Detection, quantitation, and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors: interactions of strychnine and acetylcholine at muscarinic receptors.

Novel methods of detecting and quantitating cooperative interactions between an agent and both a tritiated (muscarinic) antagonist and the endogenous agonist (acetylcholine), acting at a common (muscarinic) receptor, have been devised. In a semiquantitative protocol, binding data are transformed into affinity ratios (the ratios of the apparent affinity of the ligand in the presence of the agent to the affinity of the ligand alone), which allow estimates to be made of the potency of the agent and its cooperativity with the tritiated antagonist and with the unlabeled ligand. These parameters have been quantitated by detailed binding assays or guanosine-5'-O-(3-[35S]thio)triphosphate functional assays. The kinetic phenomena associated with the allosteric interactions have been exploited in two non-equilibrium binding assays, from which the affinity constants describing the allosteric interactions can be extracted. The different assay methods give quantitatively similar and internally consistent estimates of the parameters describing the cooperative interactions. Using these assays, strychnine has been found to act allosterically at muscarinic receptors. Strychnine has an affinity of approximately 10(5) M-1 at the unliganded m1, m2, and m4 receptors but is 5-10-fold weaker at m3 receptors. It is positively cooperative with N-methylscopolamine at m2 and m4 receptors and exhibits neutral and negative cooperativity with m1 and m3 receptors, respectively. With acetylcholine, it is negatively cooperative but the degree of cooperativity is relatively low (2-7-fold), particularly at m1 and m4 receptors. The methods and equations described should be useful in detecting and quantitating allosteric interactions of agents with the endogenous neurotransmitter at G protein-coupled receptors.