Fig 2: Kinetics of [3H]citalopram binding to SERT in the presence of 8B6 scFv, 15B8 Fab, or antiSERT-EL4 antibody.A, dissociation experiment: membranes harboring GFP-tagged SERT (1 µg/assay) were incubated with [3H]citalopram (6 nM) in the presence of 8B6 scFv (150 nM), 15B8 Fab (150 nM), or antiSERT-EL4a antibody (44 nM) in a final volume of 10 µl. After 10 min at 25 °C, the dissociation was initiated by 100-fold dilution of the reaction in buffer containing the same concentration of 8B6 scFv, 15B8 Fab, or antiSERT-EL4 antibody. Data are means ± SD from three independent experiments carried out in duplicate. The lines were drawn by fitting the data to the equation for a monoexponential decay. B, dissociation of [3H]citalopram in the presence of 10 µM S-citalopram. The experiment was done as in panel A with 5 nM [3H]citalopram, but 10 µM S-citalopram was present in in the dilution buffer to occupy the S1 site and to thereby slow dissociation of prebound [3H]citalopram. The parallel incubations were done in the absence of S-citalopram. These are not shown for the sake of clarity, but the dissociation rate constants were identical to those determined in panel A. Data are means ± SD from three independent experiments carried out in duplicate. C, association experiment: membranes harboring GFP-tagged SERT (1 µg/assay) were preincubated with 8B6 scFv (150 nM), 15B8 Fab (150 nM), or antiSERT-EL4 antibody (44 nM) for 10 min at 25 °C. Thereafter the binding reaction (final volume 0.1 ml) was initiated by the addition of [3H]citalopram (7.8 nM and 11.4 nM) and terminated by rapid filtration at the indicated time points, bound to membranes prior to the binding reaction. The lines were drawn by fitting the data to the equation for a monoexponential association. Data are means from duplicate determinations in a representative experiment, which was carried out in parallel. D–F, the apparent association rates (kapp ± SE) were obtained from experiments done as shown in panel C and plotted as a function of the [3H]citalopram concentration plots. Under control conditions (D), i.e., in the absence of antibody/Fabs, the resulting plot was better described by the sum of a rectangular hyperbola and a basal term (= koff) than by a straight line (p = 0.02; F-test based on the extra-sum-of-squares principle). In contrast, in the presence of antiSERT-EL4 antibody (E), 8B6 scFv (F), and 15B8 Fab (G), the linear regression (with koff as y-intercept and kon as slope) was an adequate and parsimonious description because there was not any improvement in the fit by assuming a hyperbolic relation.

Fig 3: Binding of [3H]citalopram to SERT in the presence of 8B6 scFv, 15B8 Fab, or of the antiSERT-EL4 antibody.A, concentration–response curve for 8B6 scFv, 15B8 Fab, and antiSERT-EL4 antibody in enhancing binding of [3H]citalopram to SERT. The experiments were carried out as outlined for [3H]imipramine binding in Figure 6C. The concentration of [3H]citalopram was 10 nM. The concentrations were 9.5, 19, 37.5, 75, and 150 nM for both 8B6 scFv and 15B8 Fab (denoted on the top x-axis), whereas the antiSERT-EL4 antibody concentrations were 2.7, 5.5, 11, 22, and 44 nM (denoted on the bottom x-axis). Data are means ± SD from three independent experiments carried out in duplicate. The lines were drawn by fitting the data to the equation for a rectangular hyperbola + basal binding in the absence of Fab/antibody. EC50 values were 14.7 ± 2.3 nM, 11.2 ± 2.5 nM, and 5.0 ± 2.7 nM for 8B6 scFv, 15B8 Fab, and antiSERT-EL4, respectively. B, saturation of [3H]citalopram binding to SERT in the absence and presence of 8B6 scFv (150 nM), 15B8 Fab (150 nM), or antiSERT-EL4 (44 nM). The reaction was done as in panel A with [3H]citalopram concentrations ranging from 0.4 to 36 nM. Shown are the pooled data from three experiments carried out in duplicate. The lines were drawn by fitting the data to a rectangular hyperbola. C, the spaghetti plot depicts the change in Bmax of [3H]citalopram binding in the presence of 8B6 scFv, 15B8 Fab, or antiSERT-EL4 antibody in three independent experiments carried out in duplicate (statistical significance was tested by repeated-measures ANOVA, followed by Holm–Sidak's multiple comparisons; Bmax in the presence of 8B6 scFv, 15B8 Fab, or of antiSERT-EL4 antibody differed from control Bmax, p < 0.005).

Fig 4: Binding of [3H]imipramine to SERT in the absence and presence of 8B6 scFv or of the antiSERT-EL4 antibody.A, concentration–response curve for 8B6 scFv and antiSERT-EL4 antibody in enhancing binding of [3H]imipramine to SERT. The reaction was done in a final volume of 0.2 ml containing membranes from HEK293 cells stably expressing GFP-tagged SERT (2 μg). 8B6 scFv (9.5, 19, 37.5, 75, and 150 nM, denoted on the top x-axis) and antiSERT-EL4 (2.7, 5.5, 11, 22, and 44 nM, denoted on the bottom x-axis) were preincubated with the membranes for 30 min at 25 °C. The binding reaction was carried out in the presence of 10 nM [3H]imipramine for 10 min at 25 °C, as outlined under Materials and Methods. Data are means ± SD from three independent experiments carried out in duplicate. The lines were drawn by fitting the data to the equation for a rectangular hyperbola + basal binding in the absence of Fab/antibody. EC50 values for 8B6 scFv and antiSERT-EL4 antibody were 14.9 ± 2.0 nM and 4.9 ± 0.6 nM, respectively. B, saturation of [3H]imipramine binding to SERT in the absence and presence of 8B6 scFv (150 nM) or antiSERT-EL4 (44 nM). The reaction was done as in panel A with [3H]imipramine concentrations ranging from 0.2 to 36 nM. Shown is a representative experiment performed in duplicate. The lines were drawn by fitting the data to a rectangular hyperbola. C, the spaghetti plot depicts the change in Bmax of [3H]imipramine binding in the presence of 8B6 scFv or antiSERT-EL4 antibody in three independent experiments carried out in duplicate (statistical significance was tested by repeated-measures ANOVA, followed by Holm–Sidak's multiple comparisons; Bmax in the presence of 8B6 scFv or of antiSERT-EL4 antibody differed from control Bmax, p < 0.05).

Fig 5: Electrophysiological approach to measure the kinetics of methylphenidate (MPH) binding to SERT in the absence and presence of the 15B8 Fab.A and B, time course for peak current inhibition by MPH in the absence (A) and presence of 15B8 Fab (B). HEK293 cells stably expressing GFP-tagged SERT were superfused with 30 μM 5-HT to elicit the reference peak current and subsequently superfused with 100 μM methylphenidate (MPH) for 0.2, 0.5, 1, 2, 5, and 10 s in the absence (A) or presence of 300 nM 15B8 Fab (B) prior to eliciting the test peak current with 30 μM 5-HT. Peak currents were isolated (i.e., the steady-state current was suppressed) by a high concentration of Na+ (152 mM) in the internal solution in the patch pipet. Shown is the compilation of representative peak current traces from a single cell. The time course of current decay reflects the apparent association rate of methylphenidate binding and blockage of SERT. The dashed lines were drawn by fitting the decline in peak current amplitude to an equation for a monoexponential decay to a residual activity. C and D, the experiments were carried out as outlined in panels A and B with 10, 30, 100, and 300 μM methylphenidate (MPH) in the absence (C) and presence of 300 nM 15B8 Fab 8 (D). The peak current reduction was normalized by dividing test currents by the reference current and plotted as a function of time. Data are means ± SD from three to seven independent recordings for each concentration of methylphenidate. E and F, time course of peak current recovery after methylphenidate washout. HEK293 cells stably expressing GFP-tagged SERT were superfused with 100 μM methylphenidate (MPH) for 5 s in the absence (E) or presence (F) of 300 nM 15B8 Fab. Then, the peak currents were elicited by rapid application of 30 μM 5-HT with 0, 0.2, 0.5, 1, 2, 5, and 10 s delay to observe its recovery. The dashed lines denote the monoexponential fit for peak current recovery resulting from the dissociation of methylphenidate. The koff of methylphenidate was extracted from this fit. G, the time course of the peak current recovery after washout of methylphenidate (MPH) was determined as depicted in Figure 9, E and F in the absence of SERT (black) and presence of 15B8 Fab-bound (purple). The solid curves were generated by fitting the data to the equation for a monoexponential rise from a basal residual peak current to obtain koff. The data are means ± SD from 13 independent recordings for both conditions. H, the values for kapp were extracted from panels C and D and plotted as a function of methylphenidate (MPH) concentrations. Dissociation rates (koff) were taken from panel G to constrain the y-intercepts of the lines in the linear regression. The kon of methylphenidate from the slope of the lines in the absence (black: control) and presence of 15B8 Fab (300 nM, purple). The rates for methylphenidate association (kon) in the absence and presence of 15B8 Fab were 8.95 ± 1.03 × 103 M−1 s−1 and 1.96 ± 0.06 × 104 M−1 s−1, respectively.