d-Tubocurarine (dTC) is a potent competitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds non-equivalently to the two agonist sites (K-d values of 30 nM and 8 mu M). When nAChR-rich membranes equilibrated with [H-3]dTC are irradiated with 254 nm UV light, [H-3]dTC is covalently incorporated into the alpha-, gamma-, and delta-subunits in a concentration-dependent and agonist-inhibitable manner, consistent with the localization of the high and low affinity dTC binding sites at the alpha-gamma- and alpha-delta-subunit interfaces, respectively (Pedersen, S. E. and Cohen, J. B. (1990) Proc. Natl. Acad Sci. U. S. A. 87, 2785-2789). We report on the amino acids within alpha-, gamma-, and delta-subunits that are the sites of specific photoincorporation of [H-3]dTC. Subunits isolated from nAChR-rich membranes photolabeled with [H-3]dTC were subjected to enzymatic digestion, and peptides containing H-3 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. Isolated peptides were then subjected to NH2-terminal sequence analysis to identify specifically labeled residues. Within the alpha-subunit, 95% of specific incorporation was contained within a 20-kDa proteolytic fragment beginning at Ser-173, with alpha Tyr-190 the primary site of [H-3]dTC photoincorporation and alpha Cys-192 and alpha Tyr-198 labeled at lower efficiency, Within gamma- and delta-subunits, specific labeling was contained within proteolytic fragments of 14 and 21 kDa, respectively, beginning at gamma Ala-49 and delta Thr-51. gamma Trp-55 and delta Trp-57 were identified as the sites of specific [H-3]dTC photoincorporation, Sequence alignment studies reveal gamma Trp-55 and delta Trp-57 to be homologous residues at whose position in receptor subunit primary structure a unique pattern of conservation exists in all nAChR (neuronal and muscle), Specifically, all subunits that associate with an cu-subunit to form an agonist site contain a tryptophan homologous to gamma Trp-55/delta Trp-57. This pattern of conservation may indicate a functional significance for tryptophan at that location in all nAChR agonist sites.
