Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters

Norepinephrine is a biogenic amine neurotransmitter that has widespread effects on cardiovascular tone, alertness and sensation of pain. As a consequence, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ a modified Drosophila melanogaster dopamine transporter as a surrogate for the human norepinephrine transporter and determine the X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. We also report structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, we observe that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. Our experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters. Highlights X-ray structures of the Drosophila dopamine transporter in substrate-free and norepinephrine bound forms. Norepinephrine and dopamine bind in distinct conformations within the binding pocket. Chronic pain inhibitors S-duloxetine, milnacipran and tramadol bind in the primary binding site and overlap with the norepinephrine-binding pose. Selective norepinephrine reuptake inhibition occurs through specific interactions at the subsite C in the primary binding pocket.