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

Abstract

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.

Figure 1. Scatchard plots of transport-specific 5-[³H]HT binding to intact L-S1 cells

The concentration of 5-[³H]HT ranged from 0.1 to 0.5 μm. Specific binding levels (‘B’) were measured as displacement of total binding by either 100 nm (near the IC₅₀ value) or 100 μm imipramine (fully inhibitory concentration). Each point is the mean of six measurements.

Figure 2. Scatchard plots of [³H]imipramine binding to L-S1 cells

Specific binding levels were defined as displacement of total [³H]imipramine binding by 1 μm (IC₅₀) or 100 μm 5-HT (fully inhibitory concentration); concentration of [³H]imipramine ranged from 0.5 to 100 nm. Each point is the mean of six measurements.

Figure 3. Dependence of 5-[³H]HT binding on Na⁺ and Cl⁻

A, sodium dependence of 5-[³H]HT binding. Levels of specific binding (‘B’) were measured at both 0.1 and 0.5 μm of ³H-ligand, and with varying [Na⁺]_o (by isotonic substitution with Li⁺) and constant [Cl⁻]_o of 137.4 mm. Analogous substitutions with K⁺, Cs⁺, Rb⁺ or choline all led to comparable findings to substitutions with Li⁺. B, chloride dependence of 5-[³H]HT binding. Specific binding levels (‘B’) were measured at both 0.1 and 0.5 μm 5-[³H]HT with varying [Cl⁻]_o (by isotonic substitution with isethionate ion) and constant [Na⁺]_o of 128 mm. Analogous substitutions by F⁻, Br⁻ or I⁻ also led to comparable findings. Non-specific binding levels were assessed by inclusion of 100 μm imipramine. Each point is the mean ±s.d. of six values.

Figure 4. Scatchard plots of specific 5-[³H]HT binding with varying [Cl⁻]_o

Using isethionate substitution, [Cl⁻]_o at 6.8 % (9.4 mm), 37.9 % (52.1 mm) and 100 % (137.4 mm) were used for quantifying specific binding at 5-[³H]HT concentrations ranging from 0.1 to 0.5 μm. Non-specific binding levels were assessed using 100 μm imipramine. Each point is the mean of six measurements.

Figure 5. Sodium dependence of 5-HT uptake

A, initial velocity profile of 5-[³H]HT (0.1 μm) uptake as a function of [Na⁺]_o. External [Na⁺] was changed by isotonic replacement with Li⁺. A Na⁺-independent component of transport was evident at 0 mm [Na⁺]_o. Removing this Na⁺-independent component gives a velocity profile characterized with unitary Hill coefficient. B, relationship between 1/K_m,obs and [Na⁺]_o. The linearity of this relationship suggests that model 3 is most consistent with experimental findings. The K_m,obs values were attained from kinetic analyses (using 50 nm to 10 mm 5-[³H]HT) conducted with 100, 33.3 and 0 % of the physiological [Na⁺]_o level. Lineweaver-Burk analyses of the results of such kinetic analyses revealed invariant V_max,obs but changing K_m,obs as a function of [Na⁺]_o. Each point is the mean ±s.d. of six measurements.

Figure 6. Chloride dependence of 5-HT uptake

A, initial velocity profile of 5-[³H]HT (0.1 μm) uptake as a function of [Cl⁻]_o. External [Cl⁻] was changed by isotonic replacement with isethionate. No Cl⁻-independent component of transport was evident, and the velocity profile was characterized with a unitary Hill coefficient. B, relationship between 1/K_m,obs and [Cl⁻]_o. The hyperbolic nature of this relationship and its intersection with the origin suggests that model 2 is most consistent with experimental findings. The K_m,obs values were attained from kinetic analyses (using 50 nm to 10 mm 5-[³H]HT) conducted with 100, 37.9 and 6.8 % of the physiological [Cl⁻]_o level. Lineweaver-Burk analyses of the results of such kinetic analyses revealed invariant V_max,obs but changing K_m,obs as a function of [Cl⁻]_o. Each point is the mean ±s.d. of six analyses.