Figure 4: Crosslinking studies and binding site determination of vortioxetine and imipramine.

(a) Schematic illustration outlining the photocrosslinking experiments. Briefly, HEK293T cells expressing WT or azF mutants of hSERT were incubated with 100 nM of [³H]vortioxetine or [³H]imipramine and exposed to ultraviolet (UV) light. Cells were lysed, the transporter immunopurified (IP) and run on an SDS–PAGE gel, before transferring to PVDF membrane for immunoblotting (IB). Each lane of the membrane was cut into three segments (20–50, 50–100 and >100 kDa, respectively) and the radioactivity of each segment was quantified. As control, non-UV-exposed samples were in all cases assayed in parallel. (b,c) Results from the photocrosslinking experiments between hSERT azF mutants, and [³H]imipramine (b) and [³H]vortioxetine (c). The bars represent the radioactivity signal expressed as counts per minute (c.p.m.) detected in PVDF membrane segments corresponding to 50–100 kDa that contain hSERT. For the 20–50 and >100 kDa segments, the radioactivity signal was in all cases indistinguishable between UV-exposed and control samples (Supplementary Fig. 4). Asterisks (*) denotes significant difference in crosslinking signal compared with non-UV-exposed samples (P<0.05; two-way analysis of variance with Sidak multiple comparisons test). Inserts show that crosslink of [³H]imipramine to Y95azF (b) and [³H]vortioxetine to F341azF (c) was fully outcompeted in the presence of 100 μM unlabelled inhibitor added before UV exposure. Data are represented as mean±s.e.m. from three to seven independent experiments. (d,e) Docking models of imipramine⁵¹ (d) and vortioxetine⁴² (e) binding in the S1 site in hSERT. Imipramine is shown in green (d), vortioxetine is shown in yellow (e) and the five S1 residues that were mutated to azF are shown as orange sticks.