QUINACRINE AND ETHIDIUM BIND TO DIFFERENT LOCI ON THE TORPEDO ACETYLCHOLINE-RECEPTOR

Fluorescence spectroscopy was used to determine whether quinacrine and ethidium, two high-affinity noncompetitive inhibitors of the Torpedo acetylcholine receptor (AcChR), bind to the same loci. The ability of three nitroxide spin-labels, 5-doxylstearate (5-SAL), spin-labeled and rostane (ASL), and TEMPO, to quench receptor-bound quinacrine and ethi dium fluorescence was measured. When bound to a phencyclidine-displace able site on the AcChR, quinacrine was 16.9 and 19 times more efficien tly quenched than ethidium by the highly lipophilic 5-SAL and ASL, res pectively. TEMPO, which has a limited ability to partition into Torped o plasma membranes (<1%), was only twice as efficient at quenching rec eptor-bound quinacrine than ethidium fluorescence. The relative sensit ivity of quinacrine and ethidium fluorescence to paramagnetic quenchin g was examined in three solvents, 1-butanol, sodium phosphate buffer, and acetonitrile, with TEMPO as a quencher. The results from the diffe rent solvents demonstrate that quinacrine fluorescence is intrinsicall y 1.4-3.6 times more sensitive than ethidium fluorescence to quenching by nitroxide spin-labels. Examination of the effect of high concentra tions of 5-SAL on ethidium and quinacrine dissociation constants showe d that quinacrine but not ethidium binding was competitively inhibited . Together, these results indicate that although quinacrine and ethidi um bind in a mutually exclusive manner, the two inhibitors interact at different loci on the AcChR. Whereas the ethidium binding site is at a distance from membrane lipids, probably in or near the lumen, the qu inacrine binding site appears to be at a lipid-protein interface in th e transmembrane domain and at a distance from the lumen.