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.