The interaction of (+/-)-bupropion [(+/-)-BP] with the human (h) alpha 4 beta 2 nicotinic acetylcholine receptor (AChR) was compared to that for its photoreactive analog (+/-)-2-(N-tert-butylamino)-3'-iodo-4'-azidopropiophenone [(+)-SADU-3-72]. Ca2+ influx results indicated that (+/-)-SADU-3-72 and (+/-)-BP inhibit h alpha 4 beta 2 AChRs with practically the same potency. However, (+/-)-SADU-3-72 binds to the [H-3]imipramine sites at resting and desensitized h alpha 4 beta 2 AChRs with 3 -fold higher affinity compared to that for (+/-)-BP, which is supported by molecular docking results. The docking results also indicate that each isomer of BP and SADU-3-72, in the protonated state, interacts with luminal and non-luminal sites. In the channel lumen, both ligands bind to two overlapping subsites, one that overlaps the imipramine site, and another much closer to the cytoplasmic side. The results suggest, for the first time, three differentiated nonluminal domains, including the transmembrane (TMD), extracellular (ECD), and ECD-TMD junction. In the ECD-TMD junction, BP and SADU-3-72 bind to, overlapping sites. Interestingly, only SADU-3-72 binds to intrasubunit and intersubunit sites" in the TMD, and to additional sites in the ECD. Our results are consistent with a model where BP and SADU-3-72 bind to overlapping sites in the luminal and ECD-TMD junctional domains of the ha4132, whereas only SADU-3-72 binds to additional non-luminal sites. The BP junctional site opens the door for additional inhibitory mechanisms. The pharmacological properties of (+/-)-SADU-3-72 showed in this work support further photolabeling studies to mapping the BP binding sites in the h alpha 4 beta 2 AChR. (C) 2016 Elsevier Ltd. All rights reserved.
