Hone_2013_J.Biol.Chem_288_25428

The nicotinic acetylcholine receptor (nAChR) subtype alpha6beta2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The alpha6 subunit is closely related to the alpha3 subunit, and this presents a problem in designing ligands that discriminate between alpha6beta2* and alpha3beta2* nAChRs. We used positional scanning mutagenesis of alpha-conotoxin PeIA, which targets both alpha6beta2* and alpha3beta2*, in combination with mutagenesis of the alpha6 and alpha3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed alpha6/alpha3beta2beta3 and alpha3beta2 receptors. Mutagenesis of PeIA revealed that Asn(11) was located in an important position that interacts with the alpha6 and alpha3 subunits. Substitution of Asn(11) with a positively charged amino acid essentially abolished the activity of PeIA for alpha3beta2 but not for alpha6/alpha3beta2beta3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on alpha6/alpha3beta2beta3 than alpha3beta2 receptors. Analogs with an N11R substitution were then used to show a critical interaction between the 11th position of PeIA and Glu(152) of the alpha6 subunit and Lys(152) of the alpha3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target alpha6beta2* nAChRs.