Dissecting the Catalytic Cycle of the Serotonin Transporter

Abstract

The plasmalemmal serotonin transporter (SERT) regulates serotonin homeostasis and signaling by its reuptake from the extracellular space. In accordance with this prominent role, it is a target of several psychoactive substances, ranging from inhibitors (e.g. cocaine) to substrates (e.g. amphetamines). SERT belongs to the solute carrier 6 family. Hence, it utilizes the electrochemical gradient of co-substrates for the uphill transport of serotonin into the cell. Interestingly, the stoichiometry of this secondary-active transport mechanism has been matter of debate - despite decades of investigation. In addition, the order and kinetics of (co-)substrate binding to SERT have remained enigmatic. Here, we utilized the high temporal resolution of the whole-cell patch-clamp technique to decipher the kinetic determinants of selective ligand recognition, and of (co-)substrate binding and transport - in both, the forward transport and substrate exchange mode of SERT. Based on our electrophysiological data, we provide a comprehensive kinetic model of SERT that accounts for kinetics, stoichiometry, and order of (co-)substrate binding and translocation. We find that, Cl- does not participate in coupling of serotonin transport, but is required for substrate binding. In addition, our data suggest that two Na+ ions bind to SERT in a sequential order (Na+-serotonin-Na+). These findings are incompatible with an electroneutral stoichiometry. Our data shall provide a mechanistic framework for future attempts to integrate functional data with available structural information.