Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland.

A family of five subtypes of muscarinic acetylcholine receptors (mAChR) has been identified based on their molecular structures and second signal transduction pathways. In the present study, we examined the antagonist binding profiles of 9 muscarinic antagonists (atropine, 4-DAMP, pirenzepine, oxybutynin, tiquizium, timepidium, propiverine, darifenacin and zamifenacin) for human muscarinic acetylcholine receptor subtypes (m1, m2, m3, m4 and m5) produced by using a baculovirus infection system in Sf9 insect cells, and rat tissue membrane preparations (heart and submandibular gland). In a scopolamine methyl chloride [N-methyl-3H]- ([3H]NMS) binding assay, pirenzepine and timepidium displayed the highest affinities for the m1 and m2 subtypes, respectively, and both zamifenacin and darifenacin had the highest affinities for the m3 subtype, although the selectivities among the five subtypes were less than 10-fold. Propiverine showed a slightly higher affinity for the m5 subtype, whereas none of the drugs used in this study was uniquely selective for the m4 subtype. The binding affinities of muscarinic antagonists for rat heart and submandibular gland strong correlated with those for human cloned m2 and m3 subtypes, respectively. These data suggest that [3H]NMS binding studies using rat heart and submandibular gland might be useful methods which predict the affinities of test drugs for human muscarinic M2 and M3 receptor subtypes.     

Characterization of Muscarinic Receptor Subtypes in Canine Left Ventricular Membranes

Abstract

The pharmacological characteristics of muscarinic receptor (mAChR) subtypes in canine left ventricular membranes (LVM) were determined using [³H]quinuclidinyl benzilate ([³H]QNB) and [³H] N-methyl scopolamine ([³H]NMS) as ligands. Binding of [³H]QNB and [³H]NMS was saturable with respect to the radioligand concentrations. Analysis of binding isotherms by Scatchard plot showed that [³H]QNB and [³H] NMS bound to an apparently homogeneous population of mAChRs in LVM, with K_D values of 390 ± 100 and 285 ± 34 pM and B_max values of 240 ± 20 and 133 ± 9 fmol/mg protein, (n=6), respectively. The Hill coefficients for [³H]QNB and [³H]NMS binding were 0.95 ± 0.02 and 0.99 ± 0.01, respectively. Based on the competitive inhibition of [³H] ligand binding, atropine and NMS as well as the selective M₁ antagonist PZ revealed no selectivity for these mAChRs. PZ competed with [³H]QNB or [³H]NMS for a single binding site with a K_i value of 0.23 ± 0.03 μM and 0.62 ± 0.10 μM, (n = 6), respectively, which is close to the values of M² or M³ receptors. The data indicate that the M¹ receptor subtype did not exist in canine LVM. Competition of [³H] ligand binding with selective M₂ antagonists, AF-DX 116 and methoctramine and the selective M₃ antagonists, 4-DAMP and hexahydrosiladifenidol, gave a best fit for a two-binding site model. The inhibition of carbachol-mediated phosphoinositide hydrolysis by PZ, AF-DX 116 and 4-DAMP, generated an affinity profile for this response also dissimilar to that described for the classical cardiac M₂ response. Although no other muscarinic receptor mRNA has been detected in this tissue, these data suggest the presence of a second population of muscarinic sites, which may signify an M₂ receptor diversity.     

Muscarinic stimulation of submucosal glands in swine trachea

The properties of muscarinic acetylcholine receptors (mAChR) on tracheal explants and isolated submucosal gland cells were determined using [3H]quinuclidinyl benzilate ([3H]QNB) and N-[3H]methylscopolamine ([3H]NMS) as ligands. Analysis of competitive displacement of ([3H]NMS binding by pirenzepine demonstrated the presence of M1- (27 +/- 2%) and M2G- (73 +/- 2%) receptors on isolated tracheal submucosal gland cells (TSGC's) in control. Daily administration of diisopropylfluorophosphate (DFP) inhibited cholinesterase activity by greater than 95%. After 7 days of DFP treatment, [3H]QNB binding to intact TSGC's decreased from 14.2 +/- 0.6 to 6.3 +/- 0.8 fmol/10(6) cells; similarly, [3H]NMS binding fell from 8.1 +/- 1.9 to 2.0 +/- 0.8 fmol/10(6) cells. The loss of mAChR's was predominantly of the M2G subtype with the relative proportion dropping to 33%. In addition, 90% of the receptors assumed the high-affinity state for carbachol displacement of [3H]NMS. Mucus secretion was quantitated by measuring the release of 3H-labeled mucus macromolecules from explants of tracheal submucosal glands and isolated cells. Acetylcholine (ACh), 2 X 10(-5) M, stimulated mucus secretion by 2.5 and 2.3 times the basal rate, respectively. Elimination of acetylcholinesterase (AChe) by DFP increased the ACh sensitivity by 18- and 5-fold. Tracheal explants or TSGC's obtained 2 h after an in vivo DFP treatment showed a 6- and 3-fold ACh stimulation. This ACh sensitivity decreased during the continued daily dosing with DFP such that only a 1.3- and 1.1-fold ACh stimulation was apparent after 7 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of Agonist-Induced Down-Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line

The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.     

Muscarinic acetylcholine receptor (mAChR) inhibitor from snake venom: interaction with subtypes of human mAChR.

Snake venoms can contain a variety of well-studied neurotoxins, especially nicotinic acetylcholine receptor inhibitor, normally called postsynaptic neurotoxin. Karlsson first reported muscarinic acetylcholine receptor (mAChR) inhibitor from snake venom. In a previous study in our laboratory, we found a mAChR inhibitor from Naja naja sputatrix venom that bound to rat brain synaptosomes. Brain synaptosomes contain all subtypes of mAChRs, and thus the exact selectivity of the inhibitor could not be determined. mAChR inhibitor from N. naja sputatrix venom was purified and the binding to all human mAChR subtypes (M1, M2, M3, M4, and M5) was investigated and is reported in this communication. The inhibitor bound to all subtypes of the human mAChR, but showed considerably high selectivity for the M5 subtype. It was also found that the reduction of disulfide bonds in the inhibitor eliminated the binding to the mAChR. This suggests that a specific tertiary conformation maintained by disulfide bonds is essential for binding to the mAChR. An oligo peptide, QIHDNCYNE, comparable to a part of the inhibitor molecule, was synthesized and studied for its binding to the mAChR. The synthetic peptide did not show any binding activity, suggesting this portion of the inhibitor molecule is not involved in mAChR binding. The selective binding of the M5 mAChR subtype to antagonists has not yet been reported. Therefore, the purified inhibitor reported in this communication may be a useful tool to clarify the mechanism of muscarinic cholinergic transmission.     

[3H]N-Methylscopolamine Binding Studies Reveal M2 and M3 Muscarinic Receptor Subtypes on Cerebellar Granule Cells in Primary Culture

Saturation experiments with the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) indicated that cerebellar granule cells in primary culture possess a high density of muscarinic acetylcholine receptors (mAChRs): Bmax = 1.85 +/- 0.01 pmol/mg of protein at 10 days in culture; KD = 0.128 +/- 0.01 nM. The selective M1 antagonist pirenzepine displaced [3H]NMS binding with a low affinity (Ki = 273 +/- 13 nM), whereas the M2/M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide competed with [3H]NMS with Ki values in the nanomolar range, a result suggesting that some of the mAChRs on cerebellar granule cells belong to the M3 subtype. Methoctramine, which discriminates between M2 and M3 subtypes with high and low affinity, respectively, displayed a high and low affinity for [3H]NMS binding sites (Ki(H) = 31 +/- 5 nM; Ki(L) = 2,620 +/- 320 nM). These results provide the first demonstration that both M2 and M3 mAChR subtypes may be present on cultured cerebellar cells. In addition, complete death of neurons induced by N-methyl-D-aspartate (100 microM for 1 h) reduced by 85% the specific binding of [3H]NMS, a result indicating that most mAChRs were associated with neuronal components. Finally, the evolution of the density of mAChRs, labeled by [3H]NMS, correlated with the neuronal maturation during the in vitro development of these cells.     

Binding of radioiodinated SPECT ligands to transfected cell membranes expressing single muscarinic receptor subtypes

The equilibrium dissociation constant and the kinetic rate constants were determined for the binding of (R)-[3H]3-quinuclidinyl benzilate ([3H]QNB) and [125I]3-quinuclidinyl-4-iodobenzilate ((R,R)- and (R,S)-[125I]IQNB) to transfected cell membranes expressing one single muscarinic acetylcholine receptor (mAChR) subtype. The association and dissociation kinetics for the m2 subtype were more rapid than for the m1 and m3 subtypes. The differential kinetic properties may be useful for the single photon emission computed tomographic (SPECT) evaluation of regional mAChR subtype alterations in disease states.     

Reduction of Muscarinic Acetylcholine Receptor Number and Affinity by an Endogenous Substance

We examined the soluble fraction from homogenates of 12-day embryonic chick heart for the presence of an endogenous modulator of muscarinic acetylcholine receptors (mAChR). Homogenates were separated into 100,000 g soluble and crude membrane fractions by differential centrifugation. Aliquots of membranes were incubated in the presence or absence of the soluble fraction and the muscarinic antagonist, [3H]quinuclidinyl benzilate ( [3H]QNB), and the data subjected to Scatchard analysis. In the presence of the soluble fraction, mAChR number decreased up to 70% and the affinity for [3H]QNB decreased six- to eightfold. These results suggested that an endogenous soluble factor (ESF) affected cholinergic ligand binding to the receptor. The amount of ESF extracted from less than 10 mg of brain was sufficient to reduce by 50% [3H]QNB binding to 50 fmol mAChR. ESF activity was partially purified by heat and acid treatment. The loss of receptors was dependent upon the amount of ESF added and was time dependent. QNB protected some receptors from loss due to ESF. The change in mAChR affinity for [3H]QNB was observed only if ESF was present continuously during the [3H]QNB binding assay. Ultrafiltration and gel filtration showed that ESF was less than 10,000 daltons and probably less than 700 daltons. ESF activity was blocked by EDTA. However, ESF was not a divalent cation since it was base labile, and removal of divalent cations with Chelex-100 did not inhibit ESF activity. ESF activity was also blocked by catechol, catecholamines, ascorbate, and dithiothreitol. ESF was present in embryonic but not in adult heart.     

Regulation of Neuronal Muscarinic Acetylcholine Receptor Number by Protein Glycosylation

Tunicamycin, a potent inhibitor of protein glycosylation, was used to study the role of protein glycosylation in the regulation of muscarinic acetylcholine receptor (mAChR) number in cultures of N1E-115, a murine neuroblastoma cell line. At a concentration of 0.35 microgram/ml, tunicamycin inhibited macromolecular incorporation of [3H]mannose by 75-80%, whereas incorporation of [3H]leucine was reduced by only 10%. Treatment with tunicamycin caused a 30% decrease in total membrane mAChR number within 48 h as determined by a filter-binding assay using [3H]quinuclidinyl benzilate ([3H]QNB), a highly specific muscarinic antagonist. Tunicamycin also inhibited the recovery of total membrane mAChR by 70% following carbachol-induced down-regulation. The rate of mAChR degradation (control t1/2 12-14 h) was unaffected by incubation with tunicamycin. Intact cell binding studies using [3H]QNB (a membrane-permeable ligand) to measure total cellular (internal plus cell surface) mAChR and [3H]N-methylscopolamine ([3H]NMS, a membrane-impermeable ligand) to measure cell surface mAChR were conducted to determine whether tunicamycin selectively depleted cell surface mAChR. With 12 h of treatment with tunicamycin, cell surface mAChR number declined by 35%, whereas total cellular mAChR fell by only 10%. The ratio of cell surface receptor to total receptor decreased by 45% after 24 h. These results indicate that protein glycosylation is required for the maintenance of cell surface mAChR number. Incubation with tunicamycin causes a selective depletion of cell surface mAChR, implying that protein glycosylation plays a critical role in transport and/or incorporation of mAChR into the plasma membrane.     

Asymmetric distribution and down-regulation of the muscarinic acetylcholine receptor in rat cerebral cortex

The distribution and down-regulation of the muscarinic acetylcholine receptor (mAChR) were studied in dissociated cells from right (RCC) and left (LCC) cerebral cortex. For this purpose [³H]quinuclidinyl benzilate (QNB) and [³H]pirenzepine (Pz), two muscarinic antagonists, were used. The mAChR binding sites detected with [³H]QNB were asymmetrically distributed between the two hemispheres, the majority being found in the RCC. Asymmetry was also evident in the distribution of the mAChR subtypes (M₁ and M₂) detected with [³H]Pz. Under basal conditions the RCC had roughly 50% more M₁ subtype than the LCC. The pharmacological and kinetic parameters were similar for both antagonists in RCC and LCC, indicating that the observed lateralization was due to a different density of the receptor rather than to different kinetics of binding of the two radioligands. After sustained stimulation with the agonist carbamoylcholine, the receptor sites detected with [³H]Pz, i.e. the M₁ subtype of mAChR, decreased at a higher rate in the RCC (44%) than in the LCC (25% of controls), demonstrating that the down-regulation process is more active in the right than in the left cortex, and thus implying that there is better coupling between the stimulated mAChR and its effector system in the former.     

Pharmacologic characterization of muscarinic receptors of insect brains.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.     

Characterization of Muscarinic Cholinergic Receptors in the Crab Nervous System

The selective muscarinic antagonist L-[3H]-quinuclidinyl benzilate (L-[3H]QNB) binds reversibly and with high affinity (KD = 0.3 nM) to a single population (Bmax = 105 fmol/mg protein) of specific sites in nervous tissue of the crab Cancer magister. The binding site is stereoselective; (-)QNB is over 200 times more potent than (+)QNB as an inhibitor of specific L-[3H]QNB binding. The muscarinic antagonists scopolamine and atropine are over 10,000 times more potent inhibitors of L-[3H]QNB binding than the nicotinic antagonists decamethonium and d-tubocurarine. The muscarinic agonists oxotremorine, pilocarpine, arecoline, and carbachol also compete effectively for the L-[3H]QNB binding site. This pharmacological profile strongly suggests the presence of classical muscarinic receptors in the crab nervous system. These receptors are localized to nervous tissue containing cell bodies and neuropil, whereas specific L-[3H]QNB binding is low or absent in peripheral nerve, skeletal muscle, and artery.     

Alpha adrenergic drugs inhibit [3H]-QNB binding to muscarinic receptors of rat heart, brain and parotid gland membranes

Alpha adrenergic agonists and antagonists as clonidine, guanfacine, yohimbine, phenylephrine and prazosin inhibited the [3H]-QNB binding to rat brain cortex muscarinic acetylcholine receptor (mAChR, M-1 subtype), heart (M-2 subtype) and parotid gland homogenate (M-3 subtype) in a dose-dependent competitive fashion. Ki values were between 10(-6) and 10(-3) M. Hill coefficients were about 1. No correlation was found between mAChR inhibiting capacity of these drugs and their activity on alpha adrenergic receptors. In contrast, other transmitters, as dopamine, GABA, glutamic acid, histamine, serotonin, isoproterenol and platelet activating factor (PAF) did not affect the QNB binding.     

Quaternary forms of classical muscarinic antagonists distinguish subpopulations of muscarinic receptors: correlation with gallamine-defined subpopulations

Atropine and scopolamine inhibit the binding of [3H] quinuclidinyl benzilate (QNB) to muscarinic receptors of rat forebrain in a manner that suggests homogeneity of the binding sites. Under the same conditions, the inhibition by N-methylatropine (NMA) and N-methylscopolamine (NMS) of the binding of [3H]QNB is consistent with the presence of subpopulations of receptors that differ greatly in affinities toward these quaternary ligands. The subpopulations that are defined according to the affinities of NMA and NMS correlate very well with those that are defined by the use of gallamine. It is suggested that the heterogeneity in the binding of NMS explains some of the anomalous interactions between NMS and gallamine that have been reported previously.     

Guanine nucleotide regulation of a mammalian myocardial muscarinic receptor system. Evidence for homo- and heterotropic cooperativity in ligand binding analyzed by computer-assisted curve fitting

Highly purified dog heart sarcolemmal membranes, with a content of approximately 5 pmol of muscarinic acetylcholine receptor (mAChR)/mg of protein, were analyzed for mAChR-mediated inhibition of adenylyl cyclase and ligand binding in the absence and the presence of guanine nucleotides. Adenylyl cyclase was found to be coupled to the mAChR, being attenuated approximately 30% in a GTP-dependent manner. Direct binding studies, using 3H-labeled oxotremorine M, showed high affinity binding (apparent KD = 10 nM) that was reduced on nucleotide addition. Dose-response curves for GDP, GTP, and guanyl-5'-yl imidodiphosphate showed them to be equipotent. On the basis of pirenzepine binding, only one type of mAChR, commonly referred to as M2, was detected. Direct binding of [3H]quinuclidinyl benzilate [( 3H]QNB) uncovered 50% more binding sites than 150 nM 3H-labeled oxotremorine M; addition of guanine nucleotides uncovered the existence of positive cooperativity in the binding of [3H]QNB. Agonist displacement curves of [3H]QNB binding, without and with guanine nucleotides, extended over several orders of magnitude, which is inconsistent with single site competitive kinetics. The results and their analysis by computer-assisted curve fitting indicated that the data are well fitted by a model in which a receptor is at least bivalent and exists in two states: one with and the other without cooperativity between its sites, with guanine nucleotides decreasing both the degree of cooperativity between the sites and the proportion of the receptor that is in the cooperative form. Since the guanine nucleotide effect is mediated by the Ni coupling protein, it is suggested that direct binding detects R'Ni complexes (cooperative), R"NiG complexes (cooperative but distinct from R'Ni), and R0 complexes (non-cooperative and unaffected by Ni or NiG), where R = mAChR, Ni = the inhibitory regulatory component of adenylyl cyclase unaffected by guanine nucleotide, and NiG = Ni affected by guanine nucleotide (G).     

In vivo demonstration of M3 muscarinic receptor subtype selectivity of darifenacin in mice

A novel muscarinic receptor antagonist, darifenacin, inhibited specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS) in the mouse bladder, submaxillary gland and heart in a concentration-dependent manner. The inhibitory effect was most potent in the submaxillary gland, followed by the bladder and heart. In addition, darifenacin inhibited specific [(3)H]NMS binding in the membranes of CHO-K1 cell lines expressing muscarinic M(2) and M(3) receptor subtypes, and the potency was significantly (22-fold) greater at the M(3) than at the M(2) subtype. At 0.5 to 12 h after oral administration of darifenacin, a significant increase in K(d) values for specific [(3)H]NMS binding was seen in the bladder, submaxillary gland and lung of mice, compared with control values. Also, there was a sustained decrease in the B(max) values in the submaxillary gland. These data suggest that muscarinic receptor binding of oral darifenacin is rapid in onset and of a long duration. On the other hand, oral darifenacin exerted only temporary or little binding of muscarinic receptors in the heart and colon. Pilocarpine-induced salivary secretion in mice was continuously suppressed by oral darifenacin. The time-course of suppression coincided well with that for the muscarinic receptor binding in the submaxillary gland. The antagonistic effect of darifenacin against the dose-response curves for pilocarpine appeared to be insurmountable. In conclusion, the present study has shown that oral darifenacin may exert a pronounced and long-lasting binding of muscarinic receptors in tissues expressing the M(3) subtype.     

Effect of aging on the interaction of quinuclidinyl benzilate,N-methylscopolamine,pirenzepine, and gallamine with brain muscarinic receptors

The objective of the present study was to investigate the effects of senescence on the binding characteristics of muscarinic receptors by using [³H]quinuclidinyl benzilate ([³H]QNB) and [³H]N-methylscopolamine ([³H]NMS) as ligands in young (3months), middle-age (10months) and old (24 months) male Fischer 344 rats. Muscarinic receptor density was found to decrease significantly with aging in certain brain regions, depending on the ligand employed. Moreover, the relative proportions of M₁ and M₂ muscarinic receptor subtypes was not significantly altered by aging, except in the aged striatum. Furthermore, the dissociation kinetics of [³H]NMS in the cerebral cortex and their allosteric modulation by gallamine were only slightly influenced by age.     

Recovery of oligomers and cooperativity when monomers of the M2 muscarinic cholinergic receptor are reconstituted into phospholipid vesicles

FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.