A bupropion modulatory site in the 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, such as nicotinic acetylcholine and serotonin type 3A receptors, at clinically relevant concentrations. Here, we demonstrate that bupropion also inhibits a prokaryotic homolog of pentameric ligand-gated ion channels, the Gloeobacter violaceus ligand-gated ion channel (GLIC). Using the GLIC as a model, we used molecular docking to predict binding sites for bupropion. Bupropion was found to bind to several sites within the transmembrane domain, with the predominant site being localized to the interface between transmembrane segments M1 and M3 of two 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 a 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 half-maximal inhibitory concentration 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 the GLIC. This intersubunit binding pocket is structurally conserved and almost identical to a binding pocket described for neurosteroids in γ-aminobutyric acid type A receptors. Our data thus suggest that the T214 that lines a previously identified lipophilic binding pocket in GLIC and γ-aminobutyric acid type A receptors is also a modulatory site for bupropion interaction with the GLIC.

Identification of a binding site for bupropion in Gloeobacter violaceus ligand-gated ion channel.

Bupropion is an atypical antidepressant and smoking cessation drug which 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. However, the binding sites and binding mechanisms of bupropion are still elusive. To further understand the inhibition of pLGICs by bupropion, in this work, using a prokaryotic homologue of pLGICs as a model, we examined the inhibitory potency of bupropion in Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated ion channel. Bupropion inhibited proton-induced currents in GLIC with an inhibitory potency of 14.9 ± 2.0 µM, comparable to clinically attainable concentrations previously shown to also modulate eukaryotic pLGICs. Using single amino acid substitutions in GLIC and two-electrode voltage-clamp recordings, we further determined a binding site for bupropion in the lower third of the first transmembrane segment M1 at residue T214. The sidechain of M1 T214 together with additional residues of M1 and also of M3 of the adjacent subunit have previously been shown to contribute to binding of other lipophilic molecules like allopregnanolone and pregnanolone.