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 Na ²⁺ 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 Na ²⁺ 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.