Antagonist Binding Properties Research Articles

Effect of Ions and Sequence Variants on the Antagonist Binding Properties of the Histamine H1 Receptor.

The histamine H1 receptor (H1R) is a G protein-coupled receptor (GPCR) and represents a main target in the treatment of allergic reactions as well as inflammatory reactions and depressions. Although the overall effect of antagonists on H1 function has been extensively investigated, rather little is known about the potential modulatory effect of ions or sequence variants on antagonist binding. We investigated the dynamics of a phosphate ion present in the crystal structure and of a sodium ion, for which we determined the position in the allosteric pocket by metadynamics simulations. Both types of ions exhibit significant dynamics within their binding site; however, some key contacts remain stable over the simulation time, which might be exploited to develop more potent drugs targeting these sites. The dynamics of the ions is almost unaffected by the presence or absence of doxepin, as also reflected in their small effect (less than 1 kcal·mol−1) on doxepin binding affinity. We also examined the effect of four H1R sequence variants observed in the human population on doxepin binding. These variants cause a reduction in doxepin affinity of up to 2.5 kcal·mol−1, indicating that personalized medical treatments that take into account individual mutation patterns could increase precision in the dosage of GPCR-targeting drugs.

Systematic characterization of AT1 receptor antagonists with label-free dynamic mass redistribution assays

IntroductionIn the drug discovery field, the binding affinities and binding kinetics of drug candidates are very important. Angiotensin II type 1 (AT1) receptor antagonists, e.g., candesartan, telmisartan, irbesartan, losartan and valsartan, show high affinities and long-lasting bindings to the receptor, making them preferred medications for hypertension treatment. However, the molecular binding properties of AT1 receptor antagonists are controversial. MethodsIn this work, we established a profile to study the phenotypic properties of AT1 receptor antagonists with label-free dynamic mass redistribution (DMR) assays in native human cells. With noninvasive features, DMR assay were conducted in multiple formats. Eleven antagonists were systematically evaluated with angiotensin II as an agonist probe in the Hep G2 cell line, which endogenously expresses the AT1 receptor. ResultsThe IC50 values to the AT1 receptor of individual antagonist varied with different incubation times. The antagonists showed competitive behavior with angiotensin II. Schild analysis was used to analyze the competitive behavior of the antagonist. All of the antagonist showed long-lasting possession of the AT1 receptor, except telmisartan. The systematic evaluation of the antagonists implied that 11 antagonists showed high binding affinity but distinct binding modes to AT1 receptor. DiscussionThis study demonstrated that the DMR assay has great potential for determining the pharmacological parameters of ligands. This work may serve as guidance for other receptor and ligand assays.

Investigation of the binding and functional properties of extended length D3 dopamine receptor-selective antagonists

The D3 dopamine receptor represents an important target in drug addiction in that reducing receptor activity may attenuate the self-administration of drugs and/or disrupt drug or cue-induced relapse. Medicinal chemistry efforts have led to the development of D3 preferring antagonists and partial agonists that are >100-fold selective vs. the closely related D2 receptor, as best exemplified by extended-length 4-phenylpiperazine derivatives. Based on the D3 receptor crystal structure, these molecules are known to dock to two sites on the receptor where the 4-phenylpiperazine moiety binds to the orthosteric site and an extended aryl amide moiety docks to a secondary binding pocket. The bivalent nature of the receptor binding of these compounds is believed to contribute to their D3 selectivity. In this study, we examined if such compounds might also be “bitopic” such that their aryl amide moieties act as allosteric modulators to further enhance the affinities of the full-length molecules for the receptor. First, we deconstructed several extended-length D3-selective ligands into fragments, termed “synthons”, representing either orthosteric or secondary aryl amide pharmacophores and investigated their effects on D3 receptor binding and function. The orthosteric synthons were found to inhibit radioligand binding and to antagonize dopamine activation of the D3 receptor, albeit with lower affinities than the full-length compounds. Notably, the aryl amide-based synthons had no effect on the affinities or potencies of the orthosteric synthons, nor did they have any effect on receptor activation by dopamine. Additionally, pharmacological investigation of the full-length D3-selective antagonists revealed that these compounds interacted with the D3 receptor in a purely competitive manner. Our data further support that the 4-phenylpiperazine D3-selective antagonists are bivalent and that their enhanced affinity for the D3 receptor is due to binding at both the orthosteric site as well as a secondary binding pocket. Importantly, however, their interactions at the secondary site do not allosterically modulate their binding to the orthosteric site.

Binding properties of antagonists to Cannabinoid receptors in intact cells

The implication of the cannabinoid receptor 1 (CB(1) receptor) in several pathophysiological states has sparked the development of selective antagonists. Here we compare binding of the antagonists [(3) H]-AZ12491187, [(3) H]-taranabant and [(3) H]-rimonabant to intact human embryonic kidney cells stably expressing recombinant human CB(1) receptors (CB1r cells). Unlabelled ligands decreased the total binding of the three radioligands with closely the same order of potency: i.e. AZ12288553∼AZ12491187∼taranabant>rimonabant. Nondisplaceable (i.e. nonspecific) binding to the CB1r cells was the same as total binding to the wells containing untransfected cells and it was more pronounced for [(3) H]-AZ12491187 and [(3) H]-rimonabant than for [(3) H]-taranabant. [(3) H]-Rimonabant and (to a lesser extent) [(3) H]-AZ12491187 were also prone to bind nonspecifically to the walls of the wells. Compared to the other radioligands, [(3) H]-rimonabant displayed lower potency for the CB(1) receptors in saturation binding studies and faster association and dissociation in kinetic experiments. When dissociated, the three radioligands also showed prominent rebinding to the cells in medium only. This could be relieved by the presence of excess of unlabelled ligand and of bovine serum albumin (BSA) but a combination thereof was most efficient. The long 'residence time' of AZ12491187 at the CB(1) receptor (because of slow dissociation and prominent rebinding) and its pronounced incorporation into the membranes of the cells could contribute to long-lasting in vivo CB(1) receptor blockade.

Expression of the carbohydrate recognition domain of FimH and development of a competitive binding assay

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). In the first step of this infective process, the virulence factor FimH located on type 1 pili allows UPEC to specifically adhere to oligosaccharides, which are part of glycoproteins on the urinary bladder mucosa. This initial step prevents the clearance of E. coli from the urinary tract and enables the invasion of the host cells. Because FimH antagonists can block this interaction, they exhibit a promising therapeutic potential as anti-infectives. For the evaluation of their binding properties, a reliable, target-based affinity assay is required. Here, we describe the expression and purification of the carbohydrate recognition domain of FimH (FimH-CRD) as well as the development of a competitive binding assay. FimH-CRD linked with a thrombin cleavage site to a 6His-tag is recombinantly expressed and purified by affinity chromatography. For the evaluation of FimH antagonists, a cell-free binding assay based on the interaction of a biotinylated polyacrylamide glycopolymer with the FimH-CRD was developed. Complexation of the biotinylated glycopolymer with streptavidin coupled to horseradish peroxidase allows the quantification of the binding properties of FimH antagonists. The assay format was optimized and validated by a comparison with affinity data from reported assays.

Computational analysis of ligand recognition sites of homo- and heteropentameric 5-HT 3 receptors

Inhibition of the 5-hydroxytryptamine receptor (5-HT 3R), a member of the Cys-loop superfamily of Ligand-Gated Ion Channels (LGICs), has been recognized to have important antiemetic effects. With respect to the many other drugs already in use, such as the first generation 5-HT 3R antagonist granisetron, palonosetron, a second generation antagonist, clearly demonstrates superior inhibition potency towards the 5-HT 3Rs. Five different receptor monomers, the 5-HT 3R A–E, have been identified although the A and B subunits are the only known to build functional receptors, the homopentameric 5-HT 3AR and the heteropentameric 5-HT 3B–AR (with BBABA subunit arrangement). At present, however, no three-dimensional structure has been reported for any of the 5-HT 3R subunits. To understand the binding properties of agonists and antagonists, models of the extracellular portion of the 5-HT 3R A and B subunits are built and assembled into the receptor (homo- and hetero-) pentameric structure on the basis of the known three-dimensional structure of the nicotinic-acetylcholine receptor (nACh-R). The results of docking studies of the natural agonist serotonin and the antagonists palonosetron and granisetron into the modelled homomeric and heteromeric 5-HT 3R binding interfaces, provide a possible rationalization both of the higher potency of palonosetron with respect to other antagonists, and of its previously reported allosteric binding and positive cooperativity properties.

Intact Cell Binding for In Vitro Prediction of Sedative and Non-sedative Histamine H1–Receptor Antagonists Based on Receptor Internalization

We evaluated changes in the binding properties of sedative and non-sedative histamine H1–receptor antagonists induced by internalization of H1 receptors in intact human U373 MG astrocytoma cells. Internalization of H1 receptors was induced without their degradation by treatment with 0.1 mM histamine for 30 min at 37°C, and then the intact cell binding assay was performed at 4°C. The binding properties of [3H]mepyramine, a cell-penetrating radioligand for H1 receptors, were not changed by histamine pretreatment. Displacement curves for sedative H1-receptor antagonists (diphenhydramine, chlorpheniramine, promethazine, ketotifen, azelastine and oxatomide) against [3H]mepyramine binding were not changed by histamine pretreatment. In contrast, the displacement curves for non-sedative H1-receptor antagonists (mequitazine, bepotastine, olopatadine, epinastine, carebastine, desloratadine and fexofenadine) were changed by histamine pretreatment: two types of changes, i.e. a rightward shift in the monophasic curve or an increase in the proportion of the low affinity component of the biphasic curve, were prevented under hypertonic conditions, in which clathrin-mediated receptor internalization is known to be inhibited. Thus, internalization-mediated changes in the binding properties of H1-receptor antagonists were well correlated with their sedative and nonsedative behaviors, which might confirm their permeability through the biomembrane and possibly the blood brain barrier.

On high- and low-affinity agonist sites in GABAA receptors.

GABAA receptors are activated via low-affinity binding sites for the agonists GABA or muscimol. Evidence has been provided that the amino acid residue alpha 1F64 located at the beta2(+)/alpha1(-) subunit interface forms part of this binding site. In radioactive ligand binding studies the agonist [3H]muscimol has been found to interact with the receptor via a high-affinity binding site. This site has been interpreted as a conformational variant of the low-affinity site. Alternatively, the high-affinity binding site has been located to the alpha1(+)/beta2(-) interface and the homologous residue to alpha 1F64, beta 2Y62 has been proposed to constitute an important part of this site. Here we investigated the effect of the point mutation alpha 1F64L and the homologous mutation beta 2Y62L on agonist and antagonist binding and functional properties in alpha 1 beta 2 gamma 2 GABAA receptors. While the mutation in the alpha1 subunit had drastic consequences on all studied properties, including desensitization, the mutation in the beta2 subunit had little consequence. Our observations are relevant for the relative location of high- and low-affinity agonist sites in GABAA receptors.

Comparison of the pharmacological antagonism of M 2 and M 3 muscarinic receptors expressed in isolation and in combination

We compared the binding properties of selective muscarinic antagonists with their potencies for antagonizing muscarinic responses in Chinese hamster ovary (CHO) cells expressing M 2 and M 3 muscarinic receptors in combination and in isolation. When measured by the competitive displacement of [ 3 H ] N-methylscopolamine binding to CHO cells expressing both M 2 and M 3 muscarinic receptors (CHO M 2+M 3 cells), the competition curves of the subtype-selective muscarinic antagonists were consistent with a two-site model. One site exhibited binding properties identical to those of CHO M 2 cells, whereas the other site exhibited properties like those of CHO M 3 cells. Oxotremorine-M, a muscarinic agonist, elicited a robust, pertussis toxin-insensitive stimulation of phosphoinositide hydrolysis in both CHO M 3 and CHO M 2+M 3 cells, but not in CHO M 2 cells. The pharmacological antagonism of the phosphoinositide response exhibited similar properties in both CHO M 3 and CHO M 2+M 3 cells. Oxotremorine-M elicited a pertussis toxin-sensitive, robust inhibition of forskolin-stimulated cyclic AMP (cAMP) accumulation in both CHO M 2 and CHO M 2+M 3 cells and a less robust inhibition in CHO M 3 cells. At higher concentrations, oxotremorine-M elicited an increase in cAMP accumulation over the maximal inhibition noted at lower concentrations in both CHO M 3 and CHO M 2+M 3 cells. Following pertussis toxin treatment, only the stimulatory phase of the cAMP response to oxotremorine-M was observed in CHO M 2, CHO M 3, and CHO M 2+M 3 cells. The pharmacological antagonism of the cAMP response in CHO M 2+M 3 cells resembled that expected for a response mediated independently by both M 2 and M 3 receptors.

The putative somatostatin antagonist, cyclo-(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[BZL]), may act as potent antiproliferative agonist

The cyclic somatostatin (SST) analogue, cyclo-(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[BZL]) (cSSTA), has been widely used as somatostatin antagonist. In the human neuroblastoma cell line SH-SY5Y the cyclopeptide acts as a somatostatin receptor agonist. Similar to SST, cSSTA inhibits cell proliferation, activates the protein tyrosine phosphatase SHP-2, and stimulates the activity of mitogen-activated protein kinase. These results suggest that in SH-SY5Y neuroblastoma cells somatostatin receptors may exist which exhibit altered antagonist binding properties.

Different regulation of rat 5-HT 2A and rat 5-HT 2C receptors in NIH 3T3 cells upon exposure to 5-HT and pipamperone

The 5-HT 2A and 5-HT 2C receptors belong to the same subtype of the G-protein coupled receptor family and have several agonist and antagonist ligands in common. To gain more insight into the differences in the regulation of the two receptors, we studied the effect of agonist and antagonist pre-treatment on radioligand receptor binding and 5-HT-induced inositol phosphate formation on rat 5-HT 2A and rat 5-HT 2C receptors stable expressed in NIH 3T3 cells. We compared short (15 min) and prolonged (48 h) pre-treatment of the cells with the natural agonist, 5-HT and with the antagonist pipamperone, which can be readily washed out. The rat 5-HT 2C receptor showed an agonist-induced down-regulation (decrease in B max of labelled agonist and antagonist binding) and desensitisation (decrease in 5-HT-induced inositol phosphate formation and potency of 5-HT). Antagonist pre-treatment induced an increase in rat 5-HT 2C receptor-mediated inositol phosphate formation as well as increased agonist and antagonist radioligand binding. These findings are consistent with the classical model of G-protein coupled receptor regulation. In contrast, the rat 5-HT 2A receptor expressed in the same host cell behaved differently, unlike the classical model. Pre-treatment with 5-HT for 15 min and 48 h did not change receptor levels measured by radioligand binding, but the signal transduction response (inositol phosphate formation) was significantly reduced. Pre-treatment with the antagonist pipamperone for 15 min and 48 h caused an increase in antagonist radioligand binding but a reduction in agonist radioligand binding and a decrease in inositol phosphate formation and potency of 5-HT. Hence, the rat 5-HT 2A receptor apparently undergoes agonist desensitisation without down-regulation of the total receptor number. Antagonist pre-treatment causes a paradoxical desensitisation, possibly by uncoupling of the receptor from G-proteins. The uncoupled receptor does not bind 5-HT in the nanomolar range but retains its antagonist binding properties. Paradoxical antagonist-induced desensitisation of rat 5-HT 2A receptors has also been observed in vivo.

Different antagonist binding properties of human and rat histamine H 3 receptors

Different histamine H 3-receptor antagonists have been tested in displacement studies at human and rat H 3 receptors in stably transfected cells. Based on an actual rhodopsin structure, models for receptor–antagonist interaction were developed for receptors of both species. Similarities and discrepancies in binding profiles can be explained, but not quantified by hydrophilic interactions with Asp114 and an important lipophilic binding pocket modified by two nearby amino acids.

Binding Properties of Agonists and Antagonists to Distinct Allosteric States of the Nicotinic Acetylcholine Receptor Are Incompatible with a Concerted Model

Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde, which abolishes allosteric transitions in the protein. Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low affinity to a conformation with high affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model, including the postulate of a preformed equilibrium between the different states of the nAChR in the absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying non-competitive antagonism at the nAChR.

Conserved Ca2+-antagonist-binding properties and putative folding structure of a recombinant high-affinity dihydropyridine-binding domain

Conserved Ca2+-antagonist-binding properties and putative folding structure of a recombinant high-affinity dihydropyridine-binding domain

Conserved Ca2+-antagonist-binding properties and putative folding structure of a recombinant high-affinity dihydropyridine-binding domain

Sensitivity to 1,4-dihydropyridines (DHPs) can be transferred from L-type (alpha1C) to non-L-type (alpha1A) Ca(2+) channel alpha1 subunits by the mutation of nine pore-associated non-conserved amino acid residues, yielding mutant alpha1A(DHP). To determine whether the hallmarks of reversible DHP binding to L-type Ca(2+) channels (nanomolar dissociation constants, stereoselectivity and modulation by other chemical classes of Ca(2+) antagonist drugs) were maintained in alpha1A(DHP), we analysed the pharmacological properties of (+)-[(3)H]isradipine-labelled alpha1A(DHP) Ca(2+) channels after heterologous expression. Binding of (+)-isradipine (K(i) 7.4 nM) and the non-benzoxadiazole DHPs nifedipine (K(i) 86 nM), (+/-)-nitrendipine (K(i) 33 nM) and (+/-)-nimodipine (K(i) 67 nM) to alpha1A(DHP) occurred at low nanomolar K(i) values. DHP binding was highly stereoselective [25-fold higher affinity for (+)-isradipine]. As with native channels it was stimulated by (+)-cis-diltiazem, (+)-tetrandrine and mibefradil. This suggested that the three-dimensional architecture of the channel pore was maintained within the non-L-type alpha1A subunit. To predict the three-dimensional arrangement of the DHP-binding residues we exploited the X-ray structure of a recently crystallized bacterial K(+) channel (KcsA) as a template. Our model is based on the assumption that the Ca(2+) channel S5 and S6 segments closely resemble the KcsA transmembrane folding architecture. In the absence of three-dimensional structural data for the alpha1 subunit this is currently the most reasonable approach for modelling this drug-interaction domain. Our model predicts that the previously identified DHP-binding residues form a binding pocket large enough to co-ordinate a single DHP molecule. It also implies that the four homologous Ca(2+) channel repeats are arranged in a clockwise manner.

GABA(A) receptor binding and localization in the tiger salamander retina.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the retina and also appears to act as a trophic factor regulating photoreceptor development and regeneration. Although the tiger salamander is a major model system for the study of retinal circuitry and regeneration, our understanding of GABA receptors in this species is almost exclusively based on the results of physiological studies. Therefore, we have examined the pharmacological binding properties of GABA(A) receptors and their anatomical localization in the tiger salamander retina. Radioligand-binding studies showed that specific 3H-GABA binding to GABA(A) receptors was dominated by a single high-affinity binding site (Kd = 15.6+/-6.9 nM). Specific binding of 3H-GABA was almost completely eliminated by muscimol (Ki = 105+/-62 nM) and bicuculline (Ki = 14.3+/-2.2 microM); however, SR-95531 only displaced about 40% of specific 3H-GABA binding (Ki = 35.0+/-3.8 nM). These data indicate that there are at least two subtypes of GABA(A) receptors present in the salamander retina that can be distinguished by their antagonist binding properties: one sensitive to both bicuculline and SR-95531, and one sensitive to bicuculline but insensitive to SR-95531. Because localization of GABA receptors in the salamander retina by immunocytochemistry is problematic, GABA(A) receptors were localized by fluorescent ligand binding combined with immunocytochemical labeling for cell specific markers. Binding of fluorescently labeled muscimol to GABA(A) receptors was present in both plexiform layers and on photoreceptor cell bodies. GABA(A) receptors in the outer plexiform layer were localized to both photoreceptor terminals and horizontal cell processes.

Binding properties of glycine antagonists on N-methyl-D-aspartate (NMDA) receptors — A comparison between receptors expressed in the yeast Saccharomyces cerevisiae and membranes from rat brain : [formula omitted], Michael Berger 2 and Christian R. Noe 1 1. Institut für Pharmazeutische Chemie, Marie-Curie-Straße 9, 60439 Frankfurt, Germany 2. Institute of Biochemical Pharmacology, University of Vienna, Borschkegasse 8A, A-1090 Austria

Binding properties of glycine antagonists on N-methyl-D-aspartate (NMDA) receptors — A comparison between receptors expressed in the yeast Saccharomyces cerevisiae and membranes from rat brain : [formula omitted], Michael Berger 2 and Christian R. Noe 1 1. Institut für Pharmazeutische Chemie, Marie-Curie-Straße 9, 60439 Frankfurt, Germany 2. Institute of Biochemical Pharmacology, University of Vienna, Borschkegasse 8A, A-1090 Austria

Pharmacology and regional distribution of the binding of 6-[3H]nitro-7-sulphamoylbenzo[f]-quinoxaline-2,3-dione to rat brain.

6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) is a competitive antagonist selective for alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Here we report the pharmacological characteristics and anatomical distribution of [3H]NBQX binding to rat brain. The association rate of [3H]NBQX to rat cerebrocortical membranes was rapid, with peak binding occurring within 10 min at 0 degree C. The off-rate was also rapid, with near-complete dissociation of the radioligand within 5 min of addition of 1 mM unlabelled L-glutamate. [3H]NBQX bound to a single class of sites with KD and Bmax values of 47 nM and 2.6 pmol mg-1 of protein, respectively. The rank order of inhibition of [3H]NBQX binding by AMPA receptor ligands was NBQX > > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > or = (S)-5-fluorowillardiine > or = AMPA > > L-glutamate. The chaotrope KSCN had no effect on the IC50 value of unlabelled NBQX displacement of [3H]NBQX binding. The kainate receptor-selective ligands NS102 and kainate were only very weak displacers. It is interesting that NBQX and CNQX displaced significantly more [3H]NBQX than any of the agonists tested. Autoradiographic analysis of the binding of [3H]NBQX to coronal sections showed a distribution compatible with that of [3H]AMPA binding. These data indicate that [3H]NBQX provides a useful novel tool to characterise the antagonist binding properties of AMPA receptors.

The C terminal tail of the histamine H2 receptor contains positive and negative signals important for signal transduction and receptor down-regulation.

To examine the role of the C terminal tail in H2 receptor regulation, three cDNAs, encoding truncated histamine H2 receptor mutants (H2T295, H2T307, and H2T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H2 receptor binding of the H2T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H2T307) did not affect the binding properties of H2 antagonists and histamine or histamine-induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H2T341), which was able to form a ternary complex but was unable to fully activate the Gs protein on histamine exposure. Agonist-induced but not the cyclic AMP-dependent H2 receptor down-regulation was more profound for the H2T307 receptor, indicating that different structural elements of the H2 receptor protein are involved in the cyclic AMP-dependent and independent pathways of H2 receptor down-regulation. Taken together, in this study we identified regions in the C terminal tail of the H2 receptor that act as positive and/or negative signals in H2 receptor signaling and down-regulation.

Antagonists have a greater selectivity for muscarinic receptor subtypes in intact cerebellar granule cells than in membranes

A comparison of muscarinic acetylcholine receptor (mAChR) antagonist binding properties was made between intact cerebellar granule cell cultures and membranes prepared from these cells. [ 3H]quinuclidinyl benzylate (QNB) binding displacement by four mAChR antagonists was measured and the selectivities for m 2- or m 3-mAChRs estimated by curve fitting. For each antagonist, the preparation of membranes caused a subtype selective decrease in receptor affinity, as compared to intact cell binding. The m 2-selective antagonists had lower affinities in membranes for m 2- but not for m 3-mAChR, while the m 3-selective antagonists had lower affinities for m 3- but not for m 2-mAChR. As a result, the m 2-mAChR selectivity of AF-DX 116 and methoctramine in membranes was 66- and 1.7-fold less than in intact cells, and the m 3-mAChR selectivity of 4-DAMP and pFHHSiD was 2.4- and 3.9-fold less in membranes than in intact cells. The m 3-mAChR selectivity of 4-DAMP in intact cells was unaffected by cytoskeletal depolymerization with cytochalasins and colchicine. We suggest that the changes in selectivity seen with cell disruption may be due to a loss of cellular factors which regulate receptor properties. Antagonists binding to receptors on intact cells may cause subtype-specific changes in the interaction of the mAChR with these factors. These data suggest that mAChR antagonist binding selectivity needs to be re-examined in intact cell systems.