Constitutive activity of the M1–M4 subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists

AbstractWe investigated whether muscarinic receptors of the M1–M4 receptor subtypes are constitutively active. We have found that the synthesis of cyclic AMP was enhanced by the muscarinic antagonists atropine and N-methylscopolamine (NMS) in Chinese hamster ovary (CHO) cells stably transfected with human m2 and m4 muscarinic receptor genes and in rat cardiomyocytes expressing the M2 receptor subtype, and that the production of inositol phosphates was inhibited by atropine and NMS in CHO cells stably transfected with human m1 and m3 and with rat m1 muscarinic receptor genes. The muscarinic antagonists quinuclidinyl benzilate and AF-DX 116 had no effect in some cases and acted like atropine and NMS in others. We conclude that the M1–M4 subtypes of muscarinic receptors are constitutively active in the CHO cell lines expressing them and in cardiomyocytes and that atropine and NMS act as negative antagonists on these receptor subtypes by stabilizing them in the inactive conformation

Modulation by CertainConserved AspartateResidues of the Allosteric Interaction of Gallamine at the ml Muscarinic Receptor1

ABBREVIATiONS:Asp. aspartic acid; Asn, asparagine; CHO, Chinese hamster ovary; NMS, N-methylscopolamine; Kd, equilibriumdissociation constant; KA,eqliilibnum dissociationconstant ofallosteric antagonIstfor the unhigandedreceptor; a, cooperativity factor; lC@,concentration required to inhibit 50%of ligand binding ordissociation. ABSTRACT Muscarinic acetyicholine receptors belong to a superfamily of 6-protein coupled receptors and contain within their structure several conserved aspartate residues. These residues have been implicated to play important roles in the interaction of agonists and their competitive antagonists with the receptor. In the pres entwork,we investigated whether thesameresidues might also serve as Importantcontact points for allostencantagonistsof Recently, much attention has been focused on the localiza tion of the binding site for muscarinicreceptor agonistsand their competitive antagonists. Site-directed mutagenesis of the ml muscarinic receptor indicated that four aspartate residues, which are conserved among all muscarmnicreceptor subtypes, play an important role in ligand binding and/or receptor acti vation 1989). It is believed that the primary (competitive) binding site Received for publication January 6, 1992

Subtype Differences in Pre-Coupling of Muscarinic Acetylcholine Receptors

Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex “collides” with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to Gq/11, while even-numbered receptors prefer coupling to Gi/o. We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [3H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M1 and M3 receptors also pre-couple with non-preferential Gi/o G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein

Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor

Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M1 and M4 receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of xanomeline on the binding characteristics and function of the M1 muscarinic receptor. Pretreatment of CHO cells that stably express the M1 receptor for 1 hr with increasing concentrations of xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed xanomeline-induced inhibition of [3H]NMS binding from monophasic to biphasic. The high-affinity xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound xanomeline developed with a time course that parallels the return of receptor activation by prebound xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of xanomeline, but not its persistent binding at an allosteric site. Furthermore, the long-term effects of xanomeline on the receptor are mainly due to receptor down-regulation rather than internalization

Allosteric Modulation of GPCRs of Class A by Cholesterol

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

Allosteric Modulation of Muscarinic Acetylcholine Receptors

An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity) or decrease (negative cooperativity) in the binding or action of an orthosteric agonist (e.g., acetylcholine). Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are mile-stones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer’s disease and other disorders involving impaired cognitive function

Mixed Competitive and Allosteric Antagonism by Gallamine of Muscarinic Receptor‐Mediated Second Messenger Responses in N1E‐115 Neuroblastoma Cells

Abstract: The antagonistic effects of gallamine on muscarinic receptor‐linked responses were investigated in N1E‐115 neuroblastoma cells. M1 muscarinic receptor‐mediated phos‐phoinositide hydrolysis induced by carbamylcholine was antagonized by gallamine, with a Ki value of 33 μM. By comparison, gallamine was four‐ to fivefold less potent in blocking noncardiac M2 muscarinic receptor‐mediated inhibition of cyclic AMP formation, with a Ki value of 144 μM. The resulting Arunlakshana‐Schild plots of the antagonism of both responses by gallamine were linear and exhibited slopes not differing from 1, a result indicative of a competitive mechanism. To elucidate further the nature of gallamine\u27s inhibitory actions, experiments were performed where the effects of gallamine in combination with the known competitive muscarinic antagonist, N‐methylscopolamine (NMS), were studied. In the presence of both antagonists, a supraadditive shift in the carbamylcholine dose‐response curve was demonstrated for the two responses, a result suggestive of an allosteric mode of interaction between gallamine and NMS binding sites. Confirmation that gallamine allosterically modifies the muscarinic receptor was provided by radioligand binding studies. Gallamine competition curves with either [N‐methyl‐3H]scopolamime methyl chloride ([3H]NMS) or [N‐methyl‐3H]quinuclidinyl benzilate methyl chloride ([3H]NMeQNB) were unusually shallow. Furthermore, gallamine decelerated the rate of dissociation of receptor‐bound [3H]NMS \u3e [3HJNMeQNB in a dose‐dependent manner. The present study demonstrates that whereas gallamine antagonizes carbamylcholine‐mediated responses in N1E‐115 cells in a competitive manner, an allosteric component of its action is revealed in the presence of muscarinic antagonists such as NMS. Copyright © 1989, Wiley Blackwell. All rights reserve