Vol. 61, Issue 2, 463-472, February 2002

Mapping the Agonist Binding Site of the Nicotinic Acetylcholine Receptor by Cysteine Scanning Mutagenesis: Antagonist Footprint and Secondary Structure Prediction

Deirdre Sullivan, David C. Chiara, and Jonathan B. Cohen

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts

To further define the surface of the Torpedo californica nicotinic acetylcholine receptor (nAChR) contributing to the agonist binding site structure, we used the substituted Cys accessibility method to identify novel residues and determined the "footprint" of residues protected from modification by the reversible competitive antagonist d-tubocurarine (dTC). nAChRs containing single Cys substitutions within regions of the - or -subunit primary structure known to contribute to the agonist binding site were expressed in Xenopus laevis oocytes. Cys substitutions in binding site segments A (Tyr-93 and Asn-94), C (Tyr-198), and D (Glu-57) had been shown previously to be accessible for modification. We now introduced cysteines from Asp-195 to Ile-201 and from Ala-106 to Asp-113 and identified positions accessible for modification in segments C (Asp-195, Thr-196, Pro-197, Asp-200, and Ile-201) and E (Asn-107 and Leu-109). dTC protected against alkylation in segments D (Glu-57) and E (Leu-109) but not in segment A (Tyr-93 and Asn-94). In segment C, dTC protection experiments revealed a pattern in which every other residue (196, 198, and 200, but not 197 or 201) was protected from alkylation. This pattern of protection provides evidence that bound dTC is near amino acids in segments C, D, and E but not in segment A, and identifies a -strand surface within segment C contributing to the binding site. These results are discussed in terms of a homology model, based on the molluscan acetylcholine binding protein crystal structure, of the T. californica nAChR agonist binding site.