Bupropion Binds in the Middle (M2-9) of the Torpedo Nicotinic Acetylcholine Receptor Ion Channel

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Bupropion is clinically prescribed for the treatment of depression (Wellbutrin) and for smoking cessation (Zyban). While there is consensus that the primary mechanism of action involves increased synaptic concentrations of dopamine and norepinephrine via inhibition of the respective reuptake transporters, DAT and NET, there is growing evidence that some of the therapeutic benefits of bupropion may result from non-competitive antagonism of neuronal nicotinic acetylcholine receptors (nAChRs), in particular α4β2 and α3β4 subtypes. To aid in elucidating the mechanism of bupropion action and the development of new therapeutic agents for smoking cessation and depression, our goal is to determine the specific molecular interactions between bupropion and several nAChR subtypes (Torpedo, α4β2, α3β4). We first established that bupropion dose-dependently and reversibly inhibits ACh-induced currents in Xenopus laevis oocytes injected with affinity-purified and lipid-reconstituted Torpedo nAChRs (IC50 0.34 μM). Using a photoreactive analog of bupropion ([125I]-SADU-3-72), we next established that bupropion inhibits the binding of [125I]-SADU-3-72 to the Torpedo nAChR (IC50 closed state, 3.6 μM; desensitized state, 1.2 μM) and that binding to the closed nAChR is fully inhibited by tetracaine (IC50 0.42 μM) consistent with a bupropion/[125I]-SADU-3-72 binding site within the nAChR channel. [125I]-SADU-3-72 photolabeled Torpedo nAChR subunits in an agonist-sensitive and bupropion-inhibitable (specific) manner. Finally for the closed nAChR, we identified sites of specific [125I]-SADU-3-72 labeling within δM2 (δLeu265/δM2-9) and βM2 (βLeu257/βM2-9). In the desensitized state, TCP-inhibitable (specific) labeling was identified within δM2 (δLeu265, δM2-9 with minor labeling of δSer258, δM2-2). Our results establish that bupropion binds in the middle (M2-9) of the Torpedo nAChR channel, with a slightly broader binding locus in the desensitized channel. Currently, studies are underway to identify the site(s) of [125I]-SADU-3-72 labeling in affinity-purified α4β2 and α3β4 nAChRs.