Insights into the Drosophila Serotonin Transporter: An Ionic Switch as a Regulator of Transporter Conformation

Monoamine concentrations in the synaptic cleft are primarily regulated by reuptake through monoamine transporters (MATs) located in presynaptic terminals and axonal membranes. In the serotonin transporter (SERT), the canonical alternating-access cycle is mediated by Na+, Cl−, and the substrate in a 1:1:1 stoichiometry. However, structural studies of human SERT have revealed a second Na+ binding site, whose precise role in the transport cycle remains unresolved. The influence of this additional sodium ion on intermediate states and the overall energy landscape of the MAT function is poorly understood. Here, we provide a comprehensive characterization of the conformational consequences of differential Na+ occupancy in the serotonin transporter of Drosophila melanogaster, a genetically tractable model for human disease. Using homology modeling, molecular docking, and molecular dynamics simulations, we show that occupation of only the NaS1 site (C1) favors a dynamically open, intracellularly accessible ensemble, whereas dual occupancy of NaS1 and NaS2 (C2) stabilizes a more compact transporter with the principal cavity opening redirected toward the extracellular face. These findings suggest a model in which NaS2 occupancy could act as a regulator of DmSERT conformational dynamics, switching the accessibility of the cavity and remodeling the local interaction network within the S1 pocket. From a translational perspective, the globally conserved ionic-coupling logic, combined with specific divergences in the S1 pocket (D164/A169 and M167/I172), contributes to define a structural window for the rational design of potential species-selective ligands.