The Human and Rat Serotonin Transporter in Vitro: Studies on Reverse Transport and the Action of Uptake Inhibitors and Amphetamine Derivates

Project number: Research project P 13183 Project title: Serotonin Transporter in Vitro ABSTRACT OF THE PROJECT Projekttitel: KURZBESCHREIBUNG DES FORSCHUNGSPROJEKTES The serotonin transporter (SERT) is a member of a large family of Na + /Cl - -dependent transporters, terminating serotonergic neurotransmission by removing the monoamine from the synaptic cleft. The SERT serves as a therapeutic target for a variety of antidepressant drugs, acting as non-selective (imipramine) or highly selective (fluoxetine, citalopram) inhibitors. On the other hand, the action on SERT is believed to underlie the effects of the widely abused drug 3,4-methylenedioxymetamphetamine (MDMA, ecstasy). MDMA and other amphetamine derivatives cause an acute release of serotonin in vivo and in vitro caused by reverse transport, a condition in which uptake carriers run backward and pump transmitter out of the neuron. The cloning of the rat and human SERT has allowed studies on the isolated transporter protein expressed in non-neuronal cells. Whereas there is a vast body of data on the uptake of substrate, much less information is available on the properties of reverse transport performed by this transporter. Our laboratory has developed a method which allows the investigation of this mechanism in a superfusion system at a time resolution of 30-60 seconds. The aim of the present study is a detailed in vitro investigation of the properties of SERT using a micro- superfusion system. For the present experiments HEK-293 cells expressing the rat SERT were kindly provided by Dr. P. Schloss (Max Planck Institute for Brain Research, Frankfurt, Germany), and the cDNA encoding the human SERT was a generous gift from Dr. R. Blakely (Vanderbilt University Medical Center, Nashville, TN, USA). For the human SERT permanently transfected cell lines will be established. The cells are grown on coverslips and incubated with 3 H-serotonin for the superfusion experiments. After loading, the coverslips are transferred to superfusion chambers and superfused at a constant flow and temperature. Fractions will be collected and the radioactivity in the superfusion medium will be measured.Outward transport will be induced by lowering the Na + or Cl- -concentration of the medium. The specific aims of the proposed study are: 1. Determination of the inward and outward transport rates. 2. Investigations on the actions of serotonin uptake inhibitors (antidepressants) on outward transport induced by changes in the ionic composition of the superfusion medium. 3. Investigations on the actions of serotonin releasing drugs (p-chloroamphetamine, fenfluramine, MDMA `ecstasy`). The results of the project should allow the analysis of pharmacological properties of the human and rat SERT for which up to now no efficient method of investigation existed.