Transport mechanism and pharmacology of the human GlyT1

The glycine transporter 1 (GlyT1) plays a crucial role in the regulation of both inhibitory and excitatory neurotransmission by removing glycine from the synaptic cleft. Given its close association with glutamate/glycine co-activated NMDA receptors (NMDARs), GlyT1 has emerged as a central target for the treatment of schizophrenia, which is often linked to hypofunctional NMDARs. Here, we report the cryo-EM structures of GlyT1 bound with substrate glycine and drugs ALX-5407, SSR504734, and PF-03463275. These structures, captured at three fundamental states of the transport cycle—outward-facing, occluded, and inward-facing—enable us to illustrate a comprehensive blueprint of the conformational change associated with glycine reuptake. Additionally, we identified three specific pockets accommodating drugs, providing clear insights into the structural basis of their inhibitory mechanism and selectivity. Collectively, these structures offer significant insights into the transport mechanism and recognition of substrate and anti-schizophrenia drugs, thus providing a platform to design small molecules to treat schizophrenia. [Display omitted] •Glycine-bound complex illuminates binding pocket of substrate and Na+/Cl− ions•Sarcosine-based ALX-5407 stabilizes GlyT1 in its inward-facing state•SSR-504734 and PF-03463275 stabilize outward-facing GlyT1 with varied pockets•Transport mechanism elucidated by structures in outward, occluded, and inward states This work reports the structures of human glycine transporter 1 (GlyT1), providing insights on its substrate recognition, conformational transition, and distinct binding pockets for anti-schizophrenia drugs.