relocation to a compartment subserving endocytic regulation. Associations of SERT with Hic-5 are evident in brain synaptosomes, suggesting the existence of parallel mechanisms operating to regulate SERT at serotonergic synapses.     

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

Background

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

Methodology/Principal Findings

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

Conclusions/Significance

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

Monitoring mouse serotonin transporter internalization in stem cell-derived serotonergic neurons by confocal laser scanning microscopy

In the central nervous system serotonergic neurotransmission is terminated by the rapid removal of serotonin (5-hydroxytryptamine, 5HT) out of the extra-cellular space back into the presynaptic neuron. This task is fulfilled by a specific serotonin transporter (SERT) protein which controls the concentration of extra-cellular 5HT. Consequently, one mechanism to regulate the efficacy of serotonergic neurotransmission is via modulation of the density of SERT molecules on the cell membrane.In this regard it has been shown, that chronic activation of the p38 mitogen-activated protein kinase (p38 MAPK) leads to enhanced SERT surface expression whereas activation of protein kinase C (PKC) reduces SERT surface expression. In addition, it has been reported that exposure to selective serotonin re-uptake inhibitors (SSRIs) leads to a down-regulation of SERT expression in vivo and in vitro in different cellular systems. Here, we have studied interactions between kinase- and SSRI-induced SERT internalization in mouse stem cell-derived serotonergic neurons expressing the native SERT allele in its natural surroundings. Therefore we established a method to quantify the amount of cell surface-expressed SERT molecules on individual cells by antibody detection combined with confocal laser scanning microscopy.Using this methodology we could show that activation of PKC, inhibition of the p38 MAPK as well as exposure to the SSRI citalopram each induced a significant reduction of cell surface-expressed SERT over time. Combinations of PKC activation, p38 MAPK inhibition and SSRI exposure led to a more pronounced down-regulation of SERT surface expression depending on the time of drug exposure.     

At diabetes-like concentration, glucose down-regulates the placental serotonin transport system in a cell-cycle-dependent manner

Serotonin [5-hydroxytryptamine (5HT)] is a vasoconstrictor that also acts as a developmental signal early in embryogenesis. The 5HT transporter (SERT) on the membranes of the placental trophoblast cells controls 5HT levels in the maternal bloodstream to maintain stable transplacental blood flow and simultaneously provide 5HT to the embryo. The 5HT uptake rate of placental SERT is important for both the mother and the developing embryo. The impact of glucose on the placental SERT system during diabetic pregnancy is not known. The present in vitro study investigated this important issue in human placental choriocarcinoma (JAR) cells that were cultured for 24-96 h in a medium containing either 5.5 (physiologic concentration) or 25 mmol/L D-glucose (diabetic-like concentration). The 5HT uptake rates of the cultured cells were not altered at exogenous D-glucose concentrations in the range of 5.5-15 mmol/L, but were decreased significantly at a diabetic-like concentration (>or=25 mmol/L). To understand better the role of glucose on the placental 5HT system, we first characterized SERT in JAR cells at different cell-cycle phases and then determined the expression levels of SERT on the plasma membrane and in the intracellular pools of JAR cells at the late-S and G2 phases, where the uptake rates were decreased 73% under diabetic-like glucose concentrations. Finally, the importance of self-association of SERT molecules was examined. In JAR cells co-expressing Flag- and myc-tagged SERT, myc-antibody precipitated 70% of Flag-SERT, indicating that a large percentage of SERT proteins exist as oligomers in situ. Under diabetic conditions, myc-antibody no longer precipitated Flag-SERT, suggesting a disruption in the aggregation of SERT molecules. Therefore, we propose that under uncontrolled diabetic conditions, glucose down-regulates 5HT uptake rates of placental SERT by interfering with its functional expression in a cell-cycle-dependent manner.     

Serotonin transporters (SERTs) within the rat nucleus of the solitary tract: Subcellular distribution and relation to 5HT2A receptors

Serotonergic transmission is terminated by serotonin transporter (SERT)-mediated uptake following activation of serotonin receptors, several subtypes of which are present in the medial nucleus of the solitary tract (mNTS) at the area postrema level. In this region, serotonin (5HT) is a major modulator of the baroreceptor reflex and also affects gastric motility. This serotonin is derived from multiple sources including local neurons and inputs from raphe and visceral vagal afferents. To determine the relevant functional sites for serotonin uptake in the mNTS, we examined the electron microscopic localization of SERTs using both immunoperoxidase and immunogold labeling in rat brain. In addition, we combined these methods for dual labeling of SERTs and 5HT2A receptors to detect whether the SERT in this region was located near or at a distance from the sites of activation of these G-protein coupled receptors. Intensive SERT immunolabeling was seen on plasma membranes of axons and morphologically heterogeneous axon terminals that formed symmetric or asymmetric synapses on dendrites without detectable 5HT2A immunoreactivity (IR). 5HT2A-IR was, however, located in other nearby neuronal and glial profiles, some of which apposed intensively SERT-labeled terminals or terminals containing lower intensity of SERT immunolabeling. In somatodendritic profiles, co-expression of SERT and 5HT2A receptor immunolabeling was seen near synapses and Golgi lamellae. Our results suggest that in the mNTS 5HT activates 5HT2A receptors at a distance from SERT-mediated uptake sites in diverse cell types including some that express both 5HT2A receptors and SERTs.     

Structural basis for recruitment of RILP by small GTPase Rab7

Rab7 regulates vesicle traffic from early to late endosomes, and from late endosomes to lysosomes. The crystal structure of Rab7-GTP in complex with the Rab7 binding domain of RILP reveals that Rab7 interacts with RILP specifically via two distinct areas, with the first one involving the switch and interswitch regions and the second one consisting of RabSF1 and RabSF4. Disruption of these interactions by mutations abrogates late endosomal/lysosomal targeting of Rab7 and RILP. The Rab7 binding domain of RILP forms a coiled-coil homodimer with two symmetric surfaces to interact with two separate Rab7-GTP molecules, forming a dyad configuration of Rab7-RILP(2)-Rab7. Mutations that disrupt RILP dimerization also abolish its interactions with Rab7-GTP and late endosomal/lysosomal targeting, suggesting that the dimeric form of RILP is a functional unit. Structural comparison suggests that the combined use of RabSF1 and RabSF4 with the switch regions may be a general mode of action for most Rab proteins in regulating membrane trafficking.     

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the “long loop” recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β₂-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation.     

Brain Serotonin Transporter Binding of [123I]ADAM: Within‐Subject Variation between Summer and Winter Data

The neurotransmitter serotonin (5‐HT) controls several physiological functions, and a disturbance of the 5‐HT system is implicated in many psychiatric conditions. Seasonal variation has been suggested in the 5‐HT system. We investigated within‐subject seasonal variation in brain serotonin transporter (SERT) binding with the SERT‐ligand [¹²³I]ADAM and single photon emission computed tomography (SPECT) in 12 healthy individuals. No systematic variation was found in the midbrain or thalamus areas between scans done in summer and winter. Our results suggest that factors other than season are more important in causing within‐subject variation of brain SERT binding between summer and winter. (Author correspondence: anu.koskela@helsinki.fi)     

The role of ERp44 in maturation of serotonin transporter protein

In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT molecules in oligomeric form.     

Delayed Onset of Positive Feedback Activation of Rab5 by Rabex-5 and Rabaptin-5 in Endocytosis

Background

Rabex-5 is a guanine nucleotide exchange factor (GEF) that specifically activates Rab5, i.e., converting Rab5-GDP to Rab5-GTP, through two distinct pathways to promote endosome fusion and endocytosis. The direct pathway involves a pool of membrane-associated Rabex-5 that targets to the membrane via an early endosomal targeting (EET) domain. The indirect pathway, on the other hand, involves a cytosolic pool of Rabex-5/Rabaptin-5 complex. The complex is recruited to the membrane via Rabaptin-5 binding to Rab5-GTP, suggesting a positive feedback mechanism. The relationship of these two pathways for Rab5 activation in the cell is unclear.

Methodology/Principal Findings

We dissect the relative contribution of each pathway to Rab5 activation via mathematical modeling and kinetic analysis in the cell. These studies show that the indirect pathway constitutes a positive feedback loop for converting Rab5-GDP to Rab5-GTP on the endosomal membrane and allows sensitive regulation of endosome fusion activity by the levels of Rab5 and Rabex-5 in the cell. The onset of this positive feedback effect, however, contains a threshold, which requires above endogenous levels of Rab5 or Rabex-5 in the cell. We term this novel phenomenon “delayed response”. The presence of the direct pathway reduces the delay by increasing the basal level of Rab5-GTP, thus facilitates the function of the Rabex-5/Rabaptin-5-mediated positive feedback loop.

Conclusion

Our data support the mathematical model. With the model''s guidance, the data reveal the affinity of Rabex-5/Rabaptin-5/Rab5-GTP interaction in the cell, which is quantitatively related to the Rabex-5 concentration for the onset of the indirect positive feedback pathway. The presence of the direct pathway and increased Rab5 concentration can reduce the Rabex-5 concentration required for the onset of the positive feedback loop. Thus the direct and indirect pathways cooperate in the regulation of early endosome fusion.     

Characterisation of the serotonin efflux induced by cytosolic Ca2+ and Na+ concentration increase in human platelets.

BACKGROUND/AIM: The present study aimed at elucidating the mechanism(s) of serotonin (5-HT) efflux induced by thapsigargin from human platelets in the absence of extra-cellular Ca2+. METHODS: Efflux of pre-loaded radiolabeled serotonin was generally determined by filtration techniques. Cytosolic concentrations of Ca2+, Na+ and H+ were measured with appropriate fluorescent probes. RESULTS: 5-HT efflux from control or reserpine-treated platelets--where reserpine prevents 5-HT transport into the dense granules--was proportional to thapsigargin evoked cytosolic [Ca2+]c increase. Accordingly factors as prostacyclin, aspirin and calyculin which reduced [Ca2+]c-increase also inhibited the 5-HT efflux. Thapsigargin, which also caused a remarkable increase in cytosolic [Na+]c, promoted less 5-HT release, in parallel to lower [Na+]c and [Ca2+]c increase, when added to platelet suspensions containing low [Na+]. The Na+/H+ exchanger monensin increased the [Na+]c and induced 5-HT efflux without affecting the Ca2+ level. The 5-HT efflux induced by both [Ca2+] or [Na+]c increase did not depend on pH or membrane potential changes, whereas it decreased in the absence of extra-cellular K+, and increased in the absence of Cl- or Na+. CONCLUSION: Increases in [Ca2+]c and [Na+]c independently induce serotonin efflux through the outward directed plasma membrane serotonin transporter SERT. This event might be physiologically important at the level of capillaries or narrowed arteries where platelets are subjected to high shear stress which causes [Ca2+]c increase followed by 5-HT release which might exert vasodilatation.     

Effects of combined treatment with mephedrone and methamphetamine or 3,4-methylenedioxymethamphetamine on serotonin nerve endings of the hippocampus

Aims

Mephedrone is a stimulant drug of abuse with close structural and mechanistic similarities to methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Although mephedrone does not damage dopamine nerve endings it increases the neurotoxicity of amphetamine, methamphetamine and MDMA. The effects of mephedrone on serotonin (5HT) nerve endings are not fully understood, with some investigators reporting damage while others conclude it does not. Presently, we investigate if mephedrone given alone or with methamphetamine or MDMA damages 5HT nerve endings of the hippocampus.

Main methods

The status of 5HT nerve endings in the hippocampus of female C57BL mice was assessed through measures of 5HT by HPLC and by immunoblot analysis of serotonin transporter (SERT) and tryptophan hydroxylase 2 (TPH2), selective markers of 5HT nerve endings. Astrocytosis was assessed through measures of glial fibrillary acidic protein (GFAP) (immunoblotting) and microglial activation was determined by histochemical staining with Isolectin B4.

Key findings

Mephedrone alone did not cause persistent reductions in the levels of 5HT, SERT or TPH2. Methamphetamine and MDMA alone caused mild reductions in 5HT but did not change SERT and TPH2 levels. Combined treatment with mephedrone and methamphetamine or MDMA did not change the status of 5HT nerve endings to an extent that was different from either drug alone.

Significance

Mephedrone does not cause toxicity to 5HT nerve endings of the hippocampus. When co-administered with methamphetamine or MDMA, drugs that are often co-abused with mephedrone by humans, toxicity is not increased as is the case for dopamine nerve endings when these drugs are taken together.     

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

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

Differential Regulation of the Serotonin Transporter by Vesicle-Associated Membrane Protein 2 in Cells of Neuronal versus Non-Neuronal Origin

The serotonin transporter (SERT) is a key regulator of serotonergic signalling as it mediates the re-uptake of synaptic serotonin into nerve terminals, thereby terminating or modulating its signal. It is well-known that SERT regulation is a dynamic process orchestrated by a wide array of proteins and mechanisms. However, molecular details on possible coordinated regulation of SERT activity and 5-HT release are incomplete. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, interacts with SERT. This was documented in vitro, through GST pull-down assays, by co-immunoprecipitation experiments on heterologous cells and rat hippocampal synaptosomes, and with FRET analysis in live transfected HEK-293 MSR cells. The related isoforms VAMP1 and VAMP3 also physically interact with SERT. However, comparison of the three VAMP isoforms shows that only VAMP2 possesses a functionally distinct role in relation to SERT. VAMP2 influences 5-HT uptake, cell surface expression and the delivery rate of SERT to the plasma membrane differentially in HEK-293 MSR and PC12 cells. Moreover, siRNA-mediated knock-down of endogenous VAMP2 reduces 5-HT uptake in CAD cells stably expressing low levels of heterologous SERT. Deletion and mutant analysis suggest a role for the isoform specific C-terminal domain of VAMP2 in regulating SERT function. Our data identify a novel interaction between SERT and a synaptic vesicle protein and support a link between 5-HT release and re-uptake.     

Serotonin transporter kinetics in rats selected for extreme values of platelet serotonin level

By selective breeding of Wistar rats for the extreme values of platelet serotonin (5HT) level (PSL), we have developed earlier two sublines of animals differing markedly in this parameter. Further studies, performed on the protein and mRNA levels, revealed platelet serotonin transporter (5HTt) as parameter underlying mentioned differences in PSL between sublines. In this work, we have performed full-kinetic analysis of platelet serotonin uptake (PSU) in animals from the genetically selected sublines. The results demonstrated marked differences in maximal velocity (V(max)) of the 5HT transporter, as contrasted to the lack of any difference in the Michaelis constant (K(m)). High correlation between PSL and V(max) of PSU was demonstrated, revealing that the number of membrane 5HT transporter sites is under genetic control and responsible for marked differences in PSL between high- and low-5HT sublines. These results enabled further selective breeding of animals for the extremes of V(max) of platelet 5HT transporter, and so the development of more specific model "Wistar-Zagreb 5HT rats".     

The effect of a series of organic cations upon the plasmalemmal serotonin transporter, SERT

The aim of this work was to test the effect of a series of organic cations upon the activity of the plasma membrane serotonin transporter (SERT). The experiments were performed using the JAR cell line that constitutively expresses high levels of SERT, and rat intestine, whose mucosal epithelial cells also express SERT. Initial rates of (3)H-serotonin ((3)H-5HT; 200 nM) uptake were not changed by some of the organic cations tested (guanidine, N-methylnicotinamide, choline, atenolol, caffeine and theophylline), but were slightly (15-30%) inhibited by some other organic cations, at the highest concentrations tested (thiamine (3 mM), cimetidine (1 mM) and tetraethylammonium (3 mM)). On the other hand, some other organic cations reduced, in a concentration-dependent manner, uptake of (3)H-5HT by JAR cells (IC(50)s of 0.3, 1.3, 5.4, 89.3, 460 and 748 microM for quinidine, verapamil, propranolol, amiloride, nicotine and clonidine, respectively). Quinidine, clonidine and amiloride seem to be competitive inhibitors of (3)H-5HT uptake, whereas verapamil, nicotine and propranolol appear to be uncompetitive or non-competitive inhibitors. Moreover, quinidine, verapamil and propranolol trans-inhibited (3)H-5HT uptake, whereas clonidine, nicotine and amiloride were devoid of effect. Finally, these six organic cations were able to significantly increase the serosal-to-mucosal apparent permeability (P(app)) to (3)H-5HT of rat jejunum, ileum and colon. In conclusion, human and rat SERT-mediated transport is inhibited by several distinct organic cations, some of which are therapeutic agents or drugs of abuse. Knowledge on which organic cations interfere with SERT-mediated transport of 5HT will have major implications in tissues where 5HT plays important physiological roles (eg. central nervous system, intestine and placenta).     

Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.

BACKGROUND/AIM: platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation. METHODS: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions. RESULTS: 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 kappaDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.     

The Rab5 effector EEA1 interacts directly with syntaxin-6.

The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate in trans-Golgi network to endosome as well as in endocytic membrane traffic.