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

Abstract 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 bridge the structural and functional understanding of SERT by incorporating the fluorescent non-canonical amino acid Anap through genetic code expansion. We were able to elucidate the steady-state and time-resolved conformational dynamics of purified SERT with Anap genetically encoded at the intracellular- or extracellular site. This uncovered the competitive mechanisms underlying cation binding and assigned unique binding- and allosteric coupling signatures for several inhibitors and substrates. Finally, we tracked in real-time the conformational transitions in response to the interaction with Na+ and substrate. Our methodological platform offers an unprecedented spatiotemporal resolution which enables us to resolve novel mechanistic features of SERT.