Allosteric interactions of staurosporine and other indolocarbazoles with N-[methyl-H-3] scopolamine and acetylcholine at muscarinic receptor subtypes: Identification of a second allosteric site

We have studied the interactions of five indolocarbazoles with N-[methyl-H- 3] scopolamine (NMS) and unlabeled acetylcholine at M-1-M-4 muscarinic rece ptors, using equilibrium and nonequilibrium radioligand binding studies. Th e results are consistent with an allosteric model in which the primary and allosteric ligands bind simultaneously to the receptor and modify each othe r's affinities. The compounds were generally most active at M-1 receptors. [H-3] NMS binding was enhanced by staurosporine, KT5720, and KT5823 at M-1 and M-2 receptors, and by K-252a at M-1 receptors. Go 7874 reduced [H-3] NM S affinity by up to threefold for all subtypes. A range of cooperative effe cts 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 i nhibited the dissociation of [H-3] NMS to different extents across the rece ptor subtypes, with the largest effects at M-1 receptors. In equilibrium bi nding studies the inhibitory potency of gallamine at M-1 receptors was not affected by KT5720, indicating that these agents bind to two distinct allos teric sites and have neutral cooperativity with each other. In contrast, ga llamine and staurosporine had a negatively cooperative or competitive inter action at M-1 receptors. Similarly, the potency and relative effectiveness of KT5720 for inhibiting [H-3] NMS dissociation from M-1 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 disti nct allosteric sites on the M-1 receptor, both of which can support positiv e cooperativity with acetylcholine.