Allosteric site in M2 acetylcholine receptors: evidence for a major conformational change upon binding of an orthosteric agonist instead of an antagonist

Muscarinic acetylcholine receptors contain two distinct ligand binding sites, i.e. the orthosteric site for acetylcholine and other conventional ligands, and an allosteric site located at the entrance of the ligand binding pocket. We used a set of allosteric agents to probe whether muscarinic M₂ receptors whose orthosteric site is occupied by an agonist still reveal the common allosteric site that has been identified in M₂ receptors being occupied by an orthosteric antagonist (N-methylscopolamine, NMS). Equilibrium and dissociation binding experiments were carried out in porcine heart homogenates using either the agonist [³H]oxotremorine M ([³H]OxoM) or the antagonist [³H]NMS. The affinities of the allosteric agents were determined for the radioligand-occupied receptor states and, additionally, for the radioligand-free (ground state) M₂ receptor. The archetypal agent W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide] and its bispyridinio middle chain analogue WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl]propane dibromide) had a clearly lower affinity for [³H]OxoM-liganded receptors compared with [³H]NMS-liganded and ground state receptors. In contrast, a derivative resembling only one half of W84 had equal affinities for both radioligand-occupied receptor states. Also, the agents gallamine and obidoxime did not discriminate between [³H]OxoM- and [³H]NMS-occupied receptors. The allosteric antagonistic tool obidoxime inhibited WDuo3 action in [³H]OxoM-liganded receptors with the same potency as in [³H]NMS-liganded receptors. We conclude that the common allosteric site is still present in OxoM-liganded M₂ receptors, but its spatial conformation is considerably altered compared with NMS-liganded receptors.     

Using a radioalloster to test predictions of the cooperativity model for gallamine binding to the allosteric site of muscarinic acetylcholine M(2) receptors.

The muscarinic M(2) receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift alpha of the equilibrium dissociation constant K(D) of a radioligand for the orthosteric site. According to the cooperativity model, the K(A) of alloster binding is expected to be shifted to an identical extent depending on whether the orthosteric site is occupied by the orthoster or not. Here, the novel radioalloster [(3)H]dimethyl-W84 (N,N'-bis[3-(1,3-dihydro-1, 3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,N',N'-tetramethyl-1, 6-hexanediaminium diiodide) was applied to directly measure the K(A) shift induced for the prototype allosteric modulator gallamine by binding of N-methylscopolamine (NMS) to the orthosteric site of porcine heart M(2) receptors (4 mM Na(2)HPO(4), 1 mM KH(2)PO(4), pH 7.4; 23 degrees C; data are means +/- S.E.). First, in the common way, the concentration-dependent inhibition by gallamine of [(3)H]NMS equilibrium binding was measured and analyzed using the cooperativity model, which yielded for the affinity of gallamine binding at free receptors a pK(A)= 8.35 +/- 0.09 and a cooperativity factor alpha = 46 (n = 5). The dissociation constant for gallamine binding at NMS-occupied receptors was predicted as p(alpha. K(A)) = 6.69. Labeling of the allosteric site by [(3)H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. The K(i) for gallamine at free receptors amounted to pK(i,-NMS) = 8.27 +/- 0.39 (n = 5), which is in line with the prediction of the cooperativtiy model. In the presence of 1 microM NMS, to occupy the orthosteric site, gallamine displaced [(3)H]dimethyl-W84 with pK(i, +NMS) = 6.60 +/- 0.19 (n = 3). Thus, the NMS-induced pK(i) shift amounted to 47, which matches the predicted value of alpha = 46. These results validate the cooperativity model.     

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.     

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.     

Atypical muscarinic allosteric modulation: cooperativity between modulators and their atypical binding topology in muscarinic M2 and M2/M5 chimeric receptors

The binding and function of muscarinic acetylcholine receptors can be modulated allosterically. Some allosteric muscarinic ligands are "atypical", having steep concentration-effect curves and not interacting competitively with "typical" allosteric modulators. For atypical agents, a second allosteric site has been proposed. Different approaches have been used to gain further insight into the interaction with M2 receptors of two atypical agents, tacrine and the bispyridinium compound 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bispyridinium dibromide (Duo3). Interaction studies, using radioligand binding assays and the allosteric ligands obidoxime, Mg2+, and the new tool hexamethonium to antagonize the allosteric actions of the atypical ligands, showed different modes of interaction for tacrine and Duo3 at M2 receptors. A negatively cooperative interaction was observed between hexamethonium and tacrine (but not Duo3). A tacrine dimer that exhibited increased allosteric potency relative to tacrine but behaved like a typical allosteric modulator was competitively inhibited by hexamethonium. M2/M5-receptor mutants revealed a dependence of tacrine and Duo3 affinity on different receptor epitopes. This was confirmed by docking simulations using a three-dimensional model of the M2 receptor. These showed that the allosteric site could accommodate two molecules of tacrine simultaneously but only one molecule of Duo3, which binds in different mode from typical allosteric agents. Therefore, the atypical actions of tacrine and Duo3 involve different modes of receptor interaction, but their sites of attachment seem to be the "common" allosteric binding domain at the entrance to the orthosteric ligand binding pocket of the M2-receptor. Additional complex behavior may be rationalized by allosteric interactions transmitted within a receptor dimer.     

Interaction of Mg2+ with the allosteric site of muscarinic M2 receptors

Mg²⁺-ions have been suspected to attenuate the inhibitory effect of allosteric modulators on the dissociation of orthosteric ligands from muscarinic M₂ receptors. It was aimed to gain more insight into the molecular events underlying the effect of Mg²⁺. The interaction of Mg²⁺ with the allosteric model compounds W84 (hexane-1,6-bis [dimethyl-3’-phthalimidopropylammonium bromide]) and Chin3/6 (hexane-1,6-bis[dimethyl-3’-{4-oxo-2-phenyl-3,4-dihydro-2H-quinazolin-1-yl}propylammonium bromide]) was studied in porcine heart muscarinic receptors, the primary binding site of which was occupied by the ligand [³H]N-methylscopolamine ([³H]NMS). The incubation buffer was composed of 4 mM Na₂HPO₄ and 1 mM KH₂PO₄ (pH 7.4, 23°C). The retardation of [³H]NMS dissociation (control t_1/2 = 5.6 min) induced by the allosteric test compounds was diminished by 3 mM Mg²⁺ to a greater extent than to be expected with regard to its contribution to the ionic strength of the buffer solution. Concentration-effect curves for the allosteric retardation of [³H]NMS dissociation by W84 (half maximal effective concentration EC_0.5 = 24 nM in the absence of Mg²⁺) and by Chin3/6 (EC_0.5 = 28 nM) were shifted by Mg²⁺ to the right in a parallel fashion. The curve-shift was compatible with a competitive interplay between Mg²⁺ and the modulators. The pK _b-values as a measure of the antagonistic potency of Mg²⁺, however, differed depending on the modulator, i.e. pK _b = 3.4 with W84 and pK _b = 2.8 with Chin3/6. Mg²⁺ itself was capable of slowing the dissociation of [³H]NMS; the maximal retardation of [³H]NMS dissociation was about 3fold, the concentration-effect relationship was compatible with a two-site model using the above-mentioned pK _b-values as affinity constants. Since the equilibrium-binding of [³H]NMS remained unchanged up to a Mg²⁺-concentration of 3 mM, the cation appears to inhibit the association and dissociation of [³H]NMS to the same extent in this concentration range. Taken together, the findings indicate that Mg²⁺ may bind to the allosteric region of muscarinic M₂ receptors and that more than one site is involved in this interaction. The sites of action may represent divalent cation binding sites. Received: 1 October 1997 / Accepted: 20 January 1998     

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.     

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.     

Positive cooperativity in the binding of alcuronium and N-methylscopolamine to muscarinic acetylcholine receptors

The effect of the neuromuscular blocker alcuronium on the binding of N-[3H]-methylscopolamine [( 3H]NMS) and l-[3H]quinuclidinylbenzilate ([3H]QNB) to muscarinic binding sites in rat heart atria, longitudinal smooth muscle of the ileum, cerebral cortex, cerebellum, and submaxillary glands was measured using filtration techniques. In the presence of 10(-5) M alcuronium, the binding of [3H]NMS (which was present at a subsaturating concentration of 2 x 10(-10) M) was increased 5.3-fold in the atria and smooth muscle and 3-fold in the cerebellum; no increase was observed in the brain cortex and salivary glands. The binding of [3H]NMS was inhibited at 10(-3) M and higher concentrations of alcuronium. The rates of [3H]NMS association to and dissociation from muscarinic binding sites in the atria were diminished by 10(-5) M alcuronium. Scatchard plots of [3H]NMS binding data obtained with and without 10(-5) M alcuronium indicated that the maximum number of binding sites was not altered by the drug, whereas the apparent Kd for [3H]NMS was diminished. In contrast to [3H] NMS, the effects of alcuronium on the binding of [3H]QNB were only inhibitory. The concentration of alcuronium required to diminish the binding of [3H]QNB by 50% (IC50) was 4-7 microM in the atria, ileal smooth muscle, and the cerebellum, 140 microM in the brain cortex, and 1200 microM in the parotid gland. The results suggest that the binding of low concentrations of alcuronium to muscarinic receptors in the heart, ileal smooth muscle, and cerebellum allosterically increases the affinity of muscarinic receptors towards [3H]NMS, although not [3H]QNB. At high concentrations, alcuronium inhibits the binding of muscarinic ligands, presumably by competition for the classical muscarinic binding site. Positive cooperativity induced by alcuronium appears to be specific for the m2 (cardiac) subtype of muscarinic receptors.     

Probing the molecular mechanism of interaction between 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the muscarinic M(1) receptor: direct pharmacological evidence that AC-42 is an allosteric agonist

4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M(1) receptor previously suggested to interact with an "ectopic" site on this receptor. However, the pharmacological properties of this site (i.e., whether it overlaps to any extent with the classic orthosteric site or represents a novel allosteric site) remain undetermined. In the present study, atropine or pirenzepine significantly inhibited the ability of either carbachol or AC-42 to stimulate inositol phosphate accumulation or intracellular calcium mobilization in Chinese hamster ovary (CHO) cells stably expressing the human M(1) receptor. However, the interaction between either of these antagonists and AC-42 was characterized by Schild slopes significantly less than unity. Increasing the concentrations of atropine revealed that the Schild regression was curvilinear, consistent with a negative allosteric interaction. More direct evidence for an allosteric mode of action of AC-42 was obtained in [(3)H]N-methylscopolamine ([(3)H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [(3)H]NMS binding in a manner that was quantitatively described by an allosteric model applied to both modulator data sets. Furthermore, AC-42 and gallamine significantly retarded the rate of [(3)H]NMS dissociation from CHO-hM(1) cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M(1) receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M(1) receptors in the absence of the orthosteric agonist.     

Different interactions between MT7 toxin and the human muscarinic M1 receptor in its free and N-methylscopolamine-occupied states

Muscarinic MT7 toxin is a highly selective and potent antagonist of the M(1) subtype of muscarinic receptor and acts by binding to an allosteric site. To identify the molecular determinants by which MT7 toxin interacts with this receptor in its free and NMS-occupied states, the effect on toxin potency of alanine substitution was evaluated in equilibrium and kinetic binding experiments as well as in functional assays. The determination of the crystallographic structure of an MT7-derivative (MT7-diiodoTyr51) allowed the selection of candidate residues that are accessible and present on both faces of the three toxin loops. The equilibrium binding data are consistent with negative cooperativity between N-methylscopolamine (NMS) and wild-type or modified MT7 and highlight the critical role of the tip of the central loop of the toxin (Arg34, Met35 Tyr36) in its interaction with the unoccupied receptor. Examination of the potency of wild-type and modified toxins to allosterically decrease the dissociation rate of [(3)H]NMS allowed the identification of the MT7 residues involved in its interaction with the NMS-occupied receptor. In contrast to the results with the unoccupied receptor, the most important residue for this interaction was Tyr36 in loop II, assisted by Trp10 in loop I and Arg52 in loop III. The critical role of the tips of the MT7 loops was also confirmed in functional experiments. The high specificity of the MT7-M(1) receptor interaction exploits several MT7-specific residues and reveals a different mode of interaction of the toxin with the free and NMS-occupied states of the receptor.     

Probing the pharmacophore for allosteric ligands of muscarinic M2 receptors: SAR and QSAR studies in a series of bisquaternary salts of caracurine V and related ring systems

Allosteric effects on muscarinic acetylcholine M(2) receptors were examined in a series of bisquaternary salts of the Strychnos alkaloid caracurine V (6) and related iso-caracurine V, tetrahydrocaracurine V, and bisnortoxiferine ring systems. The compounds inhibited dissociation of the orthosteric antagonist [(3)H]N-methylscopolamine (NMS) from porcine cardiac M(2) receptors with EC(0.5,diss) values from 4 to 3270 nM. The majority of compounds hardly changed [(3)H]NMS equilibrium binding, indicating similar binding affinities in free and NMS-occupied M(2) receptors. The most potent agents were found in the caracurine V, iso-caracurine V, and tetrahydrocaracurine V series and carried nonpolar alkyl groups with a maximal chain length of three carbon atoms. 3D QSAR (CoMSIA) analysis explained the wide range of binding affinities by steric and electrostatic properties of the side chains. Furthermore, the findings suggest that the spatial orientation of the "caracurine" aromatic rings compared with the bisnortoxiferine ring skeleton is favorable to optimal allostere-receptor interactions.     

Systematic development of high affinity bis(ammonio)alkane-type allosteric enhancers of muscarinic ligand binding

Bis(ammonio)alkane compounds carrying lateral phthalimidopropyl substituents on the nitrogen atoms belong to the archetypal muscarinic allosteric agents. Herein, a series of symmetrical and nonsymmetrical compounds was synthesized in which the phthalimide residues were replaced by differently substituted imide moieties. The allosteric action was measured in porcine heart muscarinic M(2) receptors using [(3)H]N-methylscopolamine (NMS) as a ligand for the orthosteric receptor site in equilibrium binding and dissociation experiments. 1,8-Naphthalimido residues conferred an up to 100-fold gain in affinity leading into the low nanomolar range, while the inhibition of NMS binding was maintained. Additional propyl chain methylation was accompanied by an allosteric elevation of orthosteric ligand binding. In general, the gain in allosteric activity achieved by ring variation plus propyl chain methylation on one side of the molecule could not be augmented by symmetrical variations. The elevation of the ligand binding can be explained by different quantitative structure-activity relationships for the affinities to the free and the orthoster-liganded receptor.     

Analogs of WIN 62,577 define a second allosteric site on muscarinic receptors

WIN 51,708 (17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) and WIN 62,577 (17-beta-hydroxy- 17-alpha-ethynyl-delta(4)-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) are potent and centrally active antagonists at rat, but not human, NK(1) receptors. The interactions of these compounds and some analogs with [(3)H]N-methyl scopolamine ([(3)H]NMS) and unlabeled acetylcholine (ACh) at M(1)-M(4) muscarinic receptors have been studied using equilibrium and nonequilibrium radioligand binding methods. The results are consistent with the predictions of the allosteric ternary complex model. The WIN compounds have log affinities for the unliganded receptor in the range 5 to 6.7, and exhibit positive, negative, or neutral cooperativity with [(3)H]NMS and ACh, depending on the receptor subtype and nature of the interacting ligands. WIN 62,577 is an allosteric enhancer of ACh affinity at M(3) receptors. Although interacting allosterically, WIN 62,577 and WIN 51,708 do not affect [(3)H]NMS dissociation from M(3) receptors. Certain analogs have higher affinities than WIN 62,577, and truncated forms of WIN 62,577, including steroids, also act allosterically. One analog, 17-beta-hydroxy-17-alpha-Delta(4)-androstano[3,2-b]pyrido[2,3-b]indole (PG987), has the unique effect of speeding [(3)H]NMS dissociation; its largest effect, 2.5-fold, is at M(3) receptors. The interaction between PG987 and other allosteric agents on [(3)H]NMS dissociation from M(3) receptors indicate that PG987 binds reversibly to a site distinct from that to which gallamine and strychnine bind: in contrast, PG987 seems to bind to the same site on M(3) receptors as KT5720, staurosporine, and WIN 51,708. Therefore, in addition to the allosteric site that binds strychnine (and probably chloromethyl brucine, another allosteric enhancer) there is a second, nonoverlapping, pharmacologically distinct allosteric site on M(3) receptors that also supports positive cooperativity with ACh.     

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.     

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.     

Elevation of ligand binding to muscarinic M(2) acetylcholine receptors by bis(ammonio)alkane-type allosteric modulators

Bis(ammonio)alkane-type compounds are archetypal muscarinic allosteric modulators. Phthalimido-substituted hexane-bis-ammonium agents were methylated in the phthalimide moieties and the lateral propyl side chains. All compounds retarded allosterically the dissociation of the orthosteric ligand [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine heart M(2) receptors. [(3)H]NMS equilibrium binding was reduced, left unaltered, or elevated, depending on the degree and position of methylation. This is the first time that an allosteric elevation of ligand binding is demonstrated for bis(ammonio)alkane-type compounds.     

Probing the selectivity of allosteric modulators of muscarinic receptors at other G-protein-coupled receptors

1. The aim of the present investigation was to analyse whether three prototype allosteric modulators of ligand binding to muscarinic receptors, i.e. alcuronium, gallamine, and the alkane-bis-ammonium compound W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropylammonium bromide]), may have allosteric effects on radioligand-binding characteristics at other G-protein-coupled receptors, such as cerebral A1 adenosine receptors (Gi-coupled), cardiac left ventricular alpha1-adrenoceptors (Gq), and beta-adrenoceptors (Gs). 2. The modulators were applied at concentrations known to be high with regard to the allosteric delay of the dissociation of the antagonist [3H]-N-methylscopolamine (NMS) from muscarinic M2-receptors: 30 micromol l(-1) W84, 30 micromol l(-1) alcuronium, 1000 micromol l(-1) gallamine. As radioligands, we used the adenosine A1-receptor ligand [3H]-cyclopentyl-dipropylxanthine (CPX), the alpha1-adrenoceptor ligand [3H]-prazosin (PRAZ), and the beta-adrenoceptor ligand (-)-[125I]-iodocyanopindolol (ICYP). Allosteric actions on ligand dissociation and the equilibrium binding were measured in the membrane fractions of rat whole forebrain (CPX) and of rat cardiac left ventricle (PRAZ, ICYP, NMS), respectively. 3. CPX and PRAZ showed a monophasic dissociation with half-lives of 5.88+/-0.15 and 12.27+/-0.46 min, respectively. In the case of CPX, neither the binding at equilibrium nor the dissociation characteristics were influenced by the allosteric agents. With PRAZ, the binding at equilibrium remained almost unaltered in the presence of W84, whereas it was reduced to 36+/-2% of the control value with alcuronium and to 42+/-2% with gallamine. The dissociation of PRAZ was not affected by W84, whereas it was moderately accelerated by alcuronium and gallamine. In the case of ICYP, the binding at equilibrium was not affected by the allosteric modulators. The dissociation of ICYP was slow, and after 3 h, more than 50% of the radioligand was still bound, so that a reliable half-life could not be calculated. ICYP dissociation was not affected by W84. In the presence of alcuronium and gallamine, the dissociation curve of ICYP revealed an initial drop from the starting level, followed by the major phase of dissociation being parallel to the control curve. 4. In summary, the allosteric action of the applied agents is not a common feature of G-protein-coupled receptors and appears to be specific for muscarinic receptors.     

Complex allosteric modulation of cardiac muscarinic receptors by protamine: potential model for putative endogenous ligands.

A large number of diverse pharmacological agents bind to a secondary domain on the muscarinic receptor, to influence allosterically the interaction of ligands at the primary binding site. Based on common structural features of these antagonists, we examined the interaction of protamine, an endogenous polycationic peptide, and of polyamines with muscarinic receptors in rat heart. Our results provide several lines of qualitative evidence that protamine allosterically modulates the conformation of muscarinic receptors, in a marked negatively cooperative manner. It decelerated the dissociation of N-[3H]methylscopolamine ([3H] NMS) initiated by atropine, in a concentration-dependent fashion. Inhibition by protamine of [3H]NMS binding at equilibrium showed a distinct plateau, which increased in magnitude at higher ligand concentrations. Scatchard analysis of saturation isotherms of [3H]NMS binding in the absence and presence of protamine indicated that protamine did not alter Bmax in a statistically significant fashion, although there was a trend of a concentration-dependent increase in this parameter. On the other hand, it caused a marked concentration-dependent decrease in the affinity of [3H]NMS, and this effect reached a ceiling limit. However, there were marked quantitative deviations of the interaction of protamine from a simple ternary allosteric model. Some of these discrepancies could be explained by the tendency of protamine to increase Bmax. The allosteric actions of protamine demonstrated in kinetic and equilibrium experiments were selective for m1 and m2 muscarinic receptors, compared with m3, m4, and m5 receptors, as studied in Chinese hamster ovary cells transfected with the genes of the different muscarinic receptors. Arginine residues play an important role in the allosteric interaction of protamine, inasmuch as poly-L-arginine qualitatively mimicked the effects of protamine. In contrast, no effects of the polyamines spermine, spermidine, and putrescine were observed on [3H]NMS binding. This is the first report on the allosteric modulation of muscarinic receptors by an endogenous peptide.     

Interactions of cocaine with primary and secondary recognition sites on muscarinic receptors.

Several lines of evidence have suggested that muscarinic receptors may possess more than one ligand binding site. In this study, the interactions of cocaine with primary and secondary (allosteric) sites on muscarinic receptors in membrane homogenates from post-mortem human brainstem were examined. (-)-Cocaine inhibited the binding of the tritiated muscarinic antagonists N-methylscopolamine (NMS) and pirenzepine to an apparent single class of sites, with Ki values of 200-300 microM. The binding of the muscarinic agonist [3H]oxotremorine-M was inhibited with a similar Ki value (200 microM). (+)-Cocaine, although not the naturally occurring stereoisomer, was 10-20-fold more potent than (-)-cocaine in competing for binding to the primary muscarinic recognition site. The binding of cocaine was unaffected by guanine nucleotides or N-ethylmaleimide, consistent with its purported action as a competitive antagonist. Cocaine was not selective for muscarinic receptor subtypes. Rosenthal analysis of the [3H]NMS saturation binding data in the presence of increasing concentrations of either (-)-cocaine or (+)-cocaine indicated that both isomers produced an apparent competitive-like reduction in the [3H]NMS affinity. Schild regression analysis of the saturation binding data resulted in curvilinear plots suggestive of cooperative or allosteric interactions of (-)-cocaine with the [3H]NMS-labeled receptors. The effects of (-)-cocaine on the kinetics of [3H]NMS binding were consistent with an allosteric interaction with the receptor. Increasing concentrations of cocaine markedly slowed the rate of [3H]NMS dissociation from the primary recognition site. The allosteric modulation of [3H] NMS binding by (-)-cocaine was abolished with increasing ionic strength. Taken together, these data demonstrate that (-)-cocaine interacts with primary and allosteric recognition sites on muscarinic receptors.