γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and its levels in the synaptic space are controlled by the GABA transporter isoforms (GATs). GATs are structurally related to biogenic amine transporters but display interactions with distinct inhibitors used as anti-epileptics. In this study, we engineer the binding pocket of Drosophila melanogaster dopamine transporter to resemble GAT1 and determine high-resolution X-ray structures of the modified transporter in the substrate-free state and in complex with GAT1 inhibitors NO711 and SKF89976a that are analogs of tiagabine, a medication prescribed for the treatment of partial seizures. We observe that the primary binding site undergoes substantial shifts in subsite architecture in the modified transporter to accommodate the two GAT1 inhibitors. We also observe that SKF89976a additionally interacts at an allosteric site in the extracellular vestibule, yielding an occluded conformation. Interchanging SKF89976a interacting residue in the extracellular loop 4 between GAT1 and dDAT suggests a role for this motif in the selective control of neurotransmitter uptake. Our findings, therefore, provide vital insights into the organizational principles dictating GAT1 activity and inhibition.

中文翻译：

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使用工程神经递质转运蛋白对 GABA 转运抑制的结构见解


γ-氨基丁酸 (GABA) 是主要的抑制性神经递质，其在突触间隙的水平由 GABA 转运蛋白亚型 (GAT) 控制。 GAT 在结构上与生物胺转运蛋白相关，但与用作抗癫痫药的不同抑制剂有相互作用。在这项研究中，我们设计了黑腹果蝇多巴胺转运蛋白的结合袋，使其类似于 GAT1，并确定了修饰转运蛋白在无底物状态下以及与 GAT1 抑制剂 NO711 和 SKF89976a（噻加宾类似物）复合物的高分辨率 X 射线结构。 ，一种用于治疗部分性癫痫发作的药物。我们观察到，修饰转运蛋白中的主要结合位点的亚位点结构发生了重大变化，以适应两种 GAT1 抑制剂。我们还观察到 SKF89976a 另外在细胞外前庭的变构位点相互作用，产生闭塞构象。 GAT1 和 dDAT 之间细胞外环 4 中 SKF89976a 相互作用残基的互换表明该基序在神经递质摄取的选择性控制中的作用。因此，我们的研究结果为决定 GAT1 活性和抑制的组织原则提供了重要的见解。