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 ≥ 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 ≥ 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.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1 Jan 1993|