Pairwise Interactions between Neuronal α7Acetylcholine Receptors and α-Conotoxin PnIB

• Published in: The Journal of biological chemistry Vol. 275; no. 7; pp. 4889 - 4896
• Main Authors: Quiram, P.A., McIntosh, J.M., Sine, S.M.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 18.02.2000
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.7.4889

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 of125I-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.