The present work uses α-conotoxin ImI (CTx ImI) to probe the neurotransmitter binding site of neuronal α7 acetylcholine receptors. We identify key residues in α7 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 α7 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 125I- α-bungarotoxin binding. The results reveal a predominant contribution by Tyr195 in α7, accompanied by smaller contributions by Thr77, 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 α7 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 α7, and between Trp-10 of CTx ImI and Thr-77 and Asn-111 of α7. 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 α7 binding site.
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