Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label

Biochemistry. 1991 Jul 16;30(28):6987-97. doi: 10.1021/bi00242a026.


The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sites on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]nicotine binds at equilibrium with Keq = 0.6 microM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the alpha- and gamma-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the alpha-subunit was labeled via both agonist sites but the gamma-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Within the alpha-subunit, 93% of the labeling was contained within a 20-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Ser-173. Sequence analysis of this peptide indicated that approximately 80% of the incorporation was into Tyr-198, approximately 13% was into Cys-192, and approximately 7% was into Tyr-190. Chymotryptic digestion of the alpha-subunit confirmed that Tyr-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radio-sequencing strategy employing omicron-phthalaldehyde, since the efficiency of photolabeling was low (approximately 1.0%) and the labeled chymotryptic peptide was not isolated in sufficient quantity to be identified by mass. [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylcholine / chemistry
  • Acetylcholine / metabolism*
  • Affinity Labels
  • Amino Acid Sequence
  • Animals
  • Chromatography, High Pressure Liquid
  • Chymotrypsin
  • Hydrolysis
  • Molecular Sequence Data
  • Nicotine* / metabolism
  • Nicotine* / pharmacology
  • Peptide Mapping*
  • Protein Conformation
  • Receptors, Nicotinic / drug effects
  • Receptors, Nicotinic / isolation & purification*
  • Receptors, Nicotinic / metabolism
  • Serine Endopeptidases
  • Torpedo
  • Tritium


  • Affinity Labels
  • Receptors, Nicotinic
  • Tritium
  • Nicotine
  • Serine Endopeptidases
  • Chymotrypsin
  • glutamyl endopeptidase
  • Acetylcholine