Two Ligand Binding Sites in Serotonin Transporter Revealed by Nanopharmacological Force Sensing

The number of ligand binding sites in neurotransmitter-sodium symporters has been determined by crystal structure analysis and molecular pharmacology with controversial results. Here, we designed molecular tools to measure the interaction forces between the serotonin transporter (SERT) and S-citalopram on the single-molecule level by means of atomic force microscopy. Force spectroscopy allows for the extraction of dynamic information under physiological conditions which is inaccessible via X-ray crystallography. Two populations of distinctly different binding strength between S-citalopram and SERT were demonstrated in Na⁺-containing buffer. In Li⁺-containing buffer, SERT showed merely low-force interactions, whereas the vestibular mutant SERT-G402H only displayed the high force population. These observations provide physical evidence for the existence of two different binding sites in SERT when tested under near-physiological conditions.