Fluorescent non-canonical amino acid provides insight into the human serotonin transporter.

The serotonin transporter (SERT), responsible for the reuptake of released serotonin, serves as a major target for antidepressants and psychostimulants. Nevertheless, refining the mechanistic models for SERT remains challenging. Here, we expand the molecular understanding of the binding of ions, substrates, and inhibitors to SERT by incorporating the fluorescent non-canonical amino acid Anap through genetic code expansion. We elucidate steady-state changes in conformational dynamics of purified SERT with Anap inserted at intracellular- or extracellular sites. This uncovers the competitive mechanisms underlying cation binding and assigns distinct binding- and allosteric coupling patterns for several inhibitors and substrates. Finally, we track in real-time conformational transitions in response to the interaction with Na or serotonin. In this work, we present a methodological platform reporting on SERT conformational dynamics, which together with other approaches will deepen our insights into the molecular mechanisms of SERT.