Positive Cooperativity of Acetylcholine and Other Agonists with Allosteric Ligands on Muscarinic Acetylcholine Receptors

Abstract

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 ...

A Fluorescence Resonance Energy Transfer-based M2 Muscarinic Receptor Sensor Reveals Rapid Kinetics of Allosteric Modulation

“…Gallamine and dimethyl-W84 have been described as negative allosteric compounds at M 2 muscarinic receptors (29,(33)(34)(35). They have been reported to reduce the equilibrium affinity of antagonists, but even more so of agonists at these receptors and to slow down both the association and, less so, the dissociation of antagonist radioligands (10).…”

Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

“…Radioligand Binding Experiments-Measurements of radioligand binding were performed essentially as described (15,22,23). Membranes corresponding to 600,000 cells were incubated at 25°C in a final incubation volume of 0.8 ml.…”

“…The present study of the complexity of the binding of muscarinic antagonists was started within the context of our investigations of allosteric modulations of muscarinic receptors (13)(14)(15). We wanted to know how the dissociation of muscarinic antagonists N-[ ]QNB from the receptors is affected by changes in the concentration of the unlabeled ligand added to the system in order to reveal ("induce") the dissociation, by the duration of the preceding radioligand association, and also by the treatment of receptors with a compound known to associate covalently with the classical binding site of muscarinic receptors (benzilylcholine mustard (BCM); see "Experimental Procedures") (16,17).…”

“…On the day of experiment, membranes were sedimented by 15 min of centrifugation at 60,000 ϫ g, and washed twice by resuspension and recentrifugation (see Ref. 15 …”

Being mindful of seven‐transmembrane receptor ‘guests’ when assessing agonist selectivity

“…For example, the modulator eburnamonine binds to the muscarinic M 2 receptor; if the guest molecule is acetylcholine, the binding is enhanced by a factor of 15. If the guest molecule is pilocarpine, the affinity is decreased by a factor of 25 (Jakubic et al, 1997). For these reasons, the signalling pathway activated by the agonist-bound receptor also controls the affinity of the receptor for the agonist.…”