Single-residue alteration in α-conotoxin PnIA switches its nAChR subtype selectivity

alpha-Conotoxins are disulfide-rich peptides that are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Despite their small size, different alpha-conotoxins are able to discriminate among different subtypes of mammalian nAChRs. In this report, the activity of two peptides from the venom of Conus pennaceus, alpha-conotoxins PnIA and PnIB, are examined. Although the toxins differ in only two residues, PnIA preferentially blocks alpha 3 beta 2 nAChRs, whereas PnIB prefers the alpha 7 subtype, Point mutation chimeras of these alpha-conotoxins were synthesized and their activities assessed on Xenopus oocytes expressing specific nAChRs. Change of a single residue, Ala10 to Leu. in PnIA (to form PnIA [A10L]) converts the parent peptide from alpha 3 beta 2-preferring to alpha 7-preferring; furthermore, PnIA [A10L] blocks the alpha 7 receptor with an IC50 (12.6 nM) that is lower than that of either parent peptide. Kinetic analysis indicates that differences in affinity among the analogues are primarily due to differences in off-rate, with PnIA [A10L]'s interaction with alpha 7 having the smallest off-rate (k(off) = 0.17 min(-1)). Thermodynamic analysis-indicates that Leu10 enhances the peptide's interaction with alpha 7, but not alpha 3 beta 2, receptors, whereas Ser11 (in PnIA [N11S]) reduces its affinity for both alpha 7 and alpha 3 beta 2, nAChRs.