Unveiling conformation-selective regulation of the norepinephrine transporter

The norepinephrine transporter (NET) plays a crucial role in synaptic neurotransmission and is implicated in major depression and attention-deficit/hyperactivity disorders, yet our understanding of its allosteric, conformation-selective regulation-crucial for developing targeted therapeutics-remains limited. Through cryo-electron microscopy analysis of NET complexes with levomilnacipran, vanoxerine, and vilazodone, we identify a previously undefined allosteric site within NET's inner vestibule that enables conformation-selective regulation. This discovery introduces a "valve model," in which specific residues partition the cytoplasmic cavity into distinct chambers, determining inhibitor binding specificity. Leveraging this structural insight through virtual screening, we identify a set of inhibitors with potent NET inhibitory activity and demonstrate their antidepressant effects. Moreover, our structural identification of inhibitor occupancy at this conformation-selective site defines a mechanistic framework for targeted therapeutic intervention. These findings advance our understanding of NET allosteric modulation, providing a structure-guided framework for developing next-generation antidepressants targeting the inward-open conformation of NET for the treatment of neuropsychiatric disorders.