Abstract

To further define the surface of the Torpedo californicanicotinic 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 α- or γ-subunit primary structure known to contribute to the agonist binding site were expressed in Xenopus laevis oocytes. Cys substitutions in binding site segments A (αTyr-93 and αAsn-94), C (αTyr-198), and D (γGlu-57) had been shown previously to be accessible for modification. We now introduced cysteines from αAsp-195 to αIle-201 and from γAla-106 to γAsp-113 and identified positions accessible for modification in segments C (αAsp-195, αThr-196, αPro-197, αAsp-200, and αIle-201) and E (γAsn-107 and γLeu-109). dTC protected against alkylation in segments D (γGlu-57) and E (γLeu-109) but not in segment A (αTyr-93 and αAsn-94). In segment C, dTC protection experiments revealed a pattern in which every other residue (α196, α198, and α200, but not α197 or α201) 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 β-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 theT. californica nAChR agonist binding site.