Fig. 1: The cocaine analogue MFZ2-12 binds to two sites in human DAT.

a The drug molecule MFZ 2-12, was covalently conjugated to the AFM cantilever tip via its N-ethylamino derivative and an azido-PEG-NHS linker (Supplementary Fig. 1). b The force measurements (Supplementary Fig. 2) revealed that, in the presence of 10 µM Zn²⁺, the binding activity of MFZ2-12 to DAT is significantly higher than that in the absence of Zn²⁺ (one-tailed t-test with equal variance assumed, n = 5 cells. The box range indicates the standard deviation, the small square in the box indicates the mean value, the full width bar indicates the median value, the whisker indicates the range of outlier with coefficient equals to 1). c Two populations of unbinding events were observed from repeatedly recorded force curves. Ten example curves are shown for each. The noise in the force curves originates from thermal fluctuation of the cantilever with a mean value of 0.6 nm (corresponding equation and calculation in the Methods section). d Statistic probability density functions (PDF) of the unbinding force show two peaks on cells expressing DAT, whereas the PDF on CHO cells without DAT has much lower binding probability (area under the curve) and a single peak different from those on DAT. All PDF measurements were conducted on at least two biological replicas with very similar results. e A mutation within the S1 site of DAT (S422A) abolished the second peak (measured in the presence of 10 µM Zn²⁺) in the force PDF. f Substitution of Na⁺ with Li⁺ reduced the population of stronger unbinding events of the wtDAT. Measurements in panel d, e, and f were performed at pulling speed of 6 µm/s. Within each panel, the same cantilever was used to compare different condition. For different panels, the cantilevers are different. Source data are provided as a Source Data file.