Characterization of a genetically reconstituted high-affinity system for serotonin transport.

Abstract

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...

Mechanistic analyses of ion dependences in a high‐affinity human serotonin transport system in transfected murine fibroblast cells

“…It was previously shown that L_S1 cells exhibit a saturable velocity profile in transporting 5_ [ÅH]HT, characterized by Michaelis-Menten parameters (Km = 0·39 ± 0·10 ìÒ, Vmax = 2·14 ± 0·55 pmol mg¢ min¢, nH = 0·95 ± 0·09) which are highly comparable to those of other serotonin transport systems (Chang et al 1989(Chang et al , 1996. The apparent lack of ligand co-operativity during transport agrees with the observed lack of ligand co-operativity during binding to the transport complex, and further suggests that each cycle of 5_HT transport translocates one 5_HT molecule.…”

“…Novel models of such transport systems have been generated by transfecting human genomic DNA into mouse fibroblast L_M cells, resulting in clonal cell lines each of which stably manifests a high-affinity neurotransmitter uptake system (Chang et al 1989). This approach has yielded two clonal cell lines, L_S1 and L_S2, which possess physiological and pharmacological characteristics of 5_ [ÅH]HT transport that are comparable to other systems of 5_HT transport (Frnka et al 1991;Chang et al 1993).…”

“…HT transport by L_S1 cells revealed that the uptake activity was temperature dependent and less active at 4°C than at 37°C (Chang et al 1989), suggesting that, at the lower temperature, 5_[ÅH]HT was probably bound to the surface of L_S1 cells and not internalized by membrane translocation. Thus, low temperature incubation was further examined as a feasible means of quantifying 5_[ÅH]HT binding to the transport complex.…”

“…Two clonal transfectants (L-S1 and L_S2) generated by this strategy exhibit high-affinity 5_HT uptake systems with functional characteristics highly comparable to those previously discerned from the CNS and blood platelets, as well as from mammalian cells heterologously expressing the cloned 5_HT transporter gene (Chang et al 1989(Chang et al , 1996Frnka et al 1991). In particular, these transfectant transport systems have been shown to be dependent on the extracellular presence of both Na¤ and Cl¦ ions, akin to other 5_HT transport systems, but the molecular means whereby these ions facilitate the overall transport cycle awaits identification.…”

“…Previously, a gene transfer strategy was described for the introduction of transmitter-specific, high-affinity transport systems into cultured mouse L_M fibroblast cells (Chang et al 1989). Two clonal transfectants (L-S1 and L_S2) generated by this strategy exhibit high-affinity 5_HT uptake systems with functional characteristics highly comparable to those previously discerned from the CNS and blood platelets, as well as from mammalian cells heterologously expressing the cloned 5_HT transporter gene (Chang et al 1989(Chang et al , 1996Frnka et al 1991).…”