Abstract

The Erythrina alkaloids erysodine and dihydro-?-erythroidine (DH?E) are potent and selective competitive inhibitors of ?4?2 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 DH?E and a range of aromatic Erythrina alkaloids on [ 3H]cytisine binding and receptor function in conjunction with homology models of the ?4?2 nAChR, mutagenesis, and functional assays. The lactone group of DH?E 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 ?4 subunit was substituted by alanine. Sensitivity to inhibition was also decreased by substituting the conserved aromatic residues ?4Trp182 (loop B), ?4Tyr230 (loop C), and ?2Trp82 (loop D) and the nonconserved ?2Thr84; however, only ?4Trp182 was predicted to contact bound antagonist, suggesting ?4Tyr230, ?2Trp82, and ?2Thr84 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 ?2Asp196, leading to the uncapping of loop C. Consistent with this, ?2D196A abolished sensitivity to inhibition by DH?E or erysodine but not by epierythratidine, which is not predicted to form ionic bonds with ?2Asp196. This residue is not conserved in subunits that comprise nAChRs with low sensitivity to inhibition by DH?E or erysodine, which highlights ?2Asp196 as a major determinant of the receptor selectivity of Erythrina alkaloids. Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics.