Allosteric binding sites on muscarinic acetylcholine receptors

In this issue of Molecular Pharmacology, Tr?nkle et al. (p. 1597) present new findings regarding the existence of a second allosteric site on the M2 muscarinic acetylcholine receptor (M2 mAChR). The M2 mAChR is a prototypic class A G protein-coupled receptor (GPCR) that has proven to be a very useful model system to study the molecular mechanisms involved in the binding of allosteric GPCR ligands. Previous studies have identified several allosteric muscarinic ligands, including the acetylcholinesterase inhibitor tacrine and the bis-pyridinium derivative 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3), which, in contrast to conventional allosteric muscarinic ligands, display concentration-effect curves with slope factors >1. By analyzing the interactions of tacrine and Duo3 with other allosteric muscarinic agents predicted to bind to the previously identified ;common' allosteric binding site, Tr?nkle et al. provide evidence suggesting that two allosteric agents and one orthosteric ligand may be able to bind to the M2 mAChR simultaneously. Moreover, studies with mutant mAChRs indicated that the M2 receptor epitopes involved in the binding of tacrine and Duo3 may not be identical. Molecular modeling and ligand docking studies suggested that the additional allosteric site probably represents a subdomain of the receptor's allosteric binding cleft. Because allosteric binding sites have been found on many other GPCRs and drugs interacting with these sites are thought to have great therapeutic potential, the study by Tr?nkle et al. should be of considerable general interest.     

Interactions of orthosteric and allosteric ligands with [3H]dimethyl-W84 at the common allosteric site of muscarinic M2 receptors

An optimized assay for the binding of [3H]dimethyl-W84 to its allosteric site on M2 muscarinic receptors has been used to directly measure the affinities of allosteric ligands. Their potencies agree with those deduced indirectly by their modulation of the equilibrium binding and kinetics of [3H]N-methylscopolamine ([3H]NMS) binding to the orthosteric site. The affinities and cooperativities of orthosteric antagonists with [3H]dimethyl-W84 have also been quantitated. These affinities agree with those measured directly in a competition assay using [3H]NMS. All these data are compatible with the predictions of the allosteric ternary complex model. The association and dissociation kinetics of [3H]dimethyl-W84 are rapid but the estimate of its association rate constant is nevertheless comparable with that found for the orthosteric radioligand, [3H]NMS. This is unexpected, given that the allosteric site to which [3H]dimethyl-W84 binds is thought to be located on the external face of the receptor and above the [3H]NMS binding site that is buried within the transmembrane helices. The atypical allosteric ligands tacrine and 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3) inhibit [3H]dimethyl-W84 binding with the same potencies and comparably steep slope factors as found for inhibition of [3H]NMS binding. Tacrine and Duo3 decrease [3H]dimethyl-W84 affinity, not the number of binding sites. It is suggested that these atypical ligands either bind to the two known spatially separated allosteric sites on muscarinic receptors with positive cooperativity or their binding to the common allosteric site modulates receptor-receptor interactions such that homotropic positive cooperativity within a dimer or higher oligomer is generated.     

First gallamine-tacrine hybrid: design and characterization at cholinesterases and the M2 muscarinic receptor

Gallamine and tacrine are allosteric antagonists at muscarinic M2 acetylcholine receptors and inhibitors of acetylcholinesterase. At both acetylcholine-binding proteins, gallamine and tacrine are known to occupy two different binding sites: in M2 receptors within the allosteric binding area and in acetylcholinesterase at its catalytic and its peripheral site. To find new ligands of both targets, we designed a gallamine-tacrine dimer and several derived hybrid compounds to address the two binding sites. Their M2 receptor allosteric and acetylcholinesterase inhibitory potential was determined. The hybrid compounds revealed an allosteric potency in the low nanomolar range exceeding the allosteric potency of gallamine and tacrine by factors of 100 and 4800, respectively. Cholinesterase inhibition was augmented by hybrid formation, and all compounds exhibited IC50 values in the lower nanomolar range. Thus, gallamine-tacrine hybrid formation is a valuable approach toward high affinity ligands concurrently targeting these acetylcholine-binding proteins.     

Identification of various allosteric interaction sites on M1 muscarinic receptor using 125I-Met35-oxidized muscarinic toxin 7

Monoiodinated, Met35-oxidized muscarinic toxin 7 (MT7ox) was synthesized, and its affinity constants for free or N-methyl scopolamine (NMS)-occupied hM1 receptor were measured directly by equilibrium and kinetic binding experiments. Identical values were obtained with the two types of assay methods, 14 pM and 0.9 nM in free or NMS-liganded receptor states, respectively, highlighting a strong negative cooperativity between this allosteric toxin and NMS. Identical results were obtained with indirect binding experiments with [3H]NMS using the ternary complex model, clearly demonstrating the reciprocal nature of this cooperativity. Furthermore, the effects of various orthosteric and allosteric agents on the dissociation kinetic of 125I-MT7ox were measured and show that, except for the MT1 toxin, all of the ligands studied [NMS, atropine, gallamine, brucine, tacrine, staurosporine, and (9S,10S,12R)-2,3,9,10,11-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT5720)] interact allosterically with muscarinic toxin 7. Equilibrium binding experiments with 125I-MT7ox and [3H]NMS were conducted to reveal the effects of these ligands on the free receptor, and affinity constants (pKx values) were calculated using the allosteric ternary complex model. Our results suggest that MT7 toxin interacts with hM1 receptor at a specific allosteric site, which may partially overlap those identified previously for "classic" or "atypical" allosteric agents and highlight the potential of this new allosteric tracer in studying allosterism at muscarinic receptors.     

A novel multivalent ligand that bridges the allosteric and orthosteric binding sites of the M2 muscarinic receptor

THRX-160209 is a potent antagonist at the M(2) muscarinic acetylcholine (ACh) receptor subtype that was designed using a multivalent strategy, simultaneously targeting the orthosteric site and a nearby site known to bind allosteric ligands. In this report, we describe three characteristics of THRX-160209 binding that are consistent with a multivalent interaction: 1) an apparent affinity of the multivalent ligand for the M2 receptor subtype (apparent pK(I) = 9.51 +/- 0.22) that was several orders of magnitude greater than its two monovalent components (apparent pK(I) values < 6.0), 2) specificity of THRX-160209 for the M2 receptor subtype compared with the closely related M4 (apparent pK(I) = 8.78 +/- 0.24) and M1, M3, and M5 receptors (apparent pK(I) values 10-fold) of the dissociation rate of tritium-labeled THRX-160209 from M2 receptors by competing monovalent ligands that are known to interact with either the orthosteric site (e.g., atropine) or a well characterized allosteric site (e.g., obidoxime) on the receptor. In complementary kinetic studies assessing allosteric modulation of the receptor, unlabeled THRX-160209 retarded dissociation of [3H]N-methyl scopolamine (NMS). The effects of THRX-160209 on retardation of [3H]NMS dissociation were competitively inhibited by obidoxime, suggesting that obidoxime and THRX-160209 bind to an overlapping region coincident with other typical muscarinic allosteric agents, such as 3-methyl-5-[7-[4-[(4S)-4-methyl-1,3-oxazolidin-2-yl]phenoxy]heptyl]-1,2-oxazole (W84) and gallamine. Taken together, these data are consistent with the hypothesis that THRX-160209 binds in a multivalent manner to the M2 receptor, simultaneously occupying the orthosteric site and a spatially distinct allosteric site.     

Clozapine interaction with the M2 and M4 subtypes of muscarinic receptors.

Available evidence indicates that the antipsychotic drug clozapine acts as a partial agonist at the muscarinic M4 and as an antagonist at the M2 receptors. We wondered whether there is indeed a fundamental difference between its action on these two receptor subtypes, and whether it interacts with their classical or allosteric binding sites. In experiments on Chinese hamster ovary cells stably expressing the M2 or M4 receptors, clozapine inhibited the binding of the specific muscarinic ligand [3H]N-methylscopolamine to either receptor subtype. The affinity of the high-affinity sites for clozapine was diminished by GTP in the way expected for agonists on both the M2 and the M4 receptor subtypes. Arunlakshana-Schild plots of data obtained in saturation binding experiments with [3H]N-methylscopolamine at different concentrations of clozapine were linear with a slope of unity. Clozapine did not alter the time course of [3H]N-methylscopolamine dissociation from muscarinic M2 or M4 receptors. It inhibited the synthesis of cyclic AMP in cells expressing the M4 receptor subtype, but did not measurably inhibit the synthesis of cyclic AMP in cells expressing the M2 receptor subtype. We conclude that clozapine has a high affinity for muscarinic M2 and M4 receptor subtypes, that it associates with the classical and not with the allosteric binding site, and that it acts as a partial agonist on both the M2 and the M4 receptor subtype.     

On the interaction of gallamine with muscarinic receptor subtypes

The interaction of gallamine with muscarinic receptor subtypes was examined using radioligand binding studies. In competition studies using [3H]N-methylscopolamine [( 3H]NMS), gallamine displayed high affinity for the rat cardiac and guinea-pig uterine M2 muscarinic receptors and for the atypical muscarinic receptor present in chicken heart. Gallamine displayed low affinity for rat glandular and human 1321 N1 astrocytoma cell M3 receptors and also for the M4 receptors of NG108-15 and PC12 cells. The compound displayed intermediate affinity for M1 receptors of rat cortex labeled using [3H]pirenzepine. The interaction of gallamine with the M1 and M2 receptors appeared to be competitive at the low concentrations required to determine affinity estimates. Thus, gallamine inhibited the binding of [3H]pirenzepine to M1 receptors and [3H]NMS to M2 receptors at concentrations that were 263- and 23-fold lower, respectively, than those required to decrease radioligand dissociation kinetics. Furthermore, gallamine, at a concentration that inhibited between 63 and 71% of specific radioligand binding, had no effect on the observed rate of association of the radioligand with either the M1 or the M2 receptor. At the M3 glandular receptor, there was little separation between the concentrations of gallamine that produced inhibition of binding and those that decreased the association and dissociation rates of [3H]NMS. It is therefore difficult to determine if the inhibition of binding seen in competition studies on the M3 receptor was produced through a competitive or an allosteric mechanism. Despite its possible allosteric properties at the M3 receptor, gallamine can be used to detect heterogeneity of muscarinic receptor subtypes in several tissues and therefore represents a useful tool for defining muscarinic receptor subtypes.     

Modes of allosteric interactions with free and [3H]N-methylscopolamine-occupied muscarinic M2 receptors as deduced from buffer-dependent potency shifts

Muscarinic M2 acetylcholine receptors contain an allosteric site that is probably located at the entrance of the ligand binding pocket above the orthosteric binding site. With the orthosteric area not occupied, allosteric agents might gain access to this site. The interaction of allosteric agents with orthoster-occupied receptors is known to depend on the buffer conditions in an alloster-specific fashion. Utilizing the buffer-dependent potency shift as an indicator, we aimed to find out for two rod-like shaped and flexible allosteric agents whether or not there is evidence for a switch in the site of attachment in free compared with [3H]N-methylscopolamine ([3H]NMS)-occupied porcine heart M2 receptors. These agents are the bispyridinium compounds WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl] propane dibromide) and Duo3 (4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide). The prototype allosteric agents gallamine and alcuronium were included. Inhibition of [3H]NMS association was taken to reflect alloster interaction with free receptors, inhibition of [3H]NMS dissociation indicated binding to [3H]NMS-occupied receptors. In Na,K,Pi buffer (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4 at 23 degrees C) compared with Mg,Tris,Cl,Pi buffer (45 mM Tris-HCl, 2.6 mM MgHPO4, pH 7.3 at 37 degrees C) WDuo3 underwent the same loss of potency for the interaction with either free or [3H]NMS-liganded receptors. The loss of potency was quantified by a potency ratio (PR), i.e. the ratio between the concentrations of the modulator leading to a half-maximal delay of [3H]NMS association or dissociation, respectively, in Mg,Tris,Cl,Pi compared with Na,K,Pi. For WDuo3 the ratios were PRass=27 and PRdiss=22, respectively. For Duo3, the interaction with free and [3H]NMS-occupied receptors only slightly depended on the composition of the incubation medium: PRass=1.3, PRdiss=2.8. In contrast to the other agents, the concentration-effect curves of which had slope factors nH not different from unity, the curves of Duo3 were steep (nH about -1.6). For alcuronium the shift factors amounted to PRass=29 and PRdiss=25, for gallamine to PRass=216 and PRdiss=159. In conclusion, there was a wide variation between the allosteric agents with regard to the respective buffer dependence of action. Yet, for a given allosteric agent, the interaction with either free or [3H]NMS-occupied receptors was always characterized by the same buffer-dependent shift. Thus, even the applied rod-shaped allosteric agents do not appear to switch to the orthosteric site in free compared with orthoster-occupied M2 receptors.     

Allosteric site on muscarinic acetylcholine receptors: identification of two amino acids in the muscarinic M2 receptor that account entirely for the M2/M5 subtype selectivities of some structurally diverse allosteric ligands in N-methylscopolamine-occupied receptors

Two epitopes have been identified recently to be responsible for the high-affinity binding of alkane-bisammonium and caracurine V type allosteric ligands to N-methylscopolamine (NMS)-occupied M2 muscarinic acetylcholine receptors, relative to M5 receptors: the amino acid M2-Thr423 at the top of transmembrane region (TM) 7 and an epitope comprising the second extracellular loop (o2) of the M2 receptor including the flanking regions of TM4 and TM5. We aimed to find out whether a single amino acid could account for the contribution of this epitope to binding affinity. Allosteric interactions were investigated in wild-type and mutant receptors in which the orthosteric binding site was occupied by [3H]NMS (5 mM Na,K,Pi buffer, pH 7.4, 23 degrees C). Using M2/M5 chimeric and point-mutated receptors, the relevant epitope was narrowed down to M2-Tyr177. A double point-mutated M2 receptor in which both M2-Tyr177 and M2-Thr423 were replaced by the corresponding amino acids of M5 revealed that these two amino acids account entirely for the (approximately 100-fold) M2/M5 selectivity of the alkane-bisammonium and the caracurine V type allosteric ligands. At NMS-free M2 receptors, the caracurine V derivative also displayed approximately 100-fold M2/M5 selectivity, but the double point mutation reduced the M2 affinity by only approximately 10-fold; thus, additional epitopes may influence selectivity for the free receptors. A three-dimensional model of the M2 receptor was used to simulate allosteric agent docking to NMS-occupied receptors. M2-Tyr177 and M2-Thr423 seem to be located near the junction of the allosteric and the orthosteric areas of the M2 receptor ligand binding cavity.     

Design, synthesis, and action of oxotremorine-related hybrid-type allosteric modulators of muscarinic acetylcholine receptors

A novel series of muscarinic receptor ligands of the hexamethonio-type was prepared which contained, on one side, the phthalimidopropane or 1,8-naphthalimido-2,2-dimethylpropane moiety typical for subtype selective allosteric antagonists and, on the other, the acetylenic fragment typical for the nonselective orthosteric muscarinic agonists oxotremorine, oxotremorine-M, and related muscarinic agonists. Binding experiments in M(2) receptors using [(3)H]N-methylscopolamine as an orthosteric probe proved an allosteric action of both groups of hybrids, 7a-10a and 8b-10b. The difference in activity between a-group and b-group hybrids corresponded with the activity difference between the allosteric parent compounds. In M(1)-M(3) muscarinic isolated organ preparations, most of the hybrids behaved as subtype selective antagonists. [(35)S]GTPgammaS binding assays using human M(2) receptors overexpressed in CHO cells revealed that a weak intrinsic efficacy was preserved in 8b-10b. Thus, attaching muscarinic allosteric antagonist moieties to orthosteric muscarinic agonists may lead to hybrid compounds in which functions of both components are mixed.     

Selectivity of class I antiarrhythmic agents, disopyramide, pirmenol, and pentisomide for peripheral muscarinic M2 and M3 receptors.

The interactions of the class I antiarrhythmic agents, disopyramide, pirmenol, and pentisomide with peripheral muscarinic receptors were investigated by binding assay with [3H]N-methylscopolamine ([3H]NMS) as a ligand. All the agents inhibited the specific [3H]NMS binding to membrane preparations obtained from guinea pig submandibular gland (SG) and urinary bladder (UB) smooth muscle. The competition curves of these agents for [3H]NMS binding to SG membranes were monophasic, indicating competition with [3H]NMS at a single site. Comparison of results with those of our previous binding experiments using guinea pig left atrial (LA) membranes, showed that pirmenol had sevenfold lower affinity for glandular-type muscarinic receptors (M3) than for cardiac-type muscarinic receptors (M2). On the other hand, the dissociation constants (Ki) for disopyramide and pentisomide in SG were comparable to the high-affinity Ki values for these agents at M2 receptors. The competition curves of the three agents for [3H]NMS binding to UB membranes were biphasic and showed high- and low-affinity states of binding. The high- and low-affinity Ki values for pirmenol in UB were similar to its Ki values at M2 and M3 receptors obtained in LA and SG, respectively. The high-affinity Ki values for disopyramide and pentisomide were consistent with the respective Ki values determined in SG, whereas the low-affinity binding sites for these agents were presumably the result of their allosteric interactions with the receptors. All agents at higher concentrations slowed the dissociation of [3H]NMS elicited by an excess of atropine in both UB and SG, thus indicating allosteric interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human astrocytoma cell line 1321 N1 contains M2-glandular type muscarinic receptors linked to phosphoinositide turnover.

1. Muscarinic receptors present in the human astrocytoma cell line 1321 N1 were characterized in radioligand binding studies and in functional studies of carbachol-stimulated phosphatidylinositol (PI) turnover. 2. In radioligand binding studies the muscarinic receptor in intact cells could be labelled using [3H]-N-methylscopolamine ([3H]-NMS) but not by [3H]-pirenzepine. In the intact cells these receptors displayed low pirenzepine affinity (pKi = 6.83) indicating that they were not of the M1 subtype. Furthermore, the 1321 N1 muscarinic receptors displayed low affinity for the two M2-cardiac selective ligands methoctramine (pKi = 5.82) and AF-DX 116 (pKi = 6.29). This pharmacology was consistent with the 1321 N1 cells containing a single population of muscarinic receptors that displayed a similar pharmacology to the M2-receptor present in exocrine gland tissue. 3. The M2-gland nature of the receptors was further indicated in the functional studies where antagonist affinities were determined from their ability to antagonize carbachol-stimulated PI turnover in 1321 N1 cells. pA2 values for pirenzepine (7.31), methoctramine (6.10) and AF-DX 116 (6.52) were similar to those determined in the binding studies. 4. From these studies we conclude that 1321 N1 astrocytoma cells contain an M2-gland muscarinic receptor which mediates muscarinic receptor-mediated stimulation of PI turnover in these cells.     

Secoverine is a non-selective muscarinic antagonist on rat heart and brain receptors

In an attempt to determine if the selectivity of secoverine observed in vivo and in isolated tissues might be due to selective association with muscarinic receptor subtypes, we analyzed the binding of secoverine to three different receptors with specific radioligands: rat cardiac receptors (M2 receptors with low affinity for atropine), and rat cerebral cortical M1 receptors and M2 receptors with high affinity for atropine. At concentrations up to 10(-6) M, secoverine interaction with muscarinic receptors was competitive and of high affinity (Ki 4.10(-9) M) for cardiac and brain receptors. A detailed analysis using, in addition to [3H]N-methylscopolamine, the agonist [3H]oxotremorine-M (selective for high affinity binding sites at cardiac receptors) and the M1-selective antagonist [3H]pirenzepine at brain receptors, revealed identical affinities towards both receptor types, making it unlikely that secoverine distinguished the different muscarinic receptor subtypes. At concentrations between 10(-6) and 10(-3) M, secoverine interaction with an additional receptor site resulted in profound changes of tracer kinetics, suggesting the formation of a ternary complex (secoverine-radioligand-muscarinic receptor). The potency of secoverine for provoking this allosteric interaction was both tracer- and tissue-dependent. It is concluded that secoverine does not differentiate between M1, brain M2 and cardiac M2 receptors or between cardiac receptors with high, low and very low affinity for agonists. At very high concentrations secoverine recognized an allosteric site on the muscarinic receptors and reduced the dissociation rates of the 3H-ligands.     

Allosteric site on muscarinic acetylcholine receptors: a single amino acid in transmembrane region 7 is critical to the subtype selectivities of caracurine V derivatives and alkane-bisammonium ligands.

Diverse muscarinic allosteric ligands exhibit greatest affinity toward the M2 receptor subtype and lowest affinity toward M5. In this study, we evaluated the potencies with which two groups of highly M2/M5 selective allosteric agents modulate the dissociation of [3H]N-methylscopolamine from M2/M5 chimeric and point-mutated receptors. These allosteric ligands included two alkane-bisammonium compounds and a series of caracurine V derivatives, which are structurally closely related to (but stereochemically different from) the prototype allosteric ligand alcuronium. Like alcuronium, the caracurine V and alkane-bisammonium compounds displayed significantly increased affinities compared with M5 toward the chimera that included the M2 second outer loop (o2) plus surrounding regions. Unlike alcuronium, the compounds had enhanced affinities for a chimera with M2 sequence in transmembrane region (TM) 7; site-directed mutagenesis in wild-type and chimeric receptors indicated that the threonine residue at M2(423) was entirely responsible for the sensitivity toward TM7. Subsequent studies demonstrated that this TM7 epitope is likewise present in the M4 receptor, as M4(436)serine. The M2(423)threonine residue is near the M2(419)asparagine identified previously to influence gallamine binding. These studies demonstrate that a stereochemical difference can be sufficient to translate into divergent epitope sensitivities. Nonetheless, these allosteric ligands seem to derive affinity from two main regions of the receptor: o2 plus flanking regions and o3/TM7. These two epitopes are sufficient to explain the M2/M5 selectivity of the presently investigated compounds; this is the first time that the subtype selectivity of muscarinic allosteric agents has been completely accounted for by distinct receptor epitopes.     

Vinburnine decelerates [3H]N-methylscopolamine binding to recombinant human muscarinic M1-M4 acetylcholine receptors

The kinetics of [3H]N-methylscopolamine binding to membranes of Chinese hamster ovary (CHO) cells expressing muscarinic M(1)-M(4) acetylcholine receptors was studied. [3H]N-methylscopolamine dissociation was used for the "single-point" analysis of allosteric modulation by vinburnine (L-eburnamonine). [3H]N-methylscopolamine dissociation was decelerated by vinburnine with EC(50) values of 29.5, 4.1, 9.5 and 15.0 microM for muscarinic M(1)-M(4) receptors, respectively. Acetylcholine doubled the EC(50) of vinburnine for muscarinic M(3) receptors. These kinetic EC(50) values correlated with equilibrium binding constants, supporting the ternary allosteric model. Vinburnine also decelerated the association of [3H]N-methylscopolamine binding, resulting in opposite cooperativity for muscarinic M(1) and M(2) receptors.     

m2-toxin: A selective ligand for M2 muscarinic receptors.

Selective ligands are needed for distinguishing the functional roles of M2 receptors in tissues containing several muscarinic receptor subtypes. Because the venom of the green mamba Dendroaspis angusticeps contains the most specific antagonists known for M1 and M4 receptors (m1-toxin and m4-toxin), it was screened for toxins that inhibit the binding of [(3)H]N-methylscopolamine ([(3)H]NMS) to cloned M2 receptors. Desalted venom had as much anti-M2 as anti-M4 activity. The most active anti-M2 toxin in the venom was isolated by gel filtration, cation-exchange chromatography, and reversed-phase HPLC, and called m2-toxin. Spectrometry yielded a mass of 7095 Da, and N-terminal sequencing of 53 amino acids showed RICHSQMSSQPPTTTFCRVNSCYRRTLRDPHDPRGT-IIVRGCGCPRMKPGTKL. This sequence is more homologous to antinicotinic than antimuscarinic toxins, but it lacks three almost invariant residues of antinicotinic toxins required for their activity. m2-Toxin fully blocked the binding of [(3)H]NMS and [(3)H]oxotremorine-M to M2 receptors with Hill coefficients near 1, and blocked 77% of the binding sites for 0.1 nM [(3)H]NMS in the rat brainstem (K(i) = 11 nM). Concentrations that fully blocked cloned M2 receptors had no effect on M4 receptors, but slightly increased [(3)H]NMS binding to M1 receptors, an allosteric effect. In accord with these results, light microscopic autoradiography of the rat brain showed that m2-toxin decreased [(3)H]NMS binding in regions rich in M2 receptors and increased binding in regions rich in M1 receptors. Thus m2-toxin is a novel M2-selective, short-chain neurotoxin that may prove useful for binding and functional studies.     

Subtype-selective positive cooperative interactions between brucine analogs and acetylcholine at muscarinic receptors: functional studies

In radioligand binding studies, it has been reported that brucine, N-chloromethyl brucine, and brucine N-oxide increased the affinity of acetylcholine for M1, M3, and M4 muscarinic receptors, respectively, in a manner consistent with the predictions of the ternary complex allosteric model. We now demonstrate an equivalent ability of these three allosteric agents to modulate the actions of acetylcholine in functional studies in membranes and in whole cells. The enhancing actions of brucine and brucine N-oxide on acetylcholine (ACh) potency at M1 and M4 receptors respectively have been confirmed in guanosine-5'-O-(3-[35S]thio)triphosphate, GTPase, cAMP, and intracellular Ca2+ mobilization assays of function. In general, neither the basal nor the maximally stimulated response to ACh is affected. The subtype-selective allosteric effects of N-chloromethyl brucine on M2 and M3 receptors were shown to be qualitatively and quantitatively the same in guanosine-5'-O-(3-[35S]thio)triphosphate functional assays, in terms of both its affinity and cooperativity with ACh, as those found in binding assays. Neutral cooperativity of N-chloromethyl brucine with ACh on M4 receptor function was also observed, thereby demonstrating its "absolute subtype selectivity": a lack of action at any concentration at M4 receptors and an action at M2 and M3 receptors. The enhancing action of N-chloromethyl brucine on neurogenically released ACh binding at M3 receptors was also detected in whole tissue as an increased contraction of the isolated guinea pig ileum to submaximal electrical stimulation. In conclusion, these functional studies confirm that brucine analogs are allosteric enhancers of ACh affinity at certain muscarinic receptor subtypes.     

Gallamine binding to muscarinic M1 and M2 receptors, studied by inhibition of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding to rat brain membranes

Whereas classic muscarinic antagonist ligands appear to recognize only a single class of muscarinic receptor sites, the recently discovered antagonist pirenzepine appears to distinguish at least two classes of sites. Its unique binding properties, demonstrated in both indirect and direct binding studies, have led to an emerging concept of high affinity (M1) and low affinity (M2) sites. This concept has been supported by pharmacologic studies of functional muscarinic responses, as well as by data suggesting different effector relationships for the two sites. Gallamine possesses muscarinic antagonist properties, and it also recognizes heterogeneity among muscarinic receptors. The purpose of this study was to define gallamine-recognized heterogeneity in terms of the pirenzepine-defined M1, M2 concept. This has been done by studying the ability of gallamine to inhibit [3H]pirenzepine binding to the M1 site, and to inhibit [3H]quinuclidinylbenzilate ([3H]QNB) binding in cerebellar membrane preparations, which contain almost exclusively the M2 site. The results show that gallamine binds with high affinity to the M2 site, with Ki = 2.4 nM, and lower affinity to the M1 site with Ki = 24 nM. Within these classes gallamine does not recognize heterogeneity. The ability of gallamine to inhibit [3H]QNB binding to cortex is best described by a two-site model comprised of 77% low affinity gallamine sites (M1) and 23% high affinity gallamine sites (M2). Thus, the heterogeneity among muscarinic receptors which is recognized by gallamine within the receptor binding paradigms of this study can be attributed to the M1, M2 subtypes as defined by pirenzepine binding. In addition, gallamine at low concentrations appears to bind as a pure competitive antagonist at these two sites, indicated by linear Schild plots with slopes of 1.0, the lack of an effect on dissociation of radioligands, and the ability to protect [3H]pirenzepine and [3H]QNB-binding sites from alkylation by propylbenzylcholine mustard. These studies do not exclude the possibility of a non-competitive interaction of gallamine with the muscarinic receptor observed by other investigators at high gallamine concentrations, and postulated to occur at a site adjacent to the primary muscarinic site. It is proposed that gallamine is capable of interacting with both the primary muscarinic site and an allosteric site. These results support the emerging concept of M1 and M2 muscarinic subclasses and suggest that gallamine and related compounds may be useful in defining muscarinic receptor subclasses, given their higher affinity for the M2 site.     

Stereoselectivity of procyclidine binding to muscarinic receptor subtypes M1, M2 and M4

The goals of the present study were: (1) to investigate the binding properties of (R)- and (S)-procyclidine and two achiral derivatives of muscarinic M1, M2 and M4 receptor subtypes and (2) to identify the interactions which allow these receptors to discriminate between the two stereoisomers. (R)-Procyclidine showed a higher affinity for human neuroblastoma NB-OK 1 muscarinic M1 and rat striatum muscarinic M4 receptors, as compared to rat cardiac M2 receptors. (S)-Procyclidine had a 130-fold lower affinity than (R)-procyclidine for M1 and M4 receptors, and a 40-fold lower affinity for M2 receptors. Pyrrinol, the achiral diphenyl derivative with the cyclohexyl group of (S)-procyclidine replaced by a phenyl group, has an eight-fold lower affinity for M1 and M4 receptors, as compared to (R)-procyclidine, and a three-fold lower affinity for M2 receptors. Hexahydro-procyclidine, the corresponding achiral dicyclohexyl compound, had a 10- to 20-fold lower affinity than (R)-procyclidine for the three receptors. The increase in binding free energy, which is observed when the phenyl and cyclohexyl groups of procyclidine are separately replaced by cyclohexyl and phenyl groups, respectively, was additive in the case of M1, M2 and M4 receptors. This indicates that the muscarinic receptor stereoselectivity was based on the coexistence of two binding sites, one preferring a phenyl rather than cyclohexyl group and the second preferring a cyclohexyl rather than a phenyl group. In addition, there were also binding sites for the hydroxy moiety and the protonated amino group of the ligands. The greater affinity and stereoselectivity of M1 and M4 muscarinic receptors for (R)-procyclidine reflected the better fit of the cyclohexyl group of (R)-procyclidine to the subsite of M1 and M4 as compared to M2 receptors.     

Muscarinic allosteric modulators: atypical structure-activity-relationships in bispyridinium-type compounds

Allosteric modulators of receptor binding are known for a variety of membrane receptors. In case of muscarinic receptors, a considerable number of structurally divergent modulators have been described. For the M2 receptor subtype which has a high sensitivity to allosteric modulation most of the allosteric agents bind to the common allosteric binding site of the receptor protein. In this study, a series of DUO compounds characterized by a bispyridinium middle chain and lateral benzyloximeether moieties of a systematically varied substitution pattern has been evaluated with regard to their allosteric potency to affect M2 receptors, whose orthosteric site was blocked by [3H]N-methylscopolamine. The variations in potency were found to be surprisingly small and the structure-activity relationships of the DUO compounds diverged from those of correspondingly substituted hexamethonio-type allosteric modulators. One has to conclude that DUO compounds bind in an "atypical" manner which is in agreement with recently reported side-directed mutagenesis and molecular modeling studies.