Pairwise interactions between neuronal alpha(7) acetylcholine receptors and alpha-conotoxin PnIB

This work uses alpha-conotoxin PnIB to probe the agonist binding site of ne uronal alpha(7) acetylcholine receptors, We mutated the 13 non-cysteine res idues in CTx PnIB, expressed alpha(7)/5-hydroxytryptamine-3 homomeric recep tors in 293 HEK cells, and measured binding of each mutant toxin to the exp ressed receptors by competition against the initial rate of I-125-alpha-bun garotoxin binding. The results reveal that residues Ser-4, Leu-5, Pro-6, Pr o-7, Ala-9, and Leu-10 endow CTx PnIB with affinity for alpha(7)/5-hydroxyt ryptamine-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 alpha(7) that converge at subunit inter faces to form the agonist binding site, The results reveal predominant cont ributions by residues Trp-149 and Tyr-93 in alpha(7) and smaller contributi ons by Ser-34, Arg-186, Tyr-188, and Tyr-195, To identify pairwise interact ions that stabilize the receptor-conotoxin complex, we measured binding of receptor and toxin mutations and analyzed the results by double mutant cycl es, The results reveal a single dominant interaction between Leu-10 of CTx PnIB and Trp-149 of alpha(7) that anchors the toxin to the binding site. We also find weaker interactions between Pro-6 of CTx PnIB and Trp-149 and be tween both Pro-g and Pro-7 and Tyr-93 of alpha(7). The overall results demo nstrate that a localized hydrophobic region in CTx PnIB interacts with cons erved aromatic residues on one of the two faces of the alpha(7) binding sit e.