Monoamine neurotransmitters such as serotonin and dopamine are loaded by vesicular monoamine transporter 2 (VMAT2) into synaptic vesicles for storage and subsequent release in neurons. Impaired VMAT2 function underlies various neuropsychiatric diseases. VMAT2 inhibitors reserpine and tetrabenazine are used to treat hypertension, movement disorders associated with Huntington’s Disease and Tardive Dyskinesia. Despite its physiological and pharmacological significance, the structural basis underlying VMAT2 substrate recognition and its inhibition by various inhibitors remains unknown. Here we present cryo-EM structures of human apo VMAT2 in addition to states bound to serotonin, tetrabenazine, and reserpine. These structures collectively capture three states, namely the lumen-facing, occluded, and cytosol-facing conformations. Notably, tetrabenazine induces a substantial rearrangement of TM2 and TM7, extending beyond the typical rocker-switch movement. These functionally dynamic snapshots, complemented by biochemical analysis, unveil the essential components responsible for ligand recognition, elucidate the proton-driven exchange cycle, and provide a framework to design improved pharmaceutics targeting VMAT2.

单胺类神经递质（如血清素和多巴胺）由囊泡单胺转运蛋白 2 （VMAT2） 加载到突触囊泡中，以便在神经元中储存和随后释放。VMAT2 功能受损是各种神经精神疾病的基础。VMAT2 抑制剂利血平和丁苯那嗪用于治疗高血压、与亨廷顿病相关的运动障碍和迟发性运动障碍。尽管具有生理和药理学意义，但 VMAT2 底物识别及其受各种抑制剂抑制的结构基础仍然未知。在这里，我们展示了人载脂蛋白 VMAT2 的冷冻电镜结构以及与血清素、丁苯那嗪和利血平结合的状态。这些结构共同捕获三种状态，即面向管腔、闭塞和面向胞质溶胶的构象。值得注意的是，丁苯那嗪诱导 TM2 和 TM7 的大量重排，超越了典型的摇杆开关运动。这些功能动态快照，辅以生化分析，揭示了负责配体识别的基本成分，阐明了质子驱动的交换循环，并为设计针对 VMAT2 的改进药剂提供了一个框架。