title: Transverse localization of the quinacrine binding site on the Torpedo acetylcholine receptor.
abstract: We demonstrated previously that a phencyclidine-displaceable quinacrine binding site exists at the lipid-protein interface of the Torpedo acetylcholine receptor (AcChR) (Valenzuela, C. F., Kerr, J. A., and Johnson, D. A. (1992) J. Biol. Chem. 267, 8238-8244). In this manuscript, we assess (1) the transverse position of this site in the lipid bilayer by examining the ability of a series of paramagnetic n-doxyl stearates (n-SALs) and iodide to quench receptor-bound quinacrine and membrane-partitioned octadecyl rhodamine B (C18-Rho) fluorescence and (2) the stoichiometry of histrionicotoxin- or phencyclidine-displaceable quinacrine binding. Initial experiments established what fraction of the n-doxyl stearates partitioned into the membranes and that the n-doxyl stearates do not interfere with quinacrine binding to the receptor at the concentrations used in the quenching studies. The n-doxyl stearate quenching experiments indicated relatively small (< 2) differences between the n-doxyl stearates to quench receptor-bound quinacrine fluorescence, with a rank order of 7-SAL > or = 5-SAL > 12-SAL > 16-SAL. This contrasts with the n-doxyl stearate quenching of the membrane-partitioned C18-Rho which showed as much as an 8.6-fold difference between the various isomers with a rank order of quenching efficiencies of 5-SAL > 7-SAL > 12-SAL > or = 16-SAL. Iodide quenching measurements indicated significant solute accessibility to membrane-partitioned C18-Rho but not to receptor-bound quinacrine. The ratios of the bimolecular quenching rate constants for free to bound quinacrine and for free rhodamine B to membrane-partitioned C18-Rho were 53.4 and 6.6, respectively. Direct titration of quinacrine into suspensions of a high concentration of AcChR-associated membranes yielded an upper limit to the binding stoichiometry of 1.4 HTX- or PCP-displaceable quinacrine binding sites/AcChR functional units. The results suggest that there is a single phencyclidine- or histrionicotoxin-displaceable quinacrine binding site located at or somewhat below the level of the C5-C7 in the phospholipid acyl chains at the lipid-protein interface.
authors.affiliation: Division of Biomedical Sciences, University of California, Riverside 92521-0121.
authors.forename: H R
authors.initials: HR
authors.lastname: Arias
authors.forename: C F
authors.initials: CF
authors.lastname: Valenzuela
authors.forename: D A
authors.initials: DA
authors.lastname: Johnson
mesh_terms: D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D010622:Phencyclidine / Q000378:metabolism; D011796:Quinacrine / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism; D013228:Stearates / Q000378:metabolism; D014101:Torpedo
chemical_list: D007454:Iodides; D011950:Receptors, Cholinergic; D012235:Rhodamines; D013228:Stearates; D011796:Quinacrine; D010622:Phencyclidine; C029773:rhodamine B