Molecular determinants for competitive inhibition of α4β2 nicotinic acetylcholine receptors

The Erythrina alkaloids erysodine and dihydro-β-erythroidine (DHβE) are potent and selective competitive inhibitors of α4β2 nicotinic acetylcholine receptors (nAChRs), but little is known about the molecular determinants of the sensitivity of this receptor subtype to inhibition by this class of antagonists. We addressed this issue by examining the effects of DHβE and a range of aromatic Erythrina alkaloids on [ ³H]cytisine binding and receptor function in conjunction with homology models of the α4β2 nAChR, mutagenesis, and functional assays. The lactone group of DHβE and a hydroxyl group at position C-16 in aromatic Erythrina alkaloids were identified as major determinants of potency, which was decreased when the conserved residue Tyr126 in loop A of the α4 subunit was substituted by alanine. Sensitivity to inhibition was also decreased by substituting the conserved aromatic residues α4Trp182 (loop B), α4Tyr230 (loop C), and β2Trp82 (loop D) and the nonconserved β2Thr84; however, only α4Trp182 was predicted to contact bound antagonist, suggesting α4Tyr230, β2Trp82, and β2Thr84 contribute allosterically to the closed state elicited by bound antagonist. In addition, homology modeling predicted strong ionic interactions between the ammonium center of the Erythrina alkaloids and β2Asp196, leading to the uncapping of loop C. Consistent with this, β2D196A abolished sensitivity to inhibition by DHβE or erysodine but not by epierythratidine, which is not predicted to form ionic bonds with β2Asp196. This residue is not conserved in subunits that comprise nAChRs with low sensitivity to inhibition by DHβE or erysodine, which highlights β2Asp196 as a major determinant of the receptor selectivity of Erythrina alkaloids.