Barker E L, Kimmel H L, Blakely R D
Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322.
Mol Pharmacol. 1994 Nov;46(5):799-807.
The serotonin transporter (SERT) is a target for many clinically significant drugs, such as cocaine, amphetamine, and antidepressants. The relationship between the structure of SERT and the binding of substrates and antagonists is virtually unknown, despite a large body of data describing the structure-activity relationships of transporter ligands. The cloning of multiple species homologs of SERT affords a unique opportunity for molecular comparisons to identify potential domains and residues involved in ligand recognition. We have conducted pharmacological comparisons of the cloned rat and human SERTs in transiently transfected HeLa cells. Serotonin uptake and radioligand binding assays revealed that rat and human SERTs show different sensitivities to some but not all transporter ligands; most tricyclic antidepressants were significantly more potent at the human SERT, relative to rat SERT, whereas d-amphetamine was a more potent inhibitor of rat SERT. Several other ligand such as fluoxetine, paroxetine, (+)-methylenedioxymethamphetamine, cocaine, and the substrate 5-hydroxytryptamine, shows no significant species selectivity. Cross-species chimeras between rat and human SERTs were constructed to track the species-specific pharmacologies through the SERT molecule. These chimeric SERTs were expressed in HeLa cells and transported serotonin similarly to parental SERTs. Using these chimeras, we have isolated a region distal to amino acid 532 the imparts species preferences for both the tricyclic imipramine and d-amphetamine. Our results support the prediction of distinct binding sites for SERT ligands and implicate a restricted region in or near putative transmembrane domain 12 of the transport as being involved in both substrate and antagonist recognition.
血清素转运体（SERT）是许多具有临床意义的药物的作用靶点，如可卡因、苯丙胺和抗抑郁药。尽管有大量描述转运体配体构效关系的数据，但SERT的结构与底物及拮抗剂结合之间的关系实际上仍不清楚。SERT多种物种同源物的克隆为分子比较提供了独特的机会，以识别参与配体识别的潜在结构域和残基。我们在瞬时转染的HeLa细胞中对克隆的大鼠和人类SERT进行了药理学比较。血清素摄取和放射性配体结合试验表明，大鼠和人类SERT对某些但并非所有转运体配体表现出不同的敏感性；相对于大鼠SERT，大多数三环类抗抑郁药对人类SERT的效力显著更高，而d-苯丙胺是大鼠SERT更有效的抑制剂。其他几种配体，如氟西汀、帕罗西汀、（+）-亚甲基二氧甲基苯丙胺、可卡因和底物5-羟色胺，没有明显的物种选择性。构建了大鼠和人类SERT之间的跨物种嵌合体，以追踪整个SERT分子的物种特异性药理学特性。这些嵌合SERT在HeLa细胞中表达，其转运血清素的方式与亲本SERT相似。利用这些嵌合体，我们分离出了一个位于氨基酸532远端的区域，该区域赋予了三环类丙咪嗪和d-苯丙胺物种偏好性。我们的结果支持了SERT配体存在不同结合位点的预测，并表明转运体假定跨膜结构域12内或其附近的一个受限区域参与了底物和拮抗剂的识别。
