2 Subunit Contribution to 4 / 7 - Conotoxin Binding to the Nicotinic Acetylcholine Receptor

The structures of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP-derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 -conotoxins. Here, we used an 3 2 model to identify 2 subunit residues in this cleft and investigated their influence on the binding of -conotoxins MII, PnIA, and GID to the 3 2 nAChR by two-electrode voltage clamp analysis. Although a 2-L119Q mutation strongly reduced the affinity of all three -conotoxins, 2-F117A, 2-V109A, and 2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of -conotoxins GID and MII was also observed when the 2-F117A mutant was combined with the 4 instead of the 3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to 2-F117A, 2V109A, and 2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 -conotoxin binding to the 3 2 model supported a direct interaction between mutated nAChR residues and -conotoxin residues 6, 7, and 10. Taken together, these data provide evidence that the subunit contributes to -conotoxin binding and selectivity and demonstrate that a small cleft leading to the agonist binding site is targeted by -conotoxins to block the nAChR.