Abstract

We have studied the interactions of five indolocarbazoles withN-[methyl-³H]scopolamine (NMS) and unlabeled acetylcholine at M₁-M₄ muscarinic receptors, using equilibrium and nonequilibrium radioligand binding studies. The results are consistent with an allosteric model in which the primary and allosteric ligands bind simultaneously to the receptor and modify each other's affinities. The compounds were generally most active at M₁ receptors. [³H]NMS binding was enhanced by staurosporine, KT5720, and KT5823 at M₁ and M₂ receptors, and by K-252a at M₁ receptors. Gö 7874 reduced [³H]NMS affinity by up to threefold for all subtypes. A range of cooperative effects with acetylcholine was seen, and, at the M₁ receptor, KT5720 had a log affinity of 6.4 and enhanced acetylcholine affinity by 40%. The compounds inhibited the dissociation of [³H]NMS to different extents across the receptor subtypes, with the largest effects at M₁ receptors. In equilibrium binding studies the inhibitory potency of gallamine at M₁ receptors was not affected by KT5720, indicating that these agents bind to two distinct allosteric sites and have neutral cooperativity with each other. In contrast, gallamine and staurosporine had a negatively cooperative or competitive interaction at M₁ receptors. Similarly, the potency and relative effectiveness of KT5720 for inhibiting [³H]NMS dissociation from M₁ receptors were not affected by gallamine or brucine, but were affected in a complex manner by staurosporine. These results demonstrate that there are at least two distinct allosteric sites on the M₁ receptor, both of which can support positive cooperativity with acetylcholine.