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

The binding of [H-3]N-methylscopolamine ([H-3]NMS) to the muscarinic acetylcholine receptor m2 subtype increased and then decreased with the increase of concentration of 9-methoxy-alpha-lapachone (LAP), a compound isolated from a medicinal plant Mansoa alliacea, in a similar way to that reported for alcuronium (ALC). The increase in the [H-3]NMS binding 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 the m2 subtype. The effect of LAP and ALC to increase the [(3)]NMS binding was dependent on the presence of salts; the increase of [H-3]NMS binding by LAP or ALC was hardly observed in a medium of 10 mM Hepes-KOH 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 dependent on the presence of salts. The [H-3]NMS binding to the m2 subtype solubilized and purified from Sf9 membranes was also increased by addition of LAP or ALC in the presence of salts but not in their absence. These results indicate that LAP and ALC directly interact with the receptor 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.