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.
Footnotes
- Received January 27, 2000.
- Accepted March 22, 2000.
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Send reprint requests to: Dr. S. Lazareno, MRC Technology, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, UK. E-mail:slazare{at}nimr.mrc.ac.uk.
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This work was funded by Sankyo Co. Ltd, Tokyo, Japan, and the Medical Research Council, UK.
- The American Society for Pharmacology and Experimental Therapeutics
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