SUMMARYIt is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M 1 -M 4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [ 3 H]Nmethylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (Ϫ)-eburnamonine on the M 2 and M 4 receptors and by brucine on the M 1 and M 3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M 1 , M 3 , and M 4 receptors, for pentylthio-TZTP on the M 2 receptors, and for arecoline on the M 3 receptors. (Ϫ)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M 2 receptors and for pilocarpine on the M 4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (Ϫ)-eburnamonine and pilocarpine and (Ϫ)-eburnamonine and oxotremorine-M on the M 2 receptors (25-and 7-fold increases in affinity, respectively) and between brucine and pentylthio-TZTP on the M 2 and brucine and carbachol on the M 1 receptors (8-fold increases in affinity). The discovery that it is possible to increase the affinity of muscarinic receptors for their agonists by allosteric modulators offers a new way to subtype-specific pharmacological enhancement of transmission at cholinergic (muscarinic) synapses.It has long been known that the affinity of muscarinic receptors for their agonists and antagonists can be diminished by compounds acting allosterically (for reviews, see Refs. 1-3). It was discovered, however, that the affinity of cardiac muscarinic receptors for the muscarinic antagonist NMS can be increased by the neuromuscular blocker alcuronium (4, 5). Subsequently, it was shown that the positive allosteric action of alcuronium on the binding of NMS is specific for the M 2 and M 4 muscarinic receptor subtypes (Ref.6, but see Refs. 7 and 8 for conflicting data concerning the M 3 and M 4 subtypes) and that it also applies to the binding ...

Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We classical-binding site (12-18). The attachment of the allosteric modulator to the allosteric-binding site brings about a change in the conformation of the classical (orthosteric)-binding site and thereby an increase or (more usually) a decrease in the affinity of the receptor for muscarinic agonists and competitive antagonists.The effects of the activation of the allosteric-binding site on the function of the classical-binding site (as reflected by changes in the association, dissociation, and equilibrium binding of muscarinic agonists and competitive antagonists) have been amply investigated (6-8). However, little attention has been paid to the possibility that the allosteric modulators might also affect the conformation and function of the G protein binding site and, consequently, the interaction between muscarinic receptors, G proteins, and the G protein-regulated effector molecules. We present evidence that the allosteric modulators alcuronium, gallamine, and strychnine acting in the absence of agonists have profound agonist-like effects on the synthesis of cAMP and inositol phosphates (IPs) in cells stably transfected with the genes for muscarinic receptors and that, apparently, muscarinic receptors can be activated not only by ligands acting via their classical-binding site, but also by ligands acting via the allosteric-binding site. We suggest that the same novel mechanism of receptor activation may operate on the other G protein-coupled receptors.Muscarinic acetylcholine receptors belong to the large group of receptors coupled with G proteins (1). Signal transduction from the M2 and M4 muscarinic receptor subtypes occurs preferentially via the guanine nucleotide-binding regulatory proteins Gi and Go, whereas the M1, M3, and M5 receptor subtypes couple preferentially with proteins of the Gq family (2, 3). Muscarinic agonists bind to the classical-binding site, which is located in the depth of the cell membrane in a barrel formed by the seven transmembrane segments of the receptor molecule (4, 5). Agonist binding brings about a change in the conformation of the intracellular receptor domain responsible for the contact with the G protein, and this change brings about the activation of the G protein. Although details of these interactions are the subject of scrutiny and debate, it is generally believed that the attachment of the signal molecule to the classical-binding site is the necessary first step in muscarinic receptor-mediated signal transduction across the cell membrane.However, it is well known that the binding properties of the classical-binding sites of muscarinic receptors are subject to allosteric reg...

Xanomeline is a functionally selective M 1 /M 4 muscarinic acetylcholine receptor agonist that nevertheless binds with high affinity to all five subtypes of muscarinic receptors. A novel mode of interaction of this ligand with the muscarinic M 1 receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M 1 and M 2 receptors expressed in Chinese hamster ovary cells and [3 H]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M 2 receptor with similar affinity as at the M 1 receptor (K I ϭ 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M 2 receptors was markedly slower than to M 1 receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5Ј-O-(3-[35 S]thio)triphosphate binding at M 1 receptors, whereas at M 2 receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon preincubation for 1 h. Development of xanomeline agonistic effects at the M 2 receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M 1 than at M 2 receptors. Our data support the notion that xanomeline interacts with multiple sites on the muscarinic receptor, resulting in divergent conformations that exhibit differential effects on ligand binding and receptor activation. These conformations are both time-and concentration-dependent and vary between the M 1 and the M 2 receptor.