Discovery and Structural and Functional Characterization of a Novel A-Superfamily Conotoxin Targeting alpha 9 alpha 10 Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels widely distributed in the central peripheral nervous system and muscles which participate in rapid synaptic transmission. The alpha 9 alpha 10 nAChR is an acetylcholine receptor subtype and is involved in chronic pain. In the present study, a new A-superfamily conotoxin Bt14.12 cloned from Conus betulinus was found to selectively inhibit alpha 9 alpha 10 nAChRs with an IC50 of 62.3 nM. Unlike alpha-conotoxins and other A-superfamily conotoxins, Bt14.12 contains a four Cys (C-C-C-C) framework with a unique disulfide bond connection "C1-C4, C2-C3 ". The structure-activity studies of Bt14.12 demonstrate that all amino acid residues contribute to its potency. Interestingly, mutation experiments show that the deletion of Asp2 or the addition of three Arg residues at the N-terminus of Bt14.12 significantly enhances its inhibitory activity (IC50 is 21.9 nM or 12.7 nM, respectively) by 2-or 4-fold compared to the wild-type Bt14.12. The NMR structure of Bt14.12 shows that it contains alpha-helix-and beta-turn-like elements, and further computational modelings of the interaction between Bt14.12 and the alpha 9 alpha 10 nAChR demonstrate that Bt14.12 possesses a distinctive mode of action and displays a different structure-activity relationship from known alpha 9 alpha 10 nAChR targeting alpha-conotoxins. Our findings provide a novel conotoxin that potently targets alpha 9 alpha 10 nAChRs and a new motif for designing potent inhibitors against alpha 9 alpha 10 nAChRs.