Identification of the potassium-binding site in serotonin transporter

Abstract

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.