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; (ii) both Nal and temperature dependences; (iv) kinetic saturability; and (v) high affinity for 5-HT (Km = 0.39 + 0. 10 jIM; V.. = 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 the expression of human gene(s) coding for the 5-HT transporter. Thus, cell lines such as L-S1 may represent novel means for screening and developing therapeutic agents specific for neurotransmitteruptake systems as well as substrates for the cloning and elucidation of the genes encoding the various neurotransmitter transporters.Inactivation of neurotransmitters, following their stimulated release into the synaptic cleft, is a key regulatory process in neurotransmission. For biogenic amine and amino acid neurotransmitters, the inactivation process is achieved mainly via transmitter-specific high-affinity transport systems present in the presynaptic neuron and/or surrounding glial cells (1-6). These transport mechanisms are also of significant clinical interest because some of their inhibitors are therapeutic agents for certain neurological disorders.Although the physiology and pharmacology of a number of neurotransmitter transport systems including that for serotonin have been extensively studied (6-13), little is known about the biochemistry and molecular biology of any of these systems. With the hope of attaining such information, we report here the use of gene transfer (14-17) in the establishment and identification of a clonal mouse fibroblast cell line, L-S1, that stably expresses a presumptively human highaffinity uptake system for serotonin (5-HT). MATERIALS AND METHODSTransfection of L-M Fibroblasts. Mouse L-M fibroblast cells (ATCC CCL 1.2) were plated in 100-mm culture dishes so that by the time of transfection the cell number would be about 106. Human genomic DNA and pSV2-neo, a plasmid bearing neomycin-resistance as a selectable marker (18), were cotransfected essentially by the method of calcium phosphate precipitation (19). After transfection, the cells were trypsinized and plated into 96-well plates. Selection of resistance to G418 (GIBCO; 400 tkg/ml) was maintained until transfectant colonies (typically three to five per well) were well established. Each well was then assayed for 5-[3H]HT uptake activities.Identificatio...