The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - i.e., the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives) and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the K_M for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted with the inward-facing state. Kinetic modeling recapitulated the experimental data and verified that uncompetitive inhibition arose from preferential binding of ECSI#6 to the K⁺-bound, inward-facing conformation of SERT. This binding mode predicted a pharmacochaperoning action of ECSI#6, which was confirmed by examining its effect on the folding-deficient mutant SERT-PG^601,602AA: pre-incubation of HEK293 cells with ECSI#6 restored export of SERT-PG^601,602AA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, administration of ECSI#6 promoted delivery of SERT-PG^601,602AA to the presynaptic specialization of serotonergic neurons. To the best of our knowledge, ECSI#6 is the first example of an uncompetitive SLC inhibitor. Pharmacochaperones endowed with the binding mode of ECSI#6 are attractive, because they can rescue misfolded transporters at concentrations, which cause modest transport inhibition.

中文翻译：

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5-羟色胺转运蛋白的非竞争性抑制机制

血清素转运蛋白 (SERT/SLC6A4) 可以说是研究最广泛的溶质载体 (SLC)。在其同名作用期间 - 即从细胞外空间回收血清素 - SERT 经历了一个构象循环。典型的抑制剂（抗抑郁药和可卡因）、部分和全部底物（安非他明及其衍生物）和非典型抑制剂（伊博加因类似物）在这个循环中优先结合不同的状态。这导致竞争性或非竞争性转运抑制。在这里，我们探讨了 N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) 对 SERT 的作用：ECSI#6 对血清素摄取的抑制作用增强增加血清素浓度。相反，K _M通过增加 ECSI#6 降低了血清素的含量。ECSI#6 与 SERT 的向外状态结合的亲和力低，但对钾结合状态的亲和力增加。电生理记录显示 ECSI#6 优先与内向状态相互作用。动力学模型概括了实验数据并证实了非竞争性抑制是由 ECSI#6 优先结合到SERT的 K ^{+结合的、面向内的构象引起的。}这种结合模式预测了 ECSI#6 的药物伴侣作用，这通过检查其对折叠缺陷突变 SERT-PG ^601,602 AA 的影响得到证实：用 ECSI#6 预孵育 HEK293 细胞恢复了 SERT-PG ^{601,602的输出}AA由内质网和底物转运。同样，在转基因果蝇中，ECSI#6 的施用促进了 SERT-PG ^601,602 AA 向 5-羟色胺能神经元的突触前特化的递送。据我们所知，ECSI#6 是第一个非竞争性 SLC 抑制剂的例子。具有 ECSI#6 结合模式的药物伴侣很有吸引力，因为它们可以在一定浓度下拯救错误折叠的转运蛋白，从而导致适度的转运抑制。