Figure 5.

Kinetic model for 8B6 scFv binding to SERT during the transport cycle.A, 8B6 scFv binding was explored during different conformational states of SERT (blue: outward-facing apo, orange: outward-facing substrate-bound, green: inward-facing apo, and yellow: inward-facing substrate-bound states). Kinetic rate constants for 8B6 scFv from Fig. 4 were implemented in the model. A is the factor describing the fold-acceleration of 8B6 scFv dissociation from SERT dwelling in the low-affinity states, where the outward-facing apo state is assumed to be the only high-affinity state for 8B6 scFv. X = 8B6 scFv, S = 5-HT. B, the synthetic current traces elicited by 30 μM 5-HT were generated by the model outlined in panel A using the ionic conditions employed in Figure 1, Figure 2, Figure 3 before and after 300 nM 8B6 scFv exposure. C, comparison of experimental (dashed) and synthetic (solid) data for the normalized reduction in the peak current amplitude by binding of 8B6 scFv to SERT. Simulations were done as shown in panel B for each indicated concentration 8B6 scFv. The experimental data are from Figure 2B. The IC₅₀ calculated from the simulated data is 32.9 nM (95% confidence interval = 12.6–101.5 nM). D, compiled synthetic traces elicited by 30 μM 5-HT after 300 nM 8B6 scFv had been allowed to bind for 1, 2, 5, 10, or 15 s. The dashed line represents the monoexponential function fitted to the peak currents generated by the simulation. This curve was used to calculate k_app. E, simulations were done as shown in panel D with the indicated concentrations of 8B6 scFv to extract the time-dependent decline in normalized peak current amplitudes. The monoexponential decay curves resulting from these simulations were plotted as solid lines. For comparison, the dashed lines show the curves generated by fitting the experimental data (taken from Fig. 4C). F, the k_app values extracted from the synthetic data shown in Panel E were plotted over the corresponding concentrations of 8B6 scFv to yield a straight line (solid). The dashed line indicates the linear regression to the experimental data (taken from Fig. 4E). The slope of the line through the synthetic points yields k_on = 1.93 × 10⁶ M⁻¹∗s⁻¹ and a y-intercept corresponding to k_off = 0.20 s⁻¹. G, synthetic (left panel) and experimental traces (right panel) to highlight the modest inhibitory effect of 8B6 scFv on the 5-HT-induced steady-state current. The synthetic current traces elicited by 30 μM 5-HT and the effect of 300 nM 8B6 scFv wash-in and washout were generated by the model outlined in panel A using the ionic conditions employed in Figure 1, Figure 2, Figure 3. H, representative trace of the inhibition of the steady-state current by the simultaneous application of 8B6 scFv and 15B8 Fab using the recording conditions employed in Figure 1, Figure 2, Figure 3. I, Spaghetti plot from four independent recordings of steady-state current amplitudes done as in panel H before and after 8B6 scFv and 15B8 Fab application. J, representative trace of the apparent decrease in the membrane capacitance of HEK293 cells stably expressing GFP-tagged SERT upon consecutive binding of 15B8 Fab and 8B6 scFv. The recording condition was as in Fig. 1, E and F. The experiment was reproduced in two additional independent recordings.