Norepinephrine is a biogenic amine neurotransmitter that has widespread effects on alertness, arousal and pain sensation. Consequently, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ the Drosophila melanogaster dopamine transporter as a surrogate for the norepinephrine transporter and determine 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.

Neurotransmitters released at the neural synapse through vesicle exocytosis are spatiotemporally controlled by the action of neurotransmitter transporters. Integral membrane proteins of the solute carrier 6 (SLC6) family are involved in the sodium and chloride coupled uptake of biogenic amine neurotransmitters including dopamine, serotonin, noradrenaline and inhibitory neurotransmitters including glycine and γ-amino butyric acid. This ion-coupled symport works through a well-orchestrated gating of substrate through alternating-access, which is mediated through movements of helices that resemble a rocking-bundle. A large array of commercially prescribed drugs and psychostimulants selectively target neurotransmitter transporters thereby modulating their levels in the synaptic space. Drug-induced changes in the synaptic neurotransmitter levels can be used to treat depression or neuropathic pain whereas in some instances prolonged usage can lead to habituation. Earlier structural studies of bacterial neurotransmitter transporter homolog LeuT and recent structure elucidation of the Drosophila dopamine transporter (dDAT) and human serotonin transporter (hSERT) have yielded a wealth of information in understanding the transport and inhibition mechanism of neurotransmitter transporters. Computational studies based on the structures of dDAT and hSERT have shed light on the dynamics of varied components of these molecular gates in affecting the uphill transport of neurotransmitters. This review seeks to address structural dynamics of neurotransmitter transporters at the extracellular and intracellular gates and the effect of inhibitors on the ligand-binding pocket. We also delve into the effect of additional factors including lipids and cytosolic domains that influence the translocation of neurotransmitters across the membrane.

Highlights• X-ray structures of the Drosophila dopamine transporter in substratefree 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. AbstractNorepinephrine 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.Keywords. L-Norepinephrine (NE), Drosophila melanogaster dopamine transporter (dDAT), dopamine (DA), serotonin (5-HT), solute carrier family, secondary active transport, neurotransmitter sodium symporters, duloxetine, milnacipran, tramadol.

Pannexins are structurally related to connexins and innexins but remain as single-membrane large-pore ion channels to release cellular ATP upon activation. There are three isoforms of pannexins, 1, 2, and 3, with diverse cellular roles, including inflammation, differentiation, neuropathic pain, and ATP release. In this study, we report the cryoEM structure of pannexin 3 that displays structural differences with pannexin1 through the presence of a second compartment in the vestibule. We also report the structures of pannexin 1 congenital mutant R217H along with a pannexin 1 pore mutant W74R/R75D that mimics pannexin 2, in the resolution range of 3.8-4.2 Å. The congenital mutant and the pannexin2 mimic induce structural changes that lead to a partially closed pore and alter ATP interaction propensities. The channel conductance of the congenital mutant displays weakened voltage sensitivity. The results signify the vital role of pore-lining residues and their dynamics in dictating pannexins' architecture and physiology.