Mechanistic analyses of ion dependences in a high-affinity human serotonin transport system in transfected murine fibroblast cells
Albert S. Chang and Dominic M.-K. Lam
The Centre for Biotechnology, Baylor College of Medicine, 4000 Research Forest Drive, The Woodlands, TX 77381, USA
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.
