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

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Abstract
A clonal cell line, L‐S1, has been identified from transfection of human genomic DNA into cultured mouse L‐M fibroblasts. Because this transfectant cell line stably expresses a high‐affinity serotonin (5‐HT) transport mechanism with kinetic and pharmacological properties comparable to those of other serotonin uptake systems, it was used to investigate the mechanistic involvement of Na+ and Cl− ions in the ligand binding and kinetic uptake processes of this system. Intact transfectant cells, when incubated at low temperature (4 °C), enabled quantitative assessment of imipramine‐displaceable 5‐[3H]HT binding to the 5‐HT transport system. This binding activity is insensitive to the presence of various ligands specific for 5‐HT receptor subtypes. Imipramine‐displaceable 5‐[3H]HT binding to intact L‐S1 cells was shown to be a Cl−‐dependent but Na+‐independent process. Chloride ions lack binding co‐operativity in facilitating ligand binding. Changes in external Cl− concentration altered the Kd but not the Bmax of binding. The overall transport activity was observed to be highly dependent 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 the Km and not the Vmax of transport. Both binding and kinetic results are consistent with kinetic modelling predictions of the Cl− ion in facilitating 5‐HT binding to the transport system, and of the Na+ ion in enabling translocation of bound 5‐HT across the plasma membrane. Thus, Na+ and Cl− ions facilitate mechanistically distinct and discernible functions in the transport cycle.