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

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.

The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport

“…8C). This apparent Cl Ϫ -dependent coordination of Asp-98 to 5-HT is consistent with previously published findings, where Cl Ϫ binding to hSERT decreased the K m of 5-HT ϳ4-fold (54,66,67). In contrast, simulations with the N101A mutant with Na ϩ reveal that Asp-98 (O∂) can coordinate the amine of 5-HT even in the absence of Cl Ϫ .…”

“…This eliminates the Asp-98 -5-HT interaction, providing a rational explanation for the observed increase in the K m for 5-HT. Likewise, the absence of a cation at the Na1 site does not alter 5-HT binding (66), suggesting that, in the absence of Na ϩ , the carboxyl side chain of Asp-98 is available to stabilize 5-HT binding, although it does not lead to productive transport. Therefore, we propose that upon 5-HT binding, Asp-98 transitions from bidentate to monodentate coordination of Na ϩ at Na1 and that this change in coordination is critical to initiate transport.…”

“…Uptake studies have shown that, in the absence of Cl Ϫ , the K m for 5-HT transport is increased significantly, indicating that Cl Ϫ binding alters the apparent affinity of 5-HT for hSERT (54,66,67). Our MD studies link this positive impact of Cl Ϫ on the K m of 5-HT to the residue Asp-98.…”

A Conserved Asparagine Residue in Transmembrane Segment 1 (TM1) of Serotonin Transporter Dictates Chloride-coupled Neurotransmitter Transport

“…1). As described previously (44), removal of external Cl Ϫ from the medium of hSERT-transfected cells resulted in a significant (5-fold) decrease in 5-HT transport V max (NaCl, 0.068 Ϯ 0.006 fmol/cell/min; sodium gluconate, 0.014 Ϯ 0.002) and a significant (3.6-fold) increase in 5-HT K m (NaCl, 0.9 Ϯ 0.2 M; sodium gluconate, 3.2 Ϯ 0.36 M, one-way ANOVA, Dunnett's post hoc, p Ͻ 0.002). Remarkably, the hSERT N101A and N101C mutants were largely insensitive to Cl Ϫ substitution with gluconate ( Fig.…”

Catechol‐O‐methyltransferase activity in CHO cells expressing norepinephrine transporter

“…However, it is unclear what kind of degradative pathways is present in these cell lines used for transfection studies and, if it exists, to what extent the ‘apparent function’ of catecholamine transporters expressed in these cell lines would be altered. While the majority of studies routinely include MAO inhibitors (Usdin et al ., 1991; Galli et al ., 1996; Eshleman et al ., 1997; Chang & Lam, 1998), the potential influence of COMT activity within the cells has been largely ignored.…”