Muscarinic Receptors: Targeting Allosteric Modulation by the Development and Application of the Radiolabeled Allosteric Agent [3H]dimethyl-W84

During the last decade it became evident that in an increasing number of G-protein-coupled receptors neurotransmitter binding and action may be modulated by allosteric modulators interacting with an allosteric site on the extracellular face of the receptor topologically distinct from the orthosteric site (1). During the last 40 years mAChRs have been a paradigm for the simultaneous binding of two structurally different agents at distinct sites in a G-protein-coupled receptor leading to a ternary complex. In the ternary complex these agents may affect each other’s binding affinity. These allosteric cooperative interactions offer a novel approach to get an insight into the molecular principles of ligand-receptor interactions and open the perspective for new strategies of drug therapy [for review see Refs. (2-7)]. Many features of allosteric modulation, e.g., defined maximum effect on function, mapping of modulator receptor interactions, subtype selective modulation of orthosteric ligand binding at the neurotransmitter site, and

data evaluation by means of the ternary complex model of allosteric interaction, were initially studied in muscarinic receptors using orthosteric radioligands. Archetypal M2 prevalent muscarinic allosteric agents known for many years include gallamine, alcuronium, and bis(ammonio) alkanetype agents such as W84. Until the development of the radioligand [3H]dimethyl-W84 (Fig. 1) for the allosteric site in muscarinic M2 receptors described in this review, the binding characteristics of allosteric agents had to be deduced from the effects they exerted on the binding of radioligands to the orthosteric site [e.g., Lazareno and Birdsall (8)]. Because of the lack of a radiolabeled allosteric agent it had not been possible to directly study the binding of agents at the allosteric site (3). Additionally, it had not been possible to reject by direct measurements the objection that allosteric actions on ligand-occupied muscarinic receptors might only be an aspect of a rather nonspecific attachment to cellular membranes, although abundant evidence had pointed to a specific and preferential interaction. With the development of [3H]dimethyl-W84 as an allosteric radioligand it was possible for the first time to test the allosteric principle in a G-proteincoupled receptor in a rigorous way by directly addressing the ‘‘other side of the coin.’’ This review focuses on the development of the radioalloster [3H]dimethyl-W84 and highlights the different stages: addressing receptor specificity of allosteric action, choice of the modulator, target characterization, labeling the receptor by the radioalloster, testing the cooperativity model, and radioalloster application to elucidate different modes of allosteric actions. Finally, a mutagenesis approach, which led to a model of the M2 receptor generated by molecular modeling, is presented that allows visualizing of the allosteric binding site used by a number of allosteric modulators including dimethyl-W84.