Fig. 4: Identifying the location of S2 site of human DAT.

a Cells expressing wtDAT were treated with 3 mM sulfo-NHS-acetate (SNA) for 30 min to acetylate K92 and K384 of DAT. After the treatment, the population of the weaker unbinding events decreased substantially (dashed blue curve in b, measured in the absence of Zn²⁺, at pulling speed of 6 µm/s). For further identification, DAT mutants with single mutation K92A or K384A were measured, both of which reduced the population of weaker unbinding events (green and magenta thin solid curves in b). c The pH value of the buffer was reduced from 7.4 to 5.5 to examine the role of H477 in supporting binding to the S2. Histidine is the only amino acid residue with a pKa value within this pH range and there is no histidine at S1 site (Supplementary Table 1). The drop in pH reduction has a markedly more pronounced effect on unbinding from the S2 site (the first peak around 12 pN of the red solid line in c, measured in the absence of Zn²⁺, at scanning speed of 1.5 µm/s) than from the S1 site (the second peak around 20 pN). d Kinetic on-rate at the S1 site of untreated wtDAT is significantly higher than those of DATs changed at K92, K384, or H477, indicating that the S2 site helps dopamine enter the S1 site. e Kinetic off-rate at the S2 site is much higher than that at the S1 site. (One-tailed t-test with equal variance assumed, n = 3–8 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). Source data are provided as a Source Data file.