The dopamine transporter has a crucial role in regulation of dopaminergic neurotransmission by uptake of dopamine into neurons and contributes to the abuse potential of psychomotor stimulants^1,2,3. Despite decades of study, the structure, substrate binding, conformational transitions and drug-binding poses of human dopamine transporter remain unknown. Here we report structures of the human dopamine transporter in its apo state, and in complex with the substrate dopamine, the attention deficit hyperactivity disorder drug methylphenidate, and the dopamine-uptake inhibitors GBR12909 and benztropine. The dopamine-bound structure in the occluded state precisely illustrates the binding position of dopamine and associated ions. The structures bound to drugs are captured in outward-facing or inward-facing states, illuminating distinct binding modes and conformational transitions during substrate transport. Unlike the outward-facing state, which is stabilized by cocaine, GBR12909 and benztropine stabilize the dopamine transporter in the inward-facing state, revealing previously unseen drug-binding poses and providing insights into how they counteract the effects of cocaine. This study establishes a framework for understanding the functioning of the human dopamine transporter and developing therapeutic interventions for dopamine transporter-related disorders and cocaine addiction.

多巴胺转运蛋白在通过将多巴胺摄取到神经元中来调节多巴胺能神经传递中起着至关重要的作用，并有助于精神运动兴奋剂的滥用^1,2,3 。尽管经过数十年的研究，人类多巴胺转运蛋白的结构、底物结合、构象转变和药物结合姿势仍然未知。在这里，我们报告了处于 apo 状态的人类多巴胺转运蛋白的结构，以及与底物多巴胺、注意力缺陷多动障碍药物哌醋甲酯以及多巴胺摄取抑制剂 GBR12909 和苯甲托品的复合物。封闭状态下的多巴胺结合结构精确地说明了多巴胺和相关离子的结合位置。与药物结合的结构以面向外或面向内的状态被捕获，阐明了底物运输过程中不同的结合模式和构象转变。与可卡因稳定的外向状态不同，GBR12909 和苯托品将多巴胺转运蛋白稳定在内向状态，揭示了以前未见过的药物结合姿势，并提供了它们如何抵消可卡因作用的见解。这项研究建立了一个框架，用于了解人类多巴胺转运蛋白的功能，并开发针对多巴胺转运蛋白相关疾病和可卡因成瘾的治疗干预措施。