Figure 9.

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 k_off 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 k_off. The data are means ± SD from 13 independent recordings for both conditions. H, the values for k_app were extracted from panels C and D and plotted as a function of methylphenidate (MPH) concentrations. Dissociation rates (k_off) were taken from panel G to constrain the y-intercepts of the lines in the linear regression. The k_on 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 (k_on) 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.