Abstract
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.
Highlights
Norepinephrine is a biogenic amine neurotransmitter that has widespread effects on alertness, arousal and pain sensation
Using X-ray structures of Drosophila dopamine transporter (dDAT) (Supplementary Table 1) in complex with popularly prescribed inhibitors of chronic pain including S-duloxetine, milnacipran, and a synthetic opioid, tramadol, we identify the importance of subsite C as the major determinant of inhibitor specificity between NET and DAT
A comparison of dDAT with human norepinephrine transporter (hNET) and hDAT reveals that serotonin-norepinephrine reuptake inhibitors (SNRIs) used in the study, duloxetine, milnacipran, and tramadol display inhibition potencies that are similar to hNET in comparison with hDAT (Supplementary Table 2)
Summary
Modified dDAT resembles hNET primary substrate-binding site. The dDAT, much like its human counterparts hDAT and hNET, is capable of interacting with both DA (Ki = 2.0 μM) and NE (Ki = 19.1 μM) with varying efficacies (Fig. 2a). Despite the inability of dDAT with these mutations in subsite B to transport catecholamines, it is used in this study as it reproduces the binding site of hNET, along with the F471L substitution in the vestibule. This construct, hereafter, referred to as dDATNET closely resembles the binding propensities of monoamine transport inhibitors with hNET than with hDAT a b
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