Pairwise Interactions between neuronal α7 acetylcholine receptors and α-conotoxin ImI

The present work uses alpha-conotoxin ImI (CTx ImI) to probe the neurotransmitter binding site of neuronal alpha(7) acetylcholine receptors. We identify key residues in alpha(7) that contribute to CTx ImI affinity, and use mutant cycles analysis to identify pairs of residues that stabilize the receptor-conotoxin complex. We first mutated key residues in the seven known loops of alpha(7) that converge at the subunit interface to form the ligand binding site. The mutant subunits were expressed in 293 HEK cells, and CTx ImI binding was measured by competition against the initial rate of I-125-alpha-bungarotoxin binding. The results reveal a predominant contribution by Tyr-195 in alpha(7), accompanied by smaller contributions by Thr-77, Tyr-93, Asn-111, Gln-117, and Trp-149, Based upon our previous identification of bioactive residues in CTx ImI, we measured binding of receptor and toxin mutations and analyzed the results using thermodynamic mutant cycles. The results reveal a single dominant interaction between Arg-7 of CTx ImI and Tyr-195 of alpha(7) that anchors the toxin to the binding site. We also find multiple weak interactions between Asp-5 of CTx ImI and Trp-149, Tyr-151, and Gly-153 of alpha(7), and between Trp-10 of CTx ImI and Thr-77 and Asn-111 of alpha(7). The overall results establish the orientation of CTx ImI as it bridges the subunit interface and demonstrate close approach of residues on opposing faces of the alpha(7) binding site.