SUMMARY1. An allosteric interaction occurs when the binding of a ligand to its site on a receptor is able to modify the binding of another ligand to a topographically distinct site on the same receptor and vice versa. The muscarinic cholinoceptors represent the best-studied examples of allosteric phenomena among the G-protein-coupled receptor superfamily.2. The simplest model describing allosteric interactions at muscarinic cholinoceptors is the ternary complex model, which allows for a three-way interaction between the receptor, a classical (orthosteric) ligand and an allosteric modulator. The interaction may be quantified using the dissociation constant of each ligand for its respective binding site on the free receptor and the 'co-operativity factor' ␣. This latter term is the ratio of affinities of a ligand for the occupied versus the unoccupied receptor and is a measure of the magnitude of the cooperativity between two concomitantly bound ligands.3. Identification of allosteric phenomena requires the utilization of both radioligand binding and functional approaches. Manifestations of allosterism include: (i) a limited ability to influence radioligand binding as the concentration of the latter is increased; (ii) alterations in the dissociation rate of orthosteric ligands; (iii) curvilinear Schild regressions; and (iv) nonadditivity of agonist/orthosteric antagonist/allosteric modulator combination concentration ratios.4. Allosteric modulators of muscarinic cholinoceptors represent a diverse range of compounds. Some of the most studied agents include gallamine, alcuronium and the bis-ammonium compounds, C7/3-phth and W84. Alcuronium has proven a most useful pharmacological tool, as it has been shown to display both positive and negative co-operativity, depending on the receptor subtype and orthosteric ligand involved in the interaction.5. Evidence has accumulated pointing to the existence of a common allosteric binding site on the muscarinic cholinoceptors, located close to the orthosteric site, but at a more extracellular level. However, the possibility of more than one accessory binding site on various receptor subtypes cannot be excluded.6. Allosteric modulators offer a number of potential therapeutic advantages, including a ceiling level to their effects and the possibility of 'absolute selectivity' of action, based on the degree of co-operativity rather than the affinity of the modulator for any one receptor subtype.

Signaling pathways for muscarinic acetylcholine receptors (mAChRs) include several enzymes and ion channels. Recent studies have revealed the importance of various isoforms of both alpha and betagamma subunits of G proteins in initiation of signaling as well as the role of the small monomeric G protein, Rho, in the activation of phospholipase D. Modulation of adenylyl cyclase activity by mAChRs appears more diverse as the interaction of various receptor subtypes with the many isoforms of the enzyme are studied. Both alpha and beta subunits of G(i/o) may be involved. Some mAChR responses arise through release of nitric oxide from nitrergic nerves, including salivary gland secretion and hippocampal slow wave activity. mAChRs utilize a variety of intracellular pathways to activate various mitogen-activated protein kinases. The kinases are involved in cholinergic regulation of kidney epithelial function, catabolism of amyloid precursor protein, hippocampal long-term potentiation, activation of phospholipase A(2), and gene induction. mAChR activation can also stimulate or inhibit cellular growth and apoptosis, dependent on prior levels of cellular activity. Modulation of ion channels by mAChR agonists appears increasingly complex, based on recent studies. K(+) channels may be activated by M(2) and M(4) mAChR stimulation, although in the rat superior cervical ganglion topographical constraints appear to limit the effect to the M(2) mAChR. Another ganglionic K(+) current, the M current, is inhibited by M(1) mAChR activation, but in murine hippocampus inhibition involves another receptor subtype. R-type Ca(2+) channels are both facilitated and inhibited by M(1) and M(2) mAChRs; facilitation being more pronounced with activation of M(1) mAChRs and inhibition with M(2) mAChRs.

Signaling pathways for muscarinic acetylcholine receptors (mAChRs) include several enzymes and ion channels. Recent studies have revealed the importance of various isoforms of both alpha and betagamma subunits of G proteins in initiation of signaling as well as the role of the small monomeric G protein, Rho, in the activation of phospholipase D. Modulation of adenylyl cyclase activity by mAChRs appears more diverse as the interaction of various receptor subtypes with the many isoforms of the enzyme are studied. Both alpha and beta subunits of G(i/o) may be involved. Some mAChR responses arise through release of nitric oxide from nitrergic nerves, including salivary gland secretion and hippocampal slow wave activity. mAChRs utilize a variety of intracellular pathways to activate various mitogen-activated protein kinases. The kinases are involved in cholinergic regulation of kidney epithelial function, catabolism of amyloid precursor protein, hippocampal long-term potentiation, activation of phospholipase A(2), and gene induction. mAChR activation can also stimulate or inhibit cellular growth and apoptosis, dependent on prior levels of cellular activity. Modulation of ion channels by mAChR agonists appears increasingly complex, based on recent studies. K(+) channels may be activated by M(2) and M(4) mAChR stimulation, although in the rat superior cervical ganglion topographical constraints appear to limit the effect to the M(2) mAChR. Another ganglionic K(+) current, the M current, is inhibited by M(1) mAChR activation, but in murine hippocampus inhibition involves another receptor subtype. R-type Ca(2+) channels are both facilitated and inhibited by M(1) and M(2) mAChRs; facilitation being more pronounced with activation of M(1) mAChRs and inhibition with M(2) mAChRs.

This study investigated the reciprocal cross-interactions between two distinct allosteric sites on the M 4 muscarinic acetylcholine receptor (mAChR) in the absence or presence of different orthosteric ligands. Initial studies revealed that two novel benzimidazole allosteric modulators, 17-␤-hydroxy-17-␣-ethy nyl-delta(4)-androstano [3,2-b] (McN-A-343), or xanomeline] revealed low degrees of negative cooperativity between WIN 62,577 and each agonist, whereas stronger negative cooperativity was observed against atropine. It is interesting that when these experiments were repeated using the prototypical modulators heptane-1,7-bis-(dimethyl-3Ј-phthalimidopropyl)-ammonium bromide (C 7 /3-phth), alcuronium, or brucine (which act at a separate allosteric site), WIN 62,577 exhibited negative cooperativity with each modulator when the orthosteric site was unoccupied, but this switched to neutral cooperativity when the receptor was occupied by [ 3 H]QNB. Dissociation kinetic experiments using [ 3 H]NMS and combination of C 7 /3-phth with WIN 62,577 also provided evidence for neutral cooperativity between the two allosteric sites when the orthosteric site is occupied. Together, these results provide insight into the nature of the interaction between two distinct allosteric sites on the M 4 mAChR and how this interaction is perturbed upon occupancy of the orthosteric site.Muscarinic acetylcholine receptors (mAChRs) are prototypical members of the family A G protein-coupled receptor superfamily and mediate the majority of the actions of acetylcholine (ACh) in both the peripheral and the central nervous systems. Although these receptors are a focus of intense research as potential therapeutic targets, a significant challenge is the issue of high sequence conservation within the orthosteric domain across all five mAChR subtypes (Hulme et al., 1990;Wess, 1993). Such sequence conservation can account for the current paucity of orthosteric mAChR agonists and antagonists that display high selectivity for one mAChR subtype to the relative exclusion of all others. This problem is particularly pertinent to studies of the central nervous system, which is known to express all five subtypes of mAChR (Ehlert et al., 1995). Recent studies using mAChR knockout mice have highlighted the role of specific mAChRs in central disorders such as cognitive dysfunction, schizophrenia, and a variety of pain states (Gomeza et al., 2001;Hamilton et al., 2001;Wess et al., 2003), and thus, the ability to better target drugs to each of the mAChRs is of ongoing therapeutic relevance. 1 Current affiliation: Metabolic Pharmaceuticals Ltd., Baker Heart Research Institute, Prahran, Victoria, Australia.Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.024711.ABBREVIATIONS: mAChR, muscarinic acetylcholine receptor; C 7 /3-phth, heptane-1,7-bis-(dimethyl-3Ј-phthalimidopropyl)-ammonium bromide; DMEM, Dulbecco's modified Eagle's medium; NMS, N-methylscopolamine; ACh, acetylcholine; CH...

The interaction between a novel G protein-coupled receptor modulator, N- (2,3-diphenyl-1,2,4-thiadiazole-5-(2H)-ylidene) methanamine hydrobromide (SCH-202676), and the M 1 muscarinic acetylcholine receptor (mAChR) was investigated. In contrast to the prototypical mAChR allosteric modulator, heptane 1,7-bis-(dimethyl-3Ј-phthalimidopropyl)-ammonium bromide (C 7 /3-phth), SCH-202676 had no effect on the dissociation kinetics of