Pairwise interactions between neuronal α₇ acetylcholine receptors and α-conotoxin PnIB

This work uses α-conotoxin PnIB to probe the agonist binding site of neuronal α₇ acetylcholine receptors. We mutated the 13 non-cysteine residues in CTx PnIB, expressed α₇/5-hydroxytryptamine-3 homomeric receptors in 293 HEK cells, and measured binding of each mutant toxin to the expressed receptors by competition against the initial rate of ¹²⁵I-α- bungarotoxin binding. The results reveal that residues Ser-4, Leu-5, Pro-6, Pro-7, Ala-9, and Leu-10 endow CTx PnIB with affinity for α₇/5- hydroxytryptamine-3 receptors; side chains of these residues cluster in a localized region within the three-dimensional structure of CTx PnIB. We next mutated key residues in the seven loops of α₇ that converge at subunit interfaces to form the agonist binding site. The results reveal predominant contributions by residues Trp-149 and Tyr-93 in α₇ and smaller contributions by Ser-34, Arg-186, Tyr-188, and Tyr-195. To identify pairwise interactions that stabilize the receptor-conotoxin complex, we measured binding of receptor and toxin mutations and analyzed the results by double mutant cycles. The results reveal a single dominant interaction between Leu- 10 of CTx PnIB and Trp-149 of α₇ that anchors the toxin to the binding site. We also find weaker interactions between Pro-6 of CTx PnIB and Trp-149 and between both Pro-6 and Pro-7 and Tyr-93 of α₇. The overall results demonstrate that a localized hydrophobic region in CTx PnIB interacts with conserved aromatic residues on one of the two faces of the α₇ binding site.