Bridge Institute, USC Michelson Center for Convergent Biosciences, University of Southern California, Los Angeles, CA, USA.
Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA.
Nature. 2022 Jun;606(7915):820-826. doi: 10.1038/s41586-022-04814-x. Epub 2022 Jun 8.
γ-Aminobutyric acid (GABA) transporter 1 (GAT1) regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins.
γ-氨基丁酸（GABA）转运蛋白 1（GAT1）通过在突触小泡释放 GABA 后从突触间隙清除抑制性神经递质 GABA，从而调节中枢神经系统的神经元兴奋。通过抑制 GABA 再摄取转运体来提高突触间隙中 GABA 的水平，是治疗癫痫等神经疾病的一种既定策略。在这里，我们确定了全长野生型人 GAT1 与临床使用的抑制剂噻加宾复合物的冷冻电子显微镜结构，其有序部分仅为 60 kDa。我们的结构表明，噻加宾通过阻断 GABA 释放途径的细胞内门，将 GAT1 锁定在内向开放构象，从而抑制神经递质摄取。我们的结果提供了对噻加宾对 GAT1 的混合抑制作用的深入了解，这是一种重要的抗惊厥药物。我们的实验数据证实了其药效学特征，表明噻加宾最初与外向开放构象中的底物结合位点结合，而我们的结构则显示药物使转运体停滞在内向开放构象，这与两步抑制机制一致。所呈现的 GAT1 结构为该重要神经递质转运体的生物学和药理学提供了关键的见解，并为神经调节剂的合理设计提供了蓝图，同时也推动了挑战性膜蛋白单颗粒冷冻电子显微镜中可能的边界。
