TRANSVERSE DISTANCE BETWEEN THE MEMBRANE AND THE AGONIST BINDING-SITES ON THE TORPEDO ACETYLCHOLINE-RECEPTOR - A FLUORESCENCE STUDY

Fluorescence dipolar resonance energy transfer between a receptor-boun d fluorescent agonist, dansyl-C,choline, and two membrane-partitioned fluorescent probes, C-18-rhodamine and C-12-eosin, was used to measure the transverse distance between the acetylcholine (ACh) binding sites on the intact Torpedo nicotinic acetylcholine receptor (nAChR) and th e surface of the lipid membrane. Control experiments demonstrated that : (1) dansyl-C-6-choline binds to cobra-alpha-toxin sensitive sites on the nAChR with a K-D approximate to 20 nM, (2) the quantum yield of d ansyl-C-6-choline increases 3.1-fold upon binding, and (3) the recepto r-bound dansyl-C-6-choline fluorescence is stable for at least 2 h. Th e calculated transverse distances between receptor-bound dansyl-C-6-ch oline and the membrane-partitioned accepters, C-12-eosin and C-18-rhod amine, were 31 and 39 Angstrom, respectively, Therefore, given the dim ensions of the extracellular domain of the receptor, the ACh binding s ites are located significantly below (similar to 25 Angstrom) the extr acellular apex of the nAChR. These results are in agreement with the r ecent proposed location for the ACh binding sites in a pocket within e ach of the two alpha-subunits, similar to 30 Angstrom above the membra ne surface (Unwin, N. (1993) J. Mol. Biol. 229: 1101-1124).