ALLOSTERIC BINDING OF 9-METHOXY-ALPHA-LAPACHONE AND ALCURONIUM TO THEMUSCARINIC ACETYLCHOLINE-RECEPTOR M2 SUBTYPE

The binding of [H-3]N-methylscopolamine ([H-3]NMS) to the muscarinic a cetylcholine receptor m2 subtype increased and then decreased with the increase of concentration of 9-methoxy-alpha-lapachone (LAP), a compo und isolated from a medicinal plant Mansoa alliacea, in a similar way to that reported for alcuronium (ALC). The increase in the [H-3]NMS bi nding by addition of ALC was observed not only for the m2 subtype but also for the m3 subtype, but the effect of LAP was observed only for t he m2 subtype. The effect of LAP and ALC to increase the [(3)]NMS bind ing was dependent on the presence of salts; the increase of [H-3]NMS b inding by LAP or ALC was hardly observed in a medium of 10 mM Hepes-KO H buffer but became apparent by addition of 100 mM NaCl or KCl, 10 mM MgCl2 or CaCl2, or 0.5 mM AlCl3. The dissociation of [H-3]NMS bound to the m2 subtype in particulate preparations was inhibited by addition of LAP as well as ALC, and unexpectedly these effects were not depende nt on the presence of salts. The [H-3]NMS binding to the m2 subtype so lubilized and purified from Sf9 membranes was also increased by additi on of LAP or ALC in the presence of salts but not in their absence. Th ese results indicate that LAP and ALC directly interact with the recep tor molecule at a site distinct from the [H-3]NMS-binding site causing inhibition of the release of prebound [3H]NMS and that the binding of cation(s) to receptors is necessary for positive cooperative binding of [H-3]NMS and LAP or ALC.