MECHANISTIC ANALYSES OF ION DEPENDENCES IN A HIGH-AFFIINITY HUMAN SEROTONIN TRANSPORT-SYSTEM IN TRANSFECTED MURINE FIBROBLAST CELLS

1. A clonal cell line, L-S1, has been identified from transfection of human genomic DNA into cultured mouse L-M fibroblasts. Because this tr ansfectant cell line stably expresses a high-affinity serotonin (5-HT) transport mechanism with kinetic and pharmacological properties compa rable to those of other serotonin uptake systems, it was used to inves tigate the mechanistic involvement of Na+ and Cl- ions in the ligand b inding and kinetic uptake processes of this system. 2. Intact transfec tant cells, when incubated at low temperature (4 degrees C), enabled q uantitative assessment of imipramine-displaceable 5-[H-3]HT binding to the 5-HT transport system. This binding activity is insensitive to th e presence of various ligands specific for 5-HT receptor subtypes. 3. Imipramine-displaceable 5-[H-3]HT binding to intact L-S1 cells was sho wn to be a Cl--dependent but Na+-independent process. Chloride ions la ck binding co-operativity in facilitating ligand binding. Changes in e xternal Cl- concentration altered the K-d but not the B-max of binding . 4. The overall transport activity was observed to be highly dependen t on both external Na+ and Cl- concentrations, characterized by a 5-HT :Na+:Cl- coupling ratio of 1 : 1 : 1 per transport cycle. Alterations in the external concentrations of both Na+ and Cl- ions altered only t he K-m and not the V-max of transport. 5. Both binding and kinetic res ults are consistent with kinetic modelling predictions of the Cl- ion in facilitating 5-HT binding to the transport system, and of the Na+ i on in enabling translocation of bound 5-HT across the plasma membrane. Thus, Na+ and Cl- ions facilitate mechanistically distinct and discer nible functions in the transport cycle.