Fig. 4: Centanafadine binding pocket of DAT.

a Overall structure of DAT in complex with centanafadine depicted in cartoon representation with two different side views. Centanafadine is illustrated as deep green spheres, while the TMs are represented as lemon yellow cartoons. b The cryo-EM density of centanafadine, along with surrounding residues, is displayed as sticks. The cryo-EM density of centanafadine in the contour level of 0.25 in ChimeraX. c The binding pocket of centanafadine in DAT, viewed from the extracellular side. The key residues in the pocket are shown as sticks or spheres and labeled. d Concentration–inhibition curves of [³H]dopamine uptake by wild-type and indicated mutants of DAT with specified concentrations of centanafadine in the range of 0.1 nM to 100 μM. The IC₅₀ values of centanafadine are: WT, 116.1 nM; V152A, 246.1 nM; and F326A, 2094 nM. Data are mean ± s.e.m. (n  =  3 biologically independent experiments). [³H]Dopamine uptake was normalized to uptake in the absence of centanafadine. Significant differences in IC₅₀ value were observed for V152A (P  =  0.0290) and F326A (P  = 0.0043) compared with WT. Two-sided unpaired t-test (threshold, α = 0.05). e Schematics generated by Ligplot illustrate the interaction between centanafadine and DAT, with the green line denoting hydrogen bond. The TMs are distinguished by background colors, enhancing visualization of the interaction landscape. Conserved residues across MATs are indicated in red, while non-conserved residues are shown in black. f Structural comparison of the binding pocket between DAT^cen in the inward-facing conformation and DAT^DA in the occluded conformation. Centanafadine, dopamine, and key residues are shown as sticks and marked.