Conformational changes are fundamental events in the transport mechanism. The serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin after its release by serotonergic neurons and is the molecular target for antidepressant drugs and psychostimulants. Despite significant progress in characterizing the structure–function relationship of SERT, its conformational mechanism has not been fully understood. We present here a cell-based method for determining conformational changes in SERT with its fluorescent substrates by fluorescence imaging analysis. This method fluorometrically measures accessibility of strategically positioned cysteine residues in the substrate permeation pathway to calculate the rate constants of reactivity with MTS reagents in live or permeabilized cells. We validated this method by investigating ligand and ion-induced conformational changes in both the extracellular and cytoplasmic pathways of SERT. Furthermore, we applied this method for examining the influence of Cl⁻ binding and vilazodone inhibition on SERT conformation. Our results showed that Cl⁻ ion, in the presence of Na⁺, facilitates the conformational conversion from outward to inward open states, and that vilazodone binding stabilizes SERT in an outward open and inward-closed conformation. The present work provided insights into the conformational mechanism of SERT and also indicated that the cell-based fluorometric method is robust, straightforward to perform, and potentially applicable to any monoamine transporters in exploring the transport mechanism and mechanism of action of therapeutic agents for the treatment of several psychiatric disorders.

The neurotransmitter sodium symporters (NSSs) play critical roles in the neurotransmission of monoamine and amino acid neurotransmitters and are the molecular targets of therapeutic agents in the treatment of several psychiatric disorders. Despite significant progress in characterizing structures and transport mechanisms, the management of conformational transitions by structural elements coupled with ion and substrate binding remains to be fully understood. In the present study, we biochemically identified a conserved GX₉P motif in the fifth transmembrane domain (TM5) of the serotonin transporter (SERT) that plays a vital role in its transport function by facilitating conformational transitions. Mutations of the conserved Gly278 or Pro288 in the GX₉P motif dramatically decreased specific transport activity by reducing the substrate binding-induced conformational transitions from an outward-open to an inward-open conformation. In addition, cysteine accessibility measurements demonstrated that the unwinding of the intracellular part of TM5 occurs during conformational transitions from an outward-open state, through an occluded state, to an inward-open state and that substrate binding triggers TM5 unwinding. Furthermore, mutations of the GX₉P motif were shown to result in destructive effects on TM5 unwinding, suggesting that the GX₉P motif controls conformational transitions through TM5 unwinding. Taken together, the present study provides new insights into the structural elements controlling conformational transitions in NSS transporters.