Substrate binding and inhibition mechanism of norepinephrine transporter

Norepinephrine transporter (NET; encoded by SLC6A2) reuptakes the majority of the released noradrenaline back to the presynaptic terminals, thereby affecting the synaptic noradrenaline level¹. Genetic mutations and dysregulation of NET are associated with a spectrum of neurological conditions in humans, making NET an important therapeutic target¹. However, the structure and mechanism of NET remain unclear. Here we provide cryogenic electron microscopy structures of the human NET (hNET) in three functional states-the apo state, and in states bound to the substrate meta-iodobenzylguanidine (MIBG) or the orthosteric inhibitor radafaxine. These structures were captured in an inward-facing conformation, with a tightly sealed extracellular gate and an open intracellular gate. The substrate MIBG binds at the centre of hNET. Radafaxine also occupies the substrate-binding site and might block the structural transition of hNET for inhibition. These structures provide insights into the mechanism of substrate recognition and orthosteric inhibition of hNET.