Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site

Biochemistry. 1988 Apr 5;27(7):2346-57. doi: 10.1021/bi00407a016.


The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent [3H]-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the alpha-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment alpha 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the alpha-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the alpha-chain that assign the amino-terminal segment alpha 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified [3H]DDF-labeled residues, which are conserved in muscle and neuronal alpha-chains but not in the other subunits, may be directly involved in agonist binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / metabolism*
  • Affinity Labels / metabolism
  • Amino Acids / analysis
  • Animals
  • Cell Membrane / metabolism
  • Cyanogen Bromide
  • Diazonium Compounds / metabolism*
  • Electric Organ / metabolism
  • Macromolecular Substances
  • Peptide Fragments / analysis
  • Receptors, Cholinergic / isolation & purification
  • Receptors, Cholinergic / metabolism*
  • Torpedo


  • Affinity Labels
  • Amino Acids
  • Diazonium Compounds
  • Macromolecular Substances
  • Peptide Fragments
  • Receptors, Cholinergic
  • 4-dimethylaminobenzenediazonium fluoroborate
  • Acetylcholine
  • Cyanogen Bromide