Mechanistic analyses of ion dependences in a high-affinity 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 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. 2. 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. 3. 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. 4. 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. 5. 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.
Chang
A S
AS
The Centre for Biotechnology, Baylor College of Medicine, 4000 Research Forest Drive, The Woodlands, TX 77381, USA. achang@research.mhmc.org
Lam
D M
DM
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007099:Imipramine / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D008959:Models, Neurological; D009419:Nerve Tissue Proteins; D014179:Neurotransmitter Uptake Inhibitors / Q000378:metabolism; D011869:Radioligand Assay; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D012964:Sodium / Q000502:physiology; D014162:Transfection / Q000235:genetics
D002352:Carrier Proteins; D002712:Chlorides; D008024:Ligands; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D014179:Neurotransmitter Uptake Inhibitors; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493231:Slc6a4 protein, mouse; D012701:Serotonin; D012964:Sodium; D007099:Imipramine