α-Conotoxin [S9A]TxID Potently Discriminates between α3β4 and α6/α3β4 Nicotinic Acetylcholine Receptors

alpha 3 beta 4 nAChRs have been implicated in various pathophysiological conditions. However, the expression profile of alpha 3 beta 4 nAChRs and alpha 6/alpha 3 beta 4 nAChRs overlap in a variety of tissues. To distinguish between these two subtypes, we redesigned peptide 1 (a-conotoxin TxID), which inhibits alpha 3 beta 4 and alpha 6/alpha 3 beta 4 nAChR subtypes. We systematically mutated 1 to evaluate analogue selectivity for alpha 3 beta 4 vs alpha 6/alpha 3 beta 4 nAChRs expressed in Xenopus laevis oocytes. One analogue, peptide 7 ([S9A]TxID), had 46-fold greater potency for alpha 3 beta 4 versus alpha 6/alpha 3 beta 4 nAChRs. Peptide 7 had IC(50)s > 10 mu M for other nAChR subtypes. Molecular dynamics simulations suggested that Ser-9 of TxID was involved in a weak hydrogen bond with beta 4 Lys-81 in the alpha 6 beta 4 binding site but not in the alpha 3 beta 4 binding site. When Ser-9 was substituted by an Ala, this hydrogen bond interaction was disrupted. These results provide further molecular insights into the selectivity of 7 and provide a guide for designing ligands that block alpha 3 beta 4 nAChRs.