Identification of the potassium binding site in serotonin transporter SERT

Clearance of serotonin (5-hydroxytryptamine, 5-HT) from the synaptic cleft after neuronal signaling is mediated by serotonin transporter SERT, which couples this process to the movement of a Na+ion down its chemical gradient. After release of 5-HT and Na+into the cytoplasm, the transporter faces a rate-limiting challenge of resetting its conformation to be primed again for 5-HT and Na+binding. Early studies of vesicles containing native SERT revealed that K+gradients can provide an additional driving force, via K+antiport. Moreover, under appropriate conditions, a H+ion can replace K+. Intracellular K+accelerates the resetting step. Structural studies of SERT have identified two binding sites for Na+ions, but the K+site remains enigmatic. Here, we show that K+antiport can drive substrate accumulation into vesicles containing SERT extracted from a heterologous expression system, allowing us to study the residues responsible for K+binding. To identify candidate binding residues, we examine many cation binding configurations using molecular dynamics simulations, predicting that K+binds to the so-called Na2+site. Site directed mutagenesis of residues in this site can eliminate the ability of both K+and H+to drive 5-HT accumulation into vesicles and, in patch clamp recordings, prevent the acceleration of turnover rates and the formation of a channel-like state by K+or H+. In conclusion, the Na2+site plays a pivotal role in orchestrating the sequential binding of Na+and then K+(or H+) ions to facilitate 5-HT uptake in SERT.