Ligand coupling mechanism of the human serotonin transporter differentiates substrates from inhibitors

Abstract

Abstract The presynaptic serotonin (5HT) transporter (SERT) clears extracellular 5HT following vesicular release to ensure temporal and spatial regulation of serotonergic signalling and neurotransmitter homeostasis. Clinically approved drugs used for the treatment of neurobehavioral disorders, including depression, anxiety, and obsessive-compulsive disorder that target SERT trap the transporter in the outward-open state thus blocking the transport cycle. In contrast, illicit drugs of abuse like amphetamines reverses SERT directionality, thereby causing 5HT efflux. Both result in an increase of extracellular 5HT levels. Stoichiometry of the transport cycle has been described by kinetic schemes, whereas the structures of the main conformations provide only static coordinates of molecular features of the process. By combining in-silico molecular dynamics modelling approaches with in-vitro and ex-vivo biochemical experiments and making use of a homologous series of 5HT analogues, we uncovered the essential coupling mechanism between the substrate and the transporter triggering the uptake process. The free energy calculations showed that only scaffold-bound substrates can correctly close the extracellular gate. Attractive forces acting on the bundle domain through long-range electrostatic interactions tilt the bundle domain towards the scaffold domain. The associated spatial requirements define substrate and inhibitor properties, enabling new possibilities for rational drug design approaches.