Regulation of M2 muscarinic acetylcholine receptor expression and signaling by prolonged exposure to allosteric modulators.

The effects of prolonged exposure of M(2) muscarinic acetylcholine receptors (mAChRs), stably expressed in Chinese hamster ovary cells, to the allosteric modulators gallamine, alcuronium, and heptane-1,7-bis (dimethyl-3'-phthalimidopropyl)-ammonium bromide (C(7)/3'-phth) were compared with the effects of the agonist carbachol (CCh) and antagonists atropine and N-methylscopolamine (NMS). Intact cell saturation binding assays using [(3)H]NMS found that pretreatment of the cells for 24 h with CCh caused a significant down-regulation of receptor number, whereas atropine, NMS, and all three allosteric modulators caused receptor up-regulation. Functional assays using a cytosensor microphysiometer to measure whole-cell metabolic rate found no acute effects of gallamine on receptor signaling, whereas atropine seemed to behave as an inverse agonist. Pretreatment of the cells with gallamine (20 microM) or atropine (20 nM) resulted in a significant enhancement of the maximal effect evoked by CCh. In contrast, CCh (100 microM) pretreatment resulted in a significant reduction in maximal receptor signaling capacity. Time-course experiments revealed that the effects of atropine and gallamine on receptor up-regulation are only visualized after at least 12-h ligand exposure, compared with the more rapid effects of CCh, which achieve steady-state down-regulation within 90 min. Additional experiments monitoring CCh-mediated M(2) mAChR internalization in the presence of gallamine revealed that part of the mechanism underlying the effects of the modulator on receptor expression may involve a change in receptor internalization properties. These findings suggest that, like orthosteric ligands, G protein-coupled receptor allosteric modulators also are able to mediate long-term effects on receptor regulation.

G-protein-coupled receptor allosterism: the promise and the problem(s).

Allosteric modulators of G-protein-coupled receptors interact with binding sites that are topographically distinct from the orthosteric site recognized by the receptor's endogenous agonist. Allosteric ligands offer a number of advantages over orthosteric drugs, including the potential for greater receptor subtype selectivity and a more 'physiological' regulation of receptor activity. However, the manifestations of allosterism at G-protein-coupled receptors are quite varied, and significant challenges remain for the optimization of screening methods to ensure the routine detection and validation of allosteric ligands.

G-protein coupled receptors as allosteric machines.

Allosterism, whereby small molecule ligands produce global changes in the conformations of receptors, is a powerful mechanism for drug effect. This is illustrated by the recent data describing CCR5 antagonists as blockers of HIV infection. Allosteric effects are described in terms of a change in the tertiary conformation of the receptor. This paper outlines some unique features of allosteric antagonists as new drug entities. These include the fact that allosteric ligands have texture in antagonism (not all allosterically blocked receptors are alike), allosteric blockade is probe dependent (not all agonists and radioligands are blocked equally), and the fact that allosteric binding involves a separate site on the receptor may have relevance to duration of effect and selectivity. Dissociation between receptor function and binding also can be encountered with allosteric ligands.

Allosteric modulation of muscarinic receptor signaling: alcuronium-induced conversion of pilocarpine from an agonist into an antagonist.

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) = 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) = 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 microM alcuronium. Measuring guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in CHO-M(2) membranes yielded similar results. Alcuronium suppressed pilocarpine-induced stimulation of [(35)S]GTPgammaS binding (pIC(50, Alc) = 5.47) without shift in EC(50), whereas it competitively shifted the response to oxotremorine M (pK(A, Alc) = 5.97). [(3)H]Oxotremorine M binding data corresponded with the functional findings. In conclusion, alcuronium converted the agonist pilocarpine into an antagonist-a novel type of functional allosteric interaction.

Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M(3) receptors.

To clarify the involvement of specific domains of muscarinic receptors in the action of allosteric modulators, muscarinic M(3) receptors (on which allosteric interactions are weak) were genetically modified to become more similar to M(2) receptors (on which allosteric interactions are strong) and were expressed in COS-7 cells. Affinity for allosteric modulator gallamine was enhanced 25- to 50-fold by modifications of the third external loop (o3) and the negative effect of gallamine on the affinity for classical antagonist N-[(3)H]methylscopolamine ([(3)H]NMS) was augmented. Affinity for alcuronium became 3-fold higher after the o3 loop of M(3) receptors was made identical with the o3 loop of M(2) receptors, and alcuronium acquired positive influence on the affinity for [(3)H]NMS. This is the first instance of inducing positive cooperativity on muscarinic receptors by genetic manipulation. Transferring whole o2 loop from M(2) to M(3) receptors substantially enhanced affinities for gallamine and alcuronium without augmenting their negative action on [(3)H]NMS binding. In contrast, effects of simply adding two negative charges into the o2 loop of M(3) receptors were small. Removal of Arg from o1 loop abolished the negative effect of gallamine but not of alcuronium on [(3)H]NMS binding at equilibrium. Data point to an important role of o3 loop in the mechanism of the positive and negative cooperativity between [(3)H]NMS and alcuronium and gallamine, respectively, and in the binding of both modulators to M(2) receptors and reveal independence between mutation-induced changes in the affinity for a modulator and in the magnitude and direction of the allosteric effect of the modulator.

Interactions between allosteric modulators and 4-DAMP and other antagonists at muscarinic receptors: potential significance of the distance between the N and carboxyl C atoms in the molecules of antagonists.

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M2 receptors, and strychnine did so also at the M4 receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M2) and scopolamine (M2), between strychnine and butylscopolamine (M4), L-hyoscyamine (M2 and M4) and scopolamine (M4), and between brucine and scopolamine (M2). Positive effects of alcuronium, strychnine and brucine on the affinity of the M2 receptors for 4-DAMP have been confirmed by direct measurements of the binding of [3H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M2 receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.

Interactions of alcuronium, TMB-8, and other allosteric ligands with muscarinic acetylcholine receptors: studies with chimeric receptors.

A series of ligands that allosterically modulate the binding of classical ligands to muscarinic receptors was evaluated at wild-type and chimeric receptors. All of the ligands studied had highest affinity toward the M(2) subtype and lowest affinity toward the M(5) subtype. The chimeric receptors were mostly M(5) sequence; the amount of M(2) sequence ranged from about 6 to just under 30%. Alcuronium and TMB-8 had much higher affinity for the chimeric receptor that included the M(2) second outer loop of the receptor plus flanking regions of TM4 and TM5 than for any of the other chimeric receptors (the affinities of which remained similar to that of the M(5) subtype). However, this chimera retained the negative cooperativity between alcuronium and the classical antagonist N-methylscopolamine that is characteristic of M(5) (these ligands are positively cooperative at M(2)). Verapamil, tetrahydroaminoacridine, and d-tubocurarine were also sensitive to that chimeric substitution, although verapamil and tetrahydroaminoacridine had even higher affinity for a chimera with M(2) sequence in TM7. None of these ligands shared gallamine's sensitivity to a region of the third outer loop, but studies in which obidoxime reversed the allosteric effects of gallamine and other ligands suggested that they nevertheless compete for a common site. In summary, although the present data are consistent with previous studies that have suggested that allosteric ligands bind to the outermost regions of muscarinic receptors, it appears that different allosteric ligands may derive subtype selectivity from different regions of the receptor.

Testing the specificity of allosteric modulators of muscarinic receptors in phylogenetically closely related histamine H1-receptors.

Gallamine, alcuronium and W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) are prototype allosteric modulators of the G-protein coupled muscarinic acetylcholine receptor family, especially of the M2-subtype. In order to probe the specificity of muscarinic allosteric modulation, we checked whether these agents interact with histamine H1-receptors which have a high homology with muscarinic receptors. Binding experiments (38 mM Na2HPO4, 12 mM KH2PO4, pH 7.5) were performed with the H1-receptor antagonist [3H]mepyramine ([3H]MEP) in guinea pig cerebellar homogenates. For the sake of comparison, binding of [3H]N-methylscopolamine ([3H]NMS) at muscarinic M2-receptors was measured in porcine cardiac homogenates under identical conditions. The modulators retarded [3H]NMS dissociation (t1/2 control=1.3 min) concentration-dependently indicating their allosteric action with half-maximum effects for gallamine at EC50,discs=27 microM, for alcuronium at EC50,diss=53 nM, and for W84 at EC50,diss=170 nM. In contrast, [3H]MEP dissociation from H1-receptors (t1/2,control=2.6 min) remained unchanged up to concentrations of 1 mM of the modulators. Equilibrium binding of [3H]NMS (KD=0.46 nM, Bmax=98 fmol/mg protein) was inhibited by gallamine, elevated by alcuronium and left almost unchanged by W84, indicating negative, positive and nearly neutral cooperativity, respectively, with the radioligand. The ternary complex model of allosteric actions yielded the equilibrium dissociation constants K(A) for the binding of the allosteric modulators to free M2-receptors: K(A,gallamine)=100 nM, K(A,alcuronium)=450 nM, K(A,W84)=69 nM. In H1-receptors, more than 1,000-fold higher concentrations than in M2-receptors were required to elicit an effect on the binding of [3H]MEP (KD=1.2 nM, Bmax=205 fmol/mg protein). Half-maximal reduction was observed at 10 mM for gallamine, 1 mM for alcuronium and 92 microM for W84. In conclusion, the muscarinic modulators have little effect on the histamine H1-receptors.

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.

Pharmacological actions of Heteromorpha trifoliata ("dombwe") on rat isolated muscle preparations.

OBJECTIVE: To investigate the effects of Heteromorpha trifoliata on rat uterine and skeletal muscle. DESIGN: Laboratory based study using experimental animals. Investigating the effects of the plant extract and agonists on isolated muscle preparations. SETTING: Department of Physiology, University of Zimbabwe. SUBJECTS: 28 Sprague-Dawley rats. MAIN OUTCOME MEASURES: Amplitude of contraction of uterine smooth muscle and skeletal muscle. RESULTS: Experiments were performed on the isolated rat uterus preparation in which strips of myometrium were placed in tissue baths filled with Kreb's solution. The aqueous extract of the root bark of Heteromorpha trifoliata ("dombwe") contracted the rat uterus. The contractions were not antagonised by atropine but were blocked by both cyproheptadine and verapamil. In addition, "dombwe" induced a contracture of the rat diaphragm muscle in the presence of alcuronium. CONCLUSIONS: The contractile effects on the uterus appear to involve stimulation of 5-HT2 receptors leading to an increase in calcium influx into the smooth muscle cell. Promotion of calcium influx could also explain the effects observed on the skeletal muscle preparation since the contracture induced by "dombwe" occurred in the presence of the nicotinic antagonist, alcuronium. In view of the effects of "dombwe" on other smooth muscle preparations (from previous work) it appears that the pharmacological profile of the crude aqueous extract of the root bark of Heteromorpha trifoliata is complex and suggestive of the presence of more than one active ingredient.

Effects of an agonist, allosteric modulator, and antagonist on guanosine-gamma-[35S]thiotriphosphate binding to liposomes with varying muscarinic receptor/Go protein stoichiometry.

We investigated whether alcuronium, an allosteric modulator of muscarinic acetylcholine receptors, can induce receptor-mediated activation of Go proteins in liposomal membranes incorporating purified M2 receptors and Go proteins and whether its action is affected by the receptor/Go protein (R/Go) ratio. The binding of guanosine-gamma-[35S]thiotriphosphate ([35S]GTPgammaS) served as the indicator of G protein activation. It was stimulated by empty receptors at high receptor densities, and the dose-response curve was shifted to the left by the agonist carbachol and to the right by the antagonist atropine. At an R/Go ratio of 300:100, the rate of [35S]GTPgammaS binding was the same in the presence or absence of 0. 1 mM carbachol. Alcuronium increased the binding of [35S]GTPgammaS at R/Go ratios of <3:100 and diminished it at R/Go ratios of >10:100, similar to previous observations on intact cells expressing muscarinic receptors at different densities. The apparent biphasicity of alcuronium action indicates that the allosteric modulator has at least two effects on muscarinic receptor/G protein interaction but its mechanistic basis is unclear. The "active state" of muscarinic receptors induced by alcuronium probably is different from that induced by carbachol. Changes in the densities of receptors and Go proteins had little effect on the kinetics of [35S]GTPgammaS binding and on receptor affinity for carbachol, provided the R/Go ratio was kept constant. This suggests that the receptors and G proteins are located in microdomains in which their concentrations remain constant, despite variations in the amounts of lipidic membranes in the system.

The effects of brucine and alcuronium on the inhibition of [3H]acetylcholine release from rat striatum by muscarinic receptor agonists.

1. Radioligand binding experiments indicate that the affinity of muscarinic receptors for their agonists may be enhanced by allosteric modulators. We have now investigated if brucine can enhance the inhibitory effects of muscarinic receptor agonists on the electrically evoked release of [3H]acetylcholine ([3H]ACh) from superfused slices of rat striatum. 2. The evoked release of [3H]ACh was inhibited by all agonists tested (i.e., furmethide, oxotremorine-M, bethanechol and oxotremorine). 3. Brucine enhanced the inhibitory effects of furmethide, oxotremorine-M and bethanechol on the evoked [3H]ACh release without altering the inhibitory effect of oxotremorine. 4. Alcuronium was applied for comparison and found to diminish the inhibitory effect of furmethide on the evoked [3H]ACh release. 5. The results demonstrate that it is possible both to enhance and diminish the functional effects of muscarinic receptor agonists by allosteric modulators. 6. The direction of the observed effects of brucine and alcuronium on [3H]ACh release fully agrees with the effects of these modulators on the affinities of human M4 receptors for furmethide, oxotremorine-M, bethanechol and oxotremorine, as described by Jakubik et al. (1997). This supports the view that the presynaptic muscarinic receptors responsible for the autoinhibition of ACh release in rat striatum belong to the M4 muscarinic receptor subtype.

Three allosteric modulators act at a common site, distinct from that of competitive antagonists, at muscarinic acetylcholine M2 receptors.

Functional studies were conducted on guinea pig atrial muscarinic acetylcholine M2 receptors with the allosteric modulators heptane-1,7-bis(dimethyl-3'-phthalimidopropyl)ammonium bromide (C7/3'-phth), gallamine and alcuronium to determine whether these ligands are able to recognize a common accessory site. The three modulators inhibited the negative inotropic response to carbachol in this tissue. When used in combination, C7/3'-phth and gallamine or C7/3'-phth and alcuronium gave dose ratios that were either additive or underadditive. In contrast, the combinations of C7/3'-phth or alcuronium with the competitive antagonists, N-methylscopolamine or atropine, yielded supra-additive dose ratios. The data could be reconciled with a model involving a ternary complex between (1) the receptor, (2) carbachol, N-methylscopolamine or atropine acting at the orthosteric binding site and (3) C7/3'-phth, alcuronium or gallamine acting at a common, allosteric site with varying degrees of heterotropic cooperativity.

Search for lead structures to develop new allosteric modulators of muscarinic receptors.

Various compounds are known to allosterically modulate the binding of ligands to muscarinic receptors. Most of these compounds have another predominant pharmacological action. Identification of the potent representatives should be useful for the development of allosteric modulators that are specific and highly active. For various reasons, a direct comparison of allosteric potencies on the basis of literature data is difficult. Therefore, a series of compounds was compared with regard to the allosteric delay of the dissociation of N-[3H]methylscopolamine from porcine heart M2 receptors under the following assay conditions: "Na,K,Pi buffer", 4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees C; "Mg,Tris,Cl,Pi buffer', 50 mM Tris-HCl, 3 mM MgHPO4,pH 7.3, 37 degrees C. Generally, the allosteric potency of the compounds was higher in the Na,K,Pi buffer, compared with the Mg,Tris,Cl,Pi buffer. However, the extent of the potency shift differed, ranging from approximately 2-fold for tacrine to approximately 100-fold for gallamine. The concentration retarding radioligand dissociation to half of the control rate (EC50) served as a measure of allosteric potency. Under both assay conditions, alcuronium was the most potent compound (EC50,Na,K,Pi = 4 nM and EC50,Mg,Tris,Cl,Pi = 55 nM), followed by alkane-bisammonium and bispyridinium compounds containing phthalimido moieties. Gallamine showed intermediate potency (EC50 values of 180 nM and 16,000 nM in Na,K,Pi buffer and Mg,Tris,Cl,Pi buffer, respectively). Obidoxime and hexamethonium, both known to antagonize allosteric actions, revealed submaximal efficacy and low potency (EC50,Na,K,Pi of approximately 100,000 nM). The relevance of these results, regarding the identification of lead structures for the development of new allosteric modulators, is discussed.

Conformational analysis, molecular shape comparison, and pharmacophore identification of different allosteric modulators of muscarinic receptors.

Structurally dissimilar compounds such as alcuronium and the newly synthesized substances derived from the bisbenzyl ether TMB4 and from hexamethonium stabilize antagonist binding to M2-cholinoceptors which is indicative of an allosteric action. In order to propose a hypothesis for the common pharmacophore and the corresponding active conformations, seven flexible compounds in a data set were individually aligned onto the most active and, additionally, rigid alcuronium molecule using a torsional angle flexible fit. An S-shape conformation was found to be a plausible general active conformation. In a subsequent molecular shape analysis the overlap and the nonoverlap steric volumes, RMS alignment as well as electrostatic field potentials were employed as possible structure--activity correlation descriptors. The corresponding 3D-QSAR formulation exhibits a correlation between allosteric modulation potency and the nonoverlap steric volume as well as the proton and oxygen anion probe electrostatic field potentials. Because of large structural diversity among the small number of compounds studied, the apparent 3D-QSAR is best thought of as a convenient representation of the common spatial pharmacophore hypothesis.

Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation.

Comparison of structurally different allosteric modulators of muscarinic receptors by self-organizing neural networks.

Similarities in the molecular structure and surface properties of the allosteric modulators of muscarinic receptors, alcuronium, gallamine, tubocurarine, and the hexamethonium compound W84, a well-known pharmacological tool, are explored. The analysis of the molecular electrostatic potential (MEP) as well as of the shape of the molecular surface is performed by self-organizing neural networks. A distorted sandwich conformation of W84 is suggested to be the active form. The importance of the MEP for binding of these compounds could be established.

Positive allosteric action of eburnamonine on cardiac muscarinic acetylcholine receptors.

It was discovered recently that alcuronium and strychnine (which is a precursor of alcuronium) allosterically increase the affinity of cardiac muscarinic receptors for the antagonist, N-methylscopolamine. We have now investigated the effects of l-eburnamonine and vincamine, which are both closely related to strychnine. In experiments on rat heart atria, l-eburnamonine was found to increase the binding of [3H]N-methylscopolamine with Ehlert's cooperativity coefficient alpha = 0.35, which indicates that the strength of its allosteric action is close to that of alcuronium and strychnine (alpha = 0.31 and 0.44, respectively). However, the affinity of l-eburnamonine for the cardiac muscarinic receptors is lower than the affinities of alcuronium and strychnine (KAR = 22.6 microM, 0.15 microM, and 3.4 microM, respectively). In spite of its extremely close similarity to l-eburnamonine, vincamine has a negative allosteric effect on the binding of [3H]N-methylscopolamine (alpha = 4.1; KAR = 22.8 microM). It is likely that a systematic investigation of the allosteric effects of the analogues of strychnine will not only yield new allosteric effectors on muscarinic receptors, but also clarify the structural features responsible for the direction (positive or negative) of their allosteric effect.