A bupropion modulatory site in Gloeobacter violaceus ligand-gated ion channel.

Bupropion is an atypical antidepressant and smoking cessation drug that causes adverse effects such as insomnia, irritability, and anxiety. Bupropion inhibits dopamine and norepinephrine reuptake transporters and eukaryotic cation-conducting pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine (nACh) and serotonin type 3A (5-HT3A) receptors, at clinically relevant concentrations. Here, we demonstrate that bupropion also inhibits a prokaryotic homolog of pLGICs, the Gloeobacter violaceus ligand-gated ion channel (GLIC). Using GLIC as a model, we used molecular docking to predict binding sites for bupropion. Several clusters of bupropion binding pose within the transmembrane domain were identified, with the predominant cluster being localized to the interface between transmembrane segments M1 and M3 of adjacent subunits. Residues W213, T214 and W217 in the first transmembrane segment, M1, and F267 and I271 in the third transmembrane segment, M3, most frequently reside within 4 Å distance from bupropion. We then used single amino acid substitutions at these positions and two-electrode voltage-clamp recordings to determine their impact on bupropion inhibitory effects. The substitution T214F alters bupropion potency by shifting the IC50 to a 13-fold higher value compared to wild-type GLIC. Residue T214 is found within a previously identified binding pocket for neurosteroids and lipids in GLIC. This intersubunit binding pocket is structurally conserved and almost identical to a binding pocket described for neurosteroids in GABAA receptors. Our data thus suggest that T214 that lines a previously identified lipophilic binding pocket in GLIC and GABAA receptors is also a modulatory site for bupropion interaction with GLIC.