Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringer Strasse 13a, Vienna, Austria.
Mol Pharmacol. 2010 Dec;78(6):1026-35. doi: 10.1124/mol.110.067538. Epub 2010 Sep 9.
The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuT(Aa)) has been used as a model for mammalian Na(+)/Cl(-)-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuT(Aa) liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERT(Y95F) mutation, which greatly reduced the affinity for [(3)H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [(3)H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP(+), paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants.
从水生栖热菌（LeuT(Aa))中提取的细菌亮氨酸转运蛋白的结构被用作哺乳动物 Na(+)/Cl(-)依赖性转运蛋白的模型，特别是 5-羟色胺转运蛋白（SERT）。LeuT(Aa)与三环抗抑郁药结合的晶体结构预测抑制剂和底物的同时结合。这与 SERT 对底物和抑制剂的相互竞争抑制不兼容。我们通过同源建模和对接研究探索了三环抗抑郁药的结合模式。随后使用两种方法来区分三个潜在对接构象簇：1) SERT(Y95F)突变，该突变大大降低了对 [(3)H]丙咪嗪的亲和力，但不影响底物结合；2) 在存在和不存在卡马西平（即与 SERT 竞争 [(3)H]丙咪嗪结合的三环丙咪嗪类似物）的情况下进行竞争结合实验。释放剂（对氯苯丙胺、亚甲二氧基苯丙胺/摇头丸）和卡马西平的结合是相互排斥的，但在卡马西平存在的情况下生成的色氨酸、MPP(+)、帕罗西汀和伊博加因的 Dixon 图产生了相交线。这些观察结果与以下模型一致：1）三环环被对接进入外前庭，二甲氨基丙基侧链指向底物结合位点；2）安非他命的结合在内、外前庭中产生结构变化，从而阻止三环环的对接；3）伊博加因（与内向构象结合）和卡马西平的同时结合表明内前庭中有第二个结合位点，这与单胺转运蛋白的拟对称折叠一致。这可能是第二个低亲和力的抗抑郁药结合位点。
