Fig. 3: Dopamine binds to two sites of human DAT.

a The dopamine molecule was covalently conjugated to the AFM cantilever tip via NHS-PEG-maleimide linker (Supplementary Fig. 8a). Two populations of unbinding events, red solid lines, measured in the absence of Zn²⁺, (at scanning speed of 1.5 µm/s) were observed from repeatedly recorded force curves. Substitution of Na⁺ in the buffer by K⁺ (b) or NMDG (c) diminished the population of the stronger binding events (around 20 pN), indicating that the stronger binding events originate from the central S1 site, which is sodium dependent. Mutation within the S1 site of DAT (V152I) also reduced the population of stronger binding events (d) while the overall binding activity is preserved. Mutation in the extracellular vestibule (G386H) results in weaker binding (shift of the first force peak to the left) but does not affect the binding force at the S1 site e. Source data are provided as a Source Data file.