The focus of this review paper is factors affecting data interpretation in ligand binding assays under equilibrium conditions. Protocols for determining Kd (the equilibrium dissociation constant) and KdA (the equilibrium inhibitor constant) for receptor ligands are discussed. The basic theory describing the interaction of a radiotracer and an unlabelled competitor ligand with a receptor is developed. Inappropriate experimental design may result in ligand depletion and non-attainment of equilibrium, distorting the calculation of Kd and KdA. Strategies, both theoretical and practical, will be given to avoid and correct such errors, thus leading to the determination of reliable values for these constants. In determining KdA from competition binding studies, two additional concepts are discussed. First, the necessity to measure an adequate specific binding signal from the bound radiotracer ligand limits the range of affinity constants that can be measured: a particular set of assay conditions may lead to an upper limit on the apparent affinity of unlabelled ligands. Second, an extension of the basic assay methodology can indicate whether the interaction between the tracer and a test ligand is mediated by a competitive or an allosteric mechanism. Finally, the review ends with a discussion of two factors that are often overlooked: buffer composition and the temperature at which the assay is conducted, and the impact these can have on affinity measurements and the understanding of drug interactions.

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Some antagonists exhibit tissue selectivity in their pharmacological antagonism of muscarinic responses. However, the affinity constants for equilibrium binding of classical antagonists to muscarinic receptors in subcellular preparations have shown only small variations in different peripheral tissues and regions of the brain. The binding curves do not deviate significantly from the simple Langmuir isotherm, indicating apparent homogeneity of the receptor population in any given region. In contrast, heterogeneity has been detected by agonist binding studies but this may arise from different environmental or coupling restraints on the agonist-induced conformational change and cannot be taken as evidence for different receptor subtypes. We report here binding studies using a new anti-muscarinic drug, pirenzepine, in which we found heterogeneity of binding that correlates well with the pharmacological activity.

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Muscarinic acetylcholine receptors (mAChRs) are known to be involved in learning and memory, but the molecular basis of their involvement is not well understood. The availability of new and specific biochemical tools has revealed a crucial role for the mitogen-activated protein kinase (MAPK) family in learning and memory. Here, we examine the link between mAChRs and MAPK in neurons. Using the MAPK kinase (MEK)-specific inhibitor PD98059, we first demonstrate a necessary role for active ERKI/II in long-term potentiation in vivo. Using phospho-specific antibodies that recognize the activated form of ERKI/II, we find that the level of ERKI/II activation in brain is regulated by mAChRs. Carbachol, a muscarinic agonist, induces prolonged activation of ERKI/II, without effect on the related kinase SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal protein kinase) in primary cortical cultures. ERKI/II activation is Src-dependent and partially phosphoinositide-3 kinase- and Ca(2+)-dependent but is PKC-independent. M1-M4 mAChR subtypes expressed in COS-7 cells can all induce ERKI/II activation using a signal transduction pathway similar to that operating in neurons. The nature of the signal transduction suggests that ERKI/II can serve as a convergence site for mAChR activation and other neurotransmitter receptors.