Probing the structure of the nicotinic acetylcholine receptor with 4-benzoylbenzoylcholine, a novel photoaffinity competitive antagonist

[H-3]4-Benzoylbenzoylcholine (Bz(2)choline) was synthesized as a photoaffinity probe for the Torpedo nicotinic acetylcholine receptor (nAChR). [H-3]Bz(2)choline acts as an nAChR competitive antagonist and binds at equilibrium with the same affinity (K-D = 1.4 mu M) to both agonist sites. Irradiation at 320 nm of nAChR-rich membranes equilibrated with [H-3]Bz(2)choline results in the covalent incorporation of [H-3]Bz(2)choline into the nAChR gamma- and delta-subunits that is inhibitable by agonist, with little specific incorporation in the alpha-subunits. To identify the sites of photoincorporation, gamma- and delta-subunits, isolated from nAChR-rich membranes photolabeled with [H-3]Bz(2)choline, were digested enzymatically, and the labeled fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography, For the gamma-subunit, Staphylococcus aureus V8 protease produced a specifically labeled peptide beginning at gamma VaI-102, whereas for the delta-subunit, endoproteinase Asp-N produced a labeled peptide beginning at delta Asp-99. Amino-terminal sequence analysis identified the homologous residues gamma Leu-109 and delta Leu-111 as the primary sites of [H-3]Bz(2)choline photoincorporation. This is the first identification by affinity labeling of non-reactive amino acids within the acetylcholine-binding sites, and these results establish that when choline esters of benzoic acid are bound to the nAChR agonist sites, the para substituent is selectively oriented toward and in proximity to amino acids gamma Leu-109/delta Leu-111.