It 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 M₁–M₄ 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 [³H]N-methylscopolamine 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 (−)-eburnamonine on the M₂ and M₄ receptors and by brucine on the M₁ and M₃ 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 M₁, M₃, and M₄ receptors, for pentylthio-TZTP on the M₂ receptors, and for arecoline on the M₃receptors. (−)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M₂receptors and for pilocarpine on the M₄ 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 (−)-eburnamonine and pilocarpine and (−)-eburnamonine and oxotremorine-M on the M₂ receptors (25- and 7-fold increases in affinity, respectively) and between brucine and pentylthio-TZTP on the M₂ and brucine and carbachol on the M₁receptors (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.

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 have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation.

G-protein coupled receptors (GPCRs) are membrane proteins that convey extracellular signals to the cellular milieu. They represent a target for more than 30% of currently marketed drugs. Here we review the effects of membrane cholesterol on the function of GPCRs of Class A. We review both the specific effects of cholesterol mediated via its direct high-affinity binding to the receptor and non-specific effects mediated by cholesterol-induced changes in the properties of the membrane. Cholesterol binds to many GPCRs at both canonical and non-canonical binding sites. It allosterically affects ligand binding to and activation of GPCRs. Additionally, it changes the oligomerization state of GPCRs. In this review, we consider a perspective of the potential for the development of new therapies that are targeted at manipulating the level of membrane cholesterol or modulating cholesterol binding sites on to GPCRs.

Xanomeline is a functionally selective M₁/M₄ 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 M₁ receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M₁ and M₂ receptors expressed in Chinese hamster ovary cells and [³H]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M₂ receptor with similar affinity as at the M₁ receptor (K_I = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M₂ receptors was markedly slower than to M₁ receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5′-O-(3-[³⁵S]thio)triphosphate binding at M₁ receptors, whereas at M₂ 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 M₂ receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M₁ than at M₂ 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 M₁ and the M₂ receptor.

Allosteric modulators are ligands that bind to a site on the receptor that is spatially separated from the orthosteric binding site for the endogenous neurotransmitter. Allosteric modulators modulate the binding affinity, potency, and efficacy of orthosteric ligands. Muscarinic acetylcholine receptors are prototypical allosterically-modulated G-protein-coupled receptors. They are a potential therapeutic target for the treatment of psychiatric, neurologic, and internal diseases like schizophrenia, Alzheimer’s disease, Huntington disease, type 2 diabetes, or chronic pulmonary obstruction. Here, we reviewed the progress made during the last decade in our understanding of their mechanisms of binding, allosteric modulation, and in vivo actions in order to understand the translational impact of studying this important class of pharmacological agents. We overviewed newly developed allosteric modulators of muscarinic receptors as well as new spin-off ideas like bitopic ligands combining allosteric and orthosteric moieties and photo-switchable ligands based on bitopic agents.