Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark.
Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
Nat Commun. 2024 Oct 27;15(1):9267. doi: 10.1038/s41467-024-53584-9.
The serotonin transporter (SERT), responsible for the reuptake of released serotonin, serves as a major target for antidepressants and psychostimulants. Nevertheless, refining the mechanistic models for SERT remains challenging. Here, we expand the molecular understanding of the binding of ions, substrates, and inhibitors to SERT by incorporating the fluorescent non-canonical amino acid Anap through genetic code expansion. We elucidate steady-state changes in conformational dynamics of purified SERT with Anap inserted at intracellular- or extracellular sites. This uncovers the competitive mechanisms underlying cation binding and assigns distinct binding- and allosteric coupling patterns for several inhibitors and substrates. Finally, we track in real-time conformational transitions in response to the interaction with Na or serotonin. In this work, we present a methodological platform reporting on SERT conformational dynamics, which together with other approaches will deepen our insights into the molecular mechanisms of SERT.
血清素转运体（SERT）负责重摄取释放的血清素，是抗抑郁药和精神兴奋剂的主要靶标。然而，精确细化 SERT 的机械模型仍然具有挑战性。在这里，我们通过遗传密码扩展将荧光非典型氨基酸 Anap 纳入其中，从而扩展了对 SERT 与离子、底物和抑制剂结合的分子理解。我们阐明了在细胞内或细胞外位点插入 Anap 的纯化 SERT 的构象动力学的稳态变化。这揭示了阳离子结合的竞争机制，并为几种抑制剂和底物分配了不同的结合和变构偶联模式。最后，我们实时跟踪与 Na 或血清素相互作用时的构象转变。在这项工作中，我们提出了一种报告 SERT 构象动力学的方法学平台，该平台与其他方法一起，将加深我们对 SERT 分子机制的理解。
