Crystal structures and experiments relying on the tools of molecular pharmacology reported conflicting results on ligand binding sites in neurotransmitter/sodium symporters (NSS). We explored the number and functionality of ligand binding sites of NSS in a physiological setting by designing novel tools for atomic force microscopy (AFM). These allow for directly measuring the interaction forces between the serotonin transporter (SERT) and the antidepressant S-citalopram (S-CIT) on the single molecule level: the AFM cantilever tips were functionalized with S-CIT via a flexible polyethylene glycol (PEG) linker. The tip chemistry was validated by specific force measurements and recognition imaging on CHO cells. Two distinct populations of characteristic binding strengths of S-CIT binding to SERT were revealed in Na⁺-containing buffer. In contrast, in Li⁺-containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT. The dissociation rate constant of both binding sites was extracted by varying the dynamics of the force-probing experiments. Competition experiments revealed that the two sites are allosterically coupled and exert reciprocal modulation.

依赖于分子药理学工具的晶体结构和实验报告了神经递质/钠同向转运体 (NSS) 中配体结合位点的相互矛盾的结果。我们通过设计用于原子力显微镜 (AFM) 的新型工具，探索了生理环境中 NSS 配体结合位点的数量和功能。这些允许在单分子水平上直接测量血清素转运蛋白 (SERT) 和抗抑郁药 S-西酞普兰 (S-CIT) 之间的相互作用力：AFM 悬臂尖端通过柔性聚乙二醇 (PEG) 用 S-CIT 功能化链接器。通过对 CHO 细胞的比力测量和识别成像验证尖端化学。^{在 Na +}中揭示了 S-CIT 与 SERT 结合的两个不同群体的特征结合强度-含缓冲区。相反，在含有 Li ⁺的缓冲液中，SERT 仅显示出低力相互作用。相反，前庭突变体 SERT-G402H 仅显示高强度群体。这些观察结果为 SERT 中存在两个结合位点提供了物证。通过改变力探测实验的动力学来提取两个结合位点的解离速率常数。竞争实验表明，这两个位点变构耦合并施加相互调制。