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-bound 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 the 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 dansyl-C-6-choline increases 3.1-fold upon binding, and (3) the receptor-bound dansyl-C-6-choline fluorescence is stable for at least 2 h. The calculated transverse distances between receptor-bound dansyl-C-6-choline and the membrane-partitioned accepters, C-12-eosin and C-18-rhodamine, were 31 and 39 Angstrom, respectively, Therefore, given the dimensions of the extracellular domain of the receptor, the ACh binding sites are located significantly below (similar to 25 Angstrom) the extracellular apex of the nAChR. These results are in agreement with the recent proposed location for the ACh binding sites in a pocket within each of the two alpha-subunits, similar to 30 Angstrom above the membrane surface (Unwin, N. (1993) J. Mol. Biol. 229: 1101-1124).