Positional scanning mutagenesis of α-conotoxin PeIA identifies critical residues that confer potency and selectivity for α6/α3ß2ß3 and α3ß2 nicotinic acetylcholine receptors.

ABSTRACT

The nicotinic acetylcholine receptor (nAChR) subtype α6ß2* (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 α6 subunit is closely related to the α3 subunit, and this presents a problem in designing ligands that discriminate between α6ß2* and α3ß2* nAChRs. We used positional scanning mutagenesis of α-conotoxin PeIA, which targets both α6ß2* and α3ß2*, in combination with mutagenesis of the α6 and α3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed α6/α3ß2ß3 and α3ß2 receptors. Mutagenesis of PeIA revealed that Asn(11) was located in an important position that interacts with the α6 and α3 subunits. Substitution of Asn(11) with a positively charged amino acid essentially abolished the activity of PeIA for α3ß2 but not for α6/α3ß2ß3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on α6/α3ß2ß3 than α3ß2 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 α6 subunit and Lys(152) of the α3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target α6ß2* nAChRs.