Single-Residue Alteration in α-Conotoxin PnIA Switches Its nAChR Subtype Selectivity

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Abstract
α-Conotoxins are disulfide-rich peptides that are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Despite their small size, different α-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, α-conotoxins PnIA and PnIB, are examined. Although the toxins differ in only two residues, PnIA preferentially blocks α3β2 nAChRs, whereas PnIB prefers the α7 subtype. Point mutation chimeras of these α-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 α3β2-preferring to α7-preferring; furthermore, PnIA [A10L] blocks the α7 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 α7 having the smallest off-rate (koff = 0.17 min-1). Thermodynamic analysis indicates that Leu10 enhances the peptide's interaction with α7, but not α3β2, receptors, whereas Ser11 (in PnIA [N11S]) reduces its affinity for both α7 and α3β2 nAChRs.