Identification of the potassium- binding site in serotonin transporter

• 成果类型: Article
• 署名作者: Hellsberg, Eva; Boytsov, Danila; Chen, Qingyang; Niello, Marco; Freissmuth, Michael; Rudnick, Gary; Zhang, Yuan-Wei; Sandtner, Walter; Forrest, Lucy R.
• 署名单位: National Institutes of Health (NIH) - USA; NIH National Institute of Neurological Disorders & Stroke (NINDS); Medical University of Vienna; Guangzhou University; Yale University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-11629
• DOI: 10.1073/pnas.2319384121
• 发表日期: 2024-04-23
• 关键词: x-ray structures; permeation pathway; sodium; neurotransmitter; 5-hydroxytryptamine; chloride; inhibition; mechanism; MODEL; leut

摘要

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.