Allosteric Modulation of Muscarinic Receptor Signaling: Alcuronium-Induced Conversion of Pilocarpine from an Agonist into an Antagonist

Abstract

Previous studies on allosteric interactions at muscarinic receptors have often focused on ligand-receptor binding interactions, because ligand binding seemed to reflect functional consequences. The prototypal allosteric agent alcuronium is known to bind with similar affinity to the M 2 subtype of muscarinic acetylcholine receptors whether or not the receptors are occupied by the agonist pilocarpine. To determine allosteric modulation of receptor signaling by alcuronium, the effects of pilocarpine were measured in contracting guinea pig left atria and on G-protein coupling in M 2 -transfected Chinese hamster ovary (CHO) cell membranes. Alcuronium dose-dependently suppressed pilocarpine-induced reduction of isometric contraction force in atria (pIC 50, Alc ϭ 5.63) without any effect on the EC 50 of pilocarpine, consistent with an allosteric mechanism. In contrast, alcuronium shifted the concentration-effect curve of the agonist oxotremorine M to the right without affecting the maximal effect, in a formally competitive manner (pK A, Alc ϭ 5.54). If pilocarpine remained receptor bound in the presence of alcuronium, this indicates that pilocarpine can no longer act as an agonist. In support of this hypothesis, pilocarpine acted as a competitive antagonist against oxotremorine M in the presence of 10 M alcuronium. Measuring guanosine 5Ј-O-(3-[

Regulation of M2Muscarinic Acetylcholine Receptor Expression and Signaling by Prolonged Exposure to Allosteric Modulators

“…Our current findings now also suggest that these modulator-induced conformations can also be "inverse agonist-like" with respect to their effects on regulation of receptor number. Indeed, Zahn et al (2002) have previously demonstrated inverse agonism for alcuronium in an assay of M 2 mAChR-mediated guanosine 5Ј-O-(3-thio)triphosphate turnover. Overall, our observations provide further support to the concept that efficacy at GPCRs encompasses a broader spectrum of receptor behaviors than the classic activation-deactivation paradigm (Kenakin, 2002).…”

Two-State Models and the Analysis of the Allosteric Effect of Gallamine at the M2Muscarinic Receptor

“…For example, alcuronium has been shown to modulate the intrinsic efficacy of pilocarpine (Zahn et al, 2002), and the allosteric effects of strychnine on acetylcholine-stimulated guanosine 5Ј-O-(3-[…”

“…So far, only parallel shifts have been reported for the effect of gallamine on the concentration-response curves of agonists, including the partial agonist BM5, suggesting that gallamine modifies the affinity of agonists without influencing their intrinsic efficacy. In contrast, strychnine and alcuronium (Zahn et al, 2002) have been shown to reduce both the potency and maximal responses of muscarinic agonists through an allosteric mechanism. It has been shown that the allosteric shift in the concentrationresponse curve of a full agonist for eliciting a downstream response is equivalent to the product of the change in affinity (p, eq.…”

Interactions of Orthosteric and Allosteric Ligands with [3H]Dimethyl-W84 at the Common Allosteric Site of Muscarinic M2 Receptors

“…This phenomenon has been intensively studied on muscarinic acetylcholine receptors (Tuček and Proška, 1995;Birdsall et al, 1996;Ellis, 1997;Holzgrabe and Mohr, 1998). Many features of allosteric modulation have been demonstrated: enhancement of agonist binding (Birdsall et al, 1997;Jakubík et al, 1997;Lazareno et al, 1998; and action , absolute subtype selectivity of allosteric action (Birdsall et al, 1997;Lazareno et al, 1998Lazareno et al, , 1999, mapping of ligand receptor interactions Trä nkle et al, 1998a), defined maximum effects on function (Lü llmann et al, 1969;Clark and Mitchelson, 1976), and modulation of receptor signaling efficacy (Zahn et al, 2002). All five muscarinic receptor subtypes are sensitive to allosteric modulation (Ellis et al, 1991) with many of the typical quaternary ammonium modulators such as gallamine, alcuronium, W84, and dimethyl-W84, exhibiting an M 2 subtype selectivity.…”