Patrick Schloss

The serotonin transporter: a primary target for antidepressant drugs

The serotoninergic system is known to modulate mood, emotion, sleep and appetite and thus is implicated in the control of numerous behavioural and physiological functions. Decreased serotoninergic neurotransmission has been proposed to play a key role in the aetiology of depression. The concentration of synaptic serotonin is controlled directly by its reuptake into the pre-synaptic terminal and, thus, drugs blocking serotonin transport have been successfully used for the treatment of depression. In addition to tricyclic antidepressants (TCAs; e.g. imipramine) which also block noradrenaline reuptake, highly specific serotonin reuptake inhibitors (SSRIs) such as fluoxetine and paroxetine have been developed, which are increasingly prescribed for depressed patients. The mode of action of these antidepressant drugs on their direct target, the serotonin transport protein, and possible regulatory mechanisms with respect to long-term alleviation of depression, although having been investigated both neurobiologically and clinically over the last years, are not yet understood. The cloning of the cDNA encoding the serotonin transporter has allowed a more precise characterization of this protein at the molecular level. This will show how antidepressants act at this target, thereby affecting the biochemical, pharmacological and electrophysiological properties of the serotoninergic system and give an introduction of how they might exert their therapeutic effect. This review gives an overview of the recent developments in this field, discusses mechanisms of antidepressant action on this target, and also possible interactions with other components of serotoninergic neurotransmission.

Immunocytochemical detection of the serotonin transporter in rat brain

The regional distribution of the serotonin uptake system was studied in rat brain using a specific polyclonal antibody raised against the putative extracellular loop between transmembrane domains 7 and 8 of the cloned rat serotonin transporter. Light microscope analysis with fluorescence and avidin-biotin-peroxidase techniques revealed a punctate staining as well as numerous labelled thin fibres, which exhibited accumulation of reaction end-product deposit over varicosities. These immunopositive processes were widely and heterogeneously distributed in the rat brain. High densities of immunoreactivity were seen within the caudate-putamen, amygdaloid complex, cortical areas, substantia nigra, ventral pallidum, Islands of Calleja, septal nuclei, interpeduncular nucleus, trigeminal motor nucleus and olfactory nuclei. We also found strong expression of serotonin transporter in the stratum oriens of area CA3 and, to a lesser extent, in the stratum oriens of CA1 and the stratum lacunosum molecular of CA1-CA3 regions of the hippocampus. Within the raphe nuclei, a moderate to high incidence of stained processes was observed, and immunopositive cell bodies were detected in the dorsal raphe nucleus. In addition, some immunoreactive fibres were present in the molecular and granular layers of the cerebellum as well as in the cochlear and olivary nuclei. In none of the regions analysed was evidence for glial staining obtained. The present immunocytochemical data reveal a widespread and heterogeneous distribution of the serotonin transporter in rat brain and suggest that serotoni transporter is preferentially sorted into axons, where it appears concentrated at varicosities and terminal boutons.

Primary structure and functional expression of a choline transporter expressed in the rat nervous system

Synthesis of the neurotransmitter acetylcholine in cholinergic nerve terminals is regulated by a sodium‐driven high‐affinity choline uptake system in the plasma membrane. We have isolated cDNAs from rat spinal cord and brainstem which encode a choline transporter (CHOT1). The predicted protein shares considerable amino acid identity and several structural features including twelve putative transmembrane regions with other neurotransmitter transporters. Expression of in vitro transcribed CHOT1 RNA in Xenopus oocytes generated Na+‐dependent choline uptake, which was not seen in control oocytes. Amplification by polymerase chain reaction (PCR) revealed significant amounts of CHOT1 mRNA in brain, cerebellum, spinal cord and, to a lesser extent, heart, but only very low expression in lung, kidney and muscle.

Modulation of a Recombinant Glycine Transporter (GLYT1b) by Activation of Protein Kinase C

Abstract: Treatment of human embryonic kidney cells (HEK 293 cells) expressing the mouse glycine transporter 1 (GLYT1b) with the protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (PMA) decreased specific [3H]glycine uptake. This down‐regulation resulted from a reduction of the maximal transport rate and was blocked by the PKC inhibitors 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7) and staurosporine. The inhibitory effect of PMA treatment was also observed after removing all five predicted phosphorylation sites for PKC in GLYT1b by site‐directed mutagenesis. These data indicate that glycine transport by GLYT1b is modulated by PKC activation; however, this regulation may involve indirect phosphorylation mechanisms.

The membrane‐bound rat serotonin transporter, SERT1, is an oligomeric protein

The synaptic actions of the neurotransmitter serotonin are terminated by a selective re‐uptake system located in the axonal membrane. To gain information about the quaternary structure of this membrane protein, we transiently expressed the recombinant rat serotonin transporter, SERT1, in human embryonic kidney 293 cells. Treatment with sulfhydryl oxidizing agents and the homobifunctional cross‐linker dimethyl suberimidate (DMS) generated adducts of 130–180 kDa and 220–270 kDa, respectively. These data indicate an oligomeric structure of SERT1.

Ketamine inhibits monoamine transporters expressed in human embryonic kidney 293 cells.

Background Ketamine has been characterized as having psychotomimetic and sympathomimetic effects. These symptoms have raised the possibility that ketamine affects monoaminergic neurotransmission. To elucidate the relation between ketamine and monoamine transporters, the authors constructed three cell lines that stably express the norepinephrine, dopamine, and serotonin transporters and investigated the effects of ketamine on these transporters. Methods Human embryonic kidney cells were transfected using the Chen‐Okayama method with the human norepinephrine rat dopamine, and rat serotonin transporter cDNA subcloned into the eukaryotic expression vector. Using cells stably expressing these transporters, the authors investigated the effects of ketamine on the uptake of these compounds and compared them with those of pentobarbital. Results Inhibition analysis showed that ketamine significantly inhibited the uptake of all three monoamine transporters in a dose‐dependent manner. The Ki (inhibition constant) values of ketamine on the norepinephrine, dopamine, and serotonin transporters were 66.8 micro Meter, 62.9 micro Meter, and 162 micro Meter, respectively. Pentobarbital, a typical general anesthetic agent with no psychotic symptoms, did not affect the uptake of monoamines, however. Further, neither the glycine transporter 1 nor the glutamate/aspartate transporter was affected by ketamine, indicating that ketamine preferentially inhibits monoamine transporters. Conclusions Ketamine inhibited monoamine transporters expressed in human embryonic kidney cells in a dose‐dependent manner. This result suggests that the ketamine‐induced inhibition of monoamine transporters might contribute to its psychotomimetic and sympathomimetic effects through potentiating monoaminergic neurotransmission.

Marker gene polymorphisms in hyperkinetic disorder--predictors of clinical response to treatment with methylphenidate?

Gene polymorphisms of the dopamine D4 receptor (DRD4) and serotonin transporter (5-HTT) are under discussion as potential genetic risk factors for hyperkinetic disorder (HD). In this disorder, treatment with the psychostimulant methylphenidate (MPH; Ritalin) induces calming effects and amelioration in only 70% of the patients. MPH blocks the reuptake of dopamine, thus enhancing synaptic dopamine which in turn antagonizes the release of prolactin (PL). Genotyping HD patients for DRD4 and 5-HTT polymorphisms and measuring PL concentrations, we report on an association between the combination DRD4*7/5HTT LL genotype and a reduced improvement in general functioning accompanied by different PL levels upon MPH treatment. Thus, our study supports the hypothesis that marker gene polymorphism may be helpful in identifying MPH non-responders.

Gene-environment interaction in hyperkinetic conduct disorder (HD + CD) as indicated by season of birth variations in dopamine receptor (DRD4) gene polymorphism.

Recently, an interaction between season of birth and the expression of candidate genes has been suggested. Season of birth variations in tryptophan hydroxylase (TPH), the serotonin transporter (5-HTTLPR) and the dopamine D4 receptor (DRD4) gene polymorphisms are different for affective disorders and schizophrenia. The DRD4 gene has been postulated as a candidate gene for attention-deficit-hyperactivity disorder (ADHD), equivalent to hyperkinetic disorder (HD). The seven-repeat long variant of this gene (DRD4*7) in comparison to the short repeat variants of the DRD4 gene polymorphism, has been found to be associated with ADHD. A seasonal pattern of birth has also been proposed for different subtypes of ADHD. Therefore, in a subgroup of children with HD and conduct disorder (CD) and in healthy controls, we investigated a possible association between the DRD4*7 allele and HD + CD in association with the season of birth. Supporting this hypothesis, we found an interaction between the seasons of birth and the expression of the DRD4 candidate gene in children with HD + CD as well as in controls, which differ significantly from each other. Depending on the season of birth, children carrying the DRD4*7R allele showed different relative risks for developing HD + CD.

Neurotransmitter transporters: new members of known families.

The identification of two gene families encoding neurotransmitter transporters was a major step towards a better understanding of these proteins and their function in neurotransmission. The recent isolation of additional members of these families underscores their high molecular diversity and implies a delicate regulation of transmitter uptake.

Neurotransmitter transporters A novel family of integral plasma membrane proteins

The re‐uptake of neurotransmitters into the nerve terminal terminates synaptic transmission at most central synapses and constitutes a key step in the modulation of synaptic efficacy. Recently, the cloning of several Na+‐driven neurotransmitter transporters has resulted in the description of a novel family of homologous membrane proteins, each with 12 transmembrane segments. These transporters constitute major targets of widely used drugs, and modulation of transporter gene expression and/or activity may represent an important substrate for plasticity in the nervous system.