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-C6-choline, and two membrane-partitioned fluorescent probes, C18-rhodamine and C12-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-C6-choline binds to cobra-alpha-toxin sensitive sites on the nAChR with a KD approximately 20 nM, (2) the quantum yield of dansyl-C6-choline increases 3.1-fold upon binding, and (3) the receptor-bound dansyl-C6-choline fluorescence is stable for at least 2 h. The calculated transverse distances between receptor-bound dansyl-C6-choline and the membrane-partitioned acceptors, C12-eosin and C18-rhodamine, were 31 and 39 A, respectively. Therefore, given the dimensions of the extracellular domain of the receptor, the ACh binding sites are located significantly below (approximately 25 A) 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, approximately 30 A above the membrane surface (Unwin, N. (1993) J. Mol. Biol. 229: 1101-1124).
Division of Biomedical Sciences, University of California, Riverside 92521-0121.
C F
CF
Valenzuela
P
P
Weign
J
J
Yguerabide
D A
DA
Johnson
D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003619:Dansyl Compounds; D004735:Energy Transfer; D005452:Fluoresceins; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D011789:Quantum Theory; D000644:Quaternary Ammonium Compounds; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000378:metabolism; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D014101:Torpedo
D003619:Dansyl Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000644:Quaternary Ammonium Compounds; D011950:Receptors, Cholinergic; D012235:Rhodamines; C074109:5-(N-dodecanoyl)aminofluorescein; C027933:(1-(5-dimethylaminonaphthalene)sulfonamido)-n-hexanoic acid-beta-N-trimethylammonium ethyl ester; C044542:octadecyl Rhodamine B chloride