Differential regulation of high-affinity agonist binding to muscarinic sites in the rat heart, cerebellum, and cerebral cortex

The muscarinic agonist [3H]cismethyldioxolane ([3H]CD) was used to characterize the effects of regulators upon high-affinity agonist binding sites of the rat heart, cerebral cortex and cerebellum. Comparative studies with sodium ions (Na+), magnesium ions (Mg++), N-ethylmaleimide (NEM) and the guanine nucleotide Gpp(NH)p revealed tissue-specific effects. Mg++ preferentially enhanced while Gpp(NH)p and NEM reduced high-affinity [3H]CD binding in the heart and cerebellum. By comparison NEM enhanced high-affinity agonist binding in the cerebral cortex while Gpp(NH)p and Mg++ had little or no effect. Kinetic studies support an allosteric mechanism for these effects and provide further evidence for muscarinic receptor subtypes in mammalian tissues.     

Differences and Similarities in Muscarinic Receptors of Rat Heart and Retina: Effects of Agonists, Guanine Nucleotides, and N-Ethylmaleimide

Muscarinic receptor stimulation inhibits cyclic AMP formation in rat atria but not in retina. We compared the properties of the muscarinic receptors in rat atrial and retinal membranes using the antagonist [3H]quinuclidinyl benzilate. In both atria and retina there is a single binding site for antagonists, while agonists appear to interact at two classes of binding sites. Muscarinic receptors in atria and retina have the same apparent affinities for several antagonists and for a series of muscarinic agonists. In both tissues N-ethylmaleimide decreases agonist affinity for the high-affinity binding sites. Muscarinic receptors in atria and retina differ, however, in several properties relating to the proportions of high- and low-affinity agonist sites. First, guanine nucleotides markedly increase the proportion of low-affinity binding sites in atria, but not in retina. Second, for all agonists there are fewer high-affinity binding sites in retina. Third, the "partial agonist" pilocarpine appears to interact with two classes of binding sites in atria, but with only a single class of sites in retina. Our data suggest that muscarinic receptors that inhibit cyclic AMP formation and those that do not share common properties that determine receptor affinity for agonists and classic antagonists. The differences between these receptors are manifest, however, in the effects of guanine nucleotides and the ability of agonists, especially those of low efficacy, to affect the proportion of high- and low-affinity sites and to effect a biological response.     

A1 Adenosine Receptor-G Protein Coupling in Bovine Brain Membranes: Effects of Guanine Nucleotides, Salt, and Solubilization

The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high-affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)-phenylisopropyladenosine (R-PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all-low-affinity state that was indistinguishable from the low-affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R-PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R-PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration-dependent manner increased antagonist ([3H]XAC) but not agonist (R-N6-[3H]phenylisopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R-PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low-affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist interactions at hepatic alpha 1- and beta-adrenergic receptors: affinity-state regulation by guanine nucleotides and temperature

We investigated the binding characteristics of agonists to alpha 1- and beta-adrenergic receptors of intact liver cells, broken rat liver cell membranes, and detergent-solubilized preparations under varying experimental conditions, focusing on the different "states" of the receptor for agonists and the regulation of these states by temperature and guanine nucleotides. While only low-affinity binding of agonists to both receptor subtypes was evident in studies performed at 37 degrees C with solubilized preparations, biphasic competition curves for agonists were observed in both intact cells and membrane preparations; the majority of sites were of low affinity. In membrane preparations, the nonhydrolyzable GTP analogue Gpp(NH)p caused a rightward shift of agonist competition curves and a loss of high-affinity binding. These results are consistent with the involvement of guanine nucleotide binding proteins in both alpha 1- and beta-adrenergic transduction pathways. When competition studies were performed at 4 degrees C, receptor sites existed predominantly in the high-affinity configuration, in intact cells and membranes, as well as in soluble preparations. In contrast to the studies conducted at 37 degrees C, no Gpp(NH)p-induced conversion to the lower affinity state could be demonstrated in studies performed with membrane preparations at 4 degrees C. Thus, the high-affinity state of alpha 1- and beta-adrenergic receptors is stabilized at 4 degrees C in intact cells, membranes, and soluble preparations. After incubations had been performed at 37 degrees C, high-affinity binding of agonists could not be restored by subsequent incubation at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanine Nucleotide Effects on 8-Cyclopentyl-1,3-[3H]Dipropylxanthine Binding to Membrane-Bound and Solubilized A1 Adenosine Receptors of Rat Brain

The effects of guanine nucleotides on binding of 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX), a highly selective A1 adenosine receptor antagonist, have been investigated in rat brain membranes and solubilized A1 receptors. GTP, which induces uncoupling of receptors from guanine nucleotide binding proteins, increased binding of [3H]DPCPX in a concentration-dependent manner. The rank order of potency for different guanine nucleotides for increasing [3H]DPCPX binding was the same as for guanine nucleotide-induced inhibition of agonist binding. Therefore, a role for a guanine nucleotide binding protein, e.g., Gi, in the regulation of antagonist binding is suggested. This was confirmed by inactivation of Gi by N-ethylmaleimide (NEM) treatment of membranes, which resulted in an increase in [3H]DPCPX binding similar to that seen with addition of GTP. Kinetic and equilibrium binding studies showed that the GTP- or NEM-induced increase in antagonist binding was not caused by an affinity change of A1 receptors for [3H]DPCPX but by an increased Bmax value. Guanine nucleotides had similar effects on membrane-bound and solubilized receptors, with the effects in the solubilized system being more pronounced. In the absence of GTP, when most receptors are in a high-affinity state for agonists, only a few receptors are labeled by [3H]DPCPX. It is suggested that [3H]DPCPX binding is inhibited when receptors are coupled to Gi. Therefore, uncoupling of A1 receptors from Gi by guanine nucleotides or by inactivation of Gi with NEM results in an increased antagonist binding.     

Reconstitution of resolved muscarinic cholinergic receptors with purified GTP-binding proteins

The association of agonists with muscarinic receptors in membranes from bovine brain was affected only slightly by guanine nucleotides. However, solubilization of these membranes with deoxycholate and subsequent removal of detergent resulted in a preparation of receptors with increased affinity for agonists and a large increase in response to guanine nucleotides. Chromatography of deoxycholate extracts of membranes on DEAE-Sephacel resulted in the separation of receptors from 95% of the guanine nucleotide-binding activity. Guanine nucleotides had no effect on the binding of agonists to these resolved receptors. The effect of guanine nucleotides was restored after the addition of either of two purified guanine nucleotide-binding proteins from bovine brain. One of these proteins, presumably brain GI, is composed of subunits with the same molecular weights (alpha, 41,000; beta, 35,000; gamma, 11,000) and functions as the inhibitory guanine nucleotide-binding protein isolated from liver. The other protein, termed Go, is a novel guanine nucleotide-binding protein that possesses a similar subunit composition (alpha, 39,000; beta, 35,000; gamma, 11,000) but whose function is not yet known. Addition of either protein to the resolved receptor preparation increased agonist affinity by at least 10-20-fold, and low concentrations of guanine nucleotides specifically reversed this effect. Reconstitution of receptors with the resolved subunits of Go demonstrates that the beta subunit alone had no effect on agonist binding, but that this subunit does appear to enhance the effects observed with the alpha subunit alone.     

Neomycin induces high-affinity agonist binding of G-protein-coupled receptors

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1-10 mM) was found to induce high-affinity binding of the chemotactic tripeptide, N-formyl-methionylleucylphenylalanine (fMet-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine-5'-O-(3-thiotriphosphate), to reduce fMet-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-affinity GTPase by fMet-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive Gi-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of Gs-protein-coupled beta-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity binding of isoproterenol to beta-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi- and Gs-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction.     

Agonist binding promotes a guanine nucleotide reversible increase in the apparent size of the bovine anterior pituitary dopamine receptors

Dopamine receptors, solubilized from bovine anterior pituitary membranes with the detergent digitonin, retained a typical dopaminergic specificity for the binding of both agonists and antagonists. The affinities of antagonists for binding to the soluble receptors are virtually identical with those observed with the membrane-bound receptors. The affinities of agonists however, correspond to those for the form of the receptors in the membranes having low affinity for those agonists (De Lean, A., Kilpatrick, B. F., and Caron, M. G. (1982) Mol. Pharmacol. 22, 290-297). Thus, after solubilization, agonist high affinity interactions with the receptor and their sensitivity to modulation by guanine nucleotides are lost. However, high affinity agonist binding and its sensitivity to guanine nucleotides can be preserved if the membrane-bound receptors are prelabeled with the agonist [3H]n-propylapomorphine prior to solubilization. In order to investigate the molecular basis for these changes in the properties of agonist binding, the solubilized receptors were characterized by chromatographic procedures. Using molecular exclusion high pressure liquid chromatography, [3H]n-propylapomorphine-prelabeled receptors elute as an apparent larger molecular species than either unlabeled or antagonist [( 3H]spiroperidol)-pre-labeled receptors. Moreover, incubation of the pooled agonist-prelabeled receptor peak with guanine nucleotides effects a decrease in the apparent size of the receptors such that upon rechromatography they elute in a position coincidental with the 3H-antagonist-pre-labeled receptor peak. Thus, occupancy of the receptors by agonists promotes the formation of a guanine nucleotide-sensitive agonist high affinity form of the receptor which is of larger apparent size presumably due to the association of the receptor with a guanine nucleotide regulatory protein.     

Agonist-specific reverse regulation of muscarinic receptors by transition metal ions and guanine nucleotides

Transition metal ions, e.g. Mn²⁺, Ni²⁺ and Co²⁺ enhance $\text{in vitro}$ agonist binding to muscarinic receptors in mouse cortex or hippocampus. This effect arises mainly from the conversion of low to high affinity binding sites. Binding properties of antagonists in these brain areas, as well as those of both agonists and antagonists of medulla-pons muscarinic receptors, are insensitive to these ions. The induced interconversion can be reversed by either of the following procedures: (i) removal of the ions; (ii) thermal exposure; (iii) addition of micromolar concentrations of guanine nucleotides.     

Anterior pituitary dopamine receptors. Demonstration of interconvertible high and low affinity states of the D-2 dopamine receptor

The interactions of dopaminergic agonists and antagonists with binding sites in bovine anterior pituitary membranes have been investigated with radioligand-binding techniques and computer-modeling procedures. 3H-labeled agonist binding is stereospecific, reversible, saturable, and of high affinity. The rank order of catecholamines, phenothiazines, and related drugs in competing for 3H-agonist binding is indicative of interactions with a D-2 dopamine receptor. Both agonist/3H-agonist and antagonist/3H-agonist competition curves are monophasic and noncooperative (nH = 1) with computer analysis indicating a single class of binding sites. Specific 3H-agonist binding can be completely inhibited by guanine nucleotides. GppNHp us the most potent nucleotide followed by GTP and GDP which are equipotent. The equilibrium binding capacity for 3H-labeled antagonists is twice that for 3H-agonists. Unlabeled antagonists inhibit 3H-antagonist binding competitively and exhibit antagonist/3H-antagonist competition curves which model best to a state of homogeneous affinity. In contrast, unlabeled agonists inhibit 3H-antagonist binding in a heterogeneous fashion displaying multiphasic (nH less than 1) competition curves which can be resolved into high and low affinity binding sites. In the presence of saturating concentrations of guanine nucleotides, however, the agonist/3H-antagonist curves model best to a single affinity state which is identical with the low affinity state seen in control curves. The binding data can be explained by postulating two states of the D-2 dopamine receptor, inducible by agonists but not antagonists and modulated by guanine nucleotides.     

Further evidence that muscarinic cholinergic receptors of 1321N1 astrocytoma cells couple to a guanine nucleotide regulatory protein that is not Ni.

Inhibitory coupling of receptors to adenylate cyclase previously has been shown to be relatively sensitive to inactivation by alkylation with N-ethylmaleimide (NEM). Modification of the inhibitory guanine nucleotide regulatory protein, Ni, has been proposed to be responsible for this effect. The effects of NEM on GTP-sensitive binding of carbachol to muscarinic cholinergic receptors has been compared in a cell line (1321N1 human astrocytoma cells) in which these receptors stimulate phosphoinositide breakdown and in a cell line (NG108-15 neuroblastoma X glioma cells) in which activation of these receptors results in inhibition of adenylate cyclase. Pretreatment of membrane preparations from 1321N1 cells with NEM resulted in a concentration-dependent decrease in the extent of pertussis toxin-catalysed [32P]ADP-ribosylation of a 41 000 Da protein previously proposed to be the alpha subunit of Ni. Under conditions where 32P-labelling of Ni in 1321N1 membranes was reduced by NEM by 90%, no effect was observed on the extent of guanine nucleotide-sensitive high-affinity binding of carbachol to muscarinic cholinergic receptors. In contrast, treatment of NG108-15 membranes with NEM under the same conditions resulted in complete loss of high-affinity guanine nucleotide sensitive binding of carbachol. These results illustrate another difference between the muscarinic receptor population of these two cell lines, and support the previous proposal that muscarinic receptors of 1321N1 cells couple to a guanine nucleotide regulatory protein that is not Ni.     

Binding of Agonists and Antagonists to Muscarinic Acetylcholine Receptors on Intact Cultured Heart Cells

The binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured cardiac cells has been compared with the binding observed in homogenized membrane preparations. The antagonists [3H]quinuclidinyl benzilate and [3H]N-methylscopolamine bind to a single class of receptor sites on intact cells with affinities similar to those seen in membrane preparations. In contrast with the heterogeneity of agonist binding sites observed in membrane preparations, the agonist carbachol binds to a homogeneous class of low-affinity sites on intact cells with an affinity identical to that found for the low-affinity agonist site in membrane preparations in the presence of guanyl nucleotides. Kinetic studies of antagonist binding to receptors in the absence and presence of agonist did not provide evidence for the existence of a transient (greater than 30 s) high-affinity agonist site that was subsequently converted to a site of lower affinity. Nathanson N. M. Binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured heart cells.     

Guanine nucleotide regulation of agonist binding to muscarinic cholinergic receptors. Relation to efficacy of agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization.

The efficacies of a series of six muscarinic cholinergic receptor agonists for stimulation of phosphoinositide breakdown and unidirectional efflux of 45Ca2+ in 1321N1 human astrocytoma cells were compared with the relative capacity of these agonists for formation of a GTP-sensitive high-affinity binding state in washed membranes. Carbachol and methacholine were 'full' agonists as regards phosphoinositide breakdown and Ca2+ mobilization, whereas bethanechol, arecoline and oxotremorine were 'partial' agonists for these two responses. Pilocarpine was the least efficacious of the six drugs tested. Except for pilocarpine, competition curves generated with the agonists and [3H]quinuclidinyl benzilate did not follow the Law of Mass Action for ligand interaction at a single site. Non-linear regression analyses of these data indicated that the data significantly better fit a two-, rather than a single-, site model with a high- and a low-affinity binding component. Competition curves generated in the presence of GTP were shifted to the right, and the extent of receptors in the high-affinity agonist-binding state was decreased. The relative efficacies of the six agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization were significantly correlated with the difference in affinities (KL/KH) between the two affinity states for each agonist. The relative efficacy of the agonists for stimulation of Ca2+ mobilization also was significantly correlated with the extent of receptors in the high-affinity state (%H) for each agonist. The results suggest that interaction with an as-yet unidentified guanine nucleotide regulatory protein is important in the mechanism whereby muscarinic receptors stimulate phosphoinositide breakdown in 1321N1 astrocytoma cells.     

Agonists and Antagonists Recognize Different but Overlapping Populations of A1 Adenosine Receptors: Modulation of Receptor Number by MgCl2, Solubilization, and Guanine Nucleotides

A1 selective agonist and antagonist radioligands bind to the same A1 adenosine receptor binding subunit, as documented by photoaffinity labelling and partial peptide maps. In this study we document that although these radioligands recognize the same A1 adenosine receptor (A1AR), they recognize different numbers of A1ARs in bovine brain membranes, with agonist number being greater than antagonist number. Neither addition of guanine nucleotides nor removal of Mg2+ ions enhanced antagonist binding in membranes. On solubilization, agonists still recognized a greater number of A1ARs but addition of guanine nucleotides or removal of Mg2+ substantially increased the number of receptors detected with antagonist radioligands. The effects of Mg2+ and guanine nucleotides were not additive, suggesting that formation of a "low agonist-receptor-G protein state" by either modulating agent was sufficient to alter the receptor conformation such that it could be recognized by antagonist. These studies suggest that a proportion of the "precoupled A1AR-G protein complex" in membranes are in a conformation that cannot be recognized by antagonists and that membrane constraints are such that ions or guanine nucleotides cannot sufficiently modulate the conformation to allow it to recognize antagonists. On removal of membrane structure by solubilization, these constraints are removed.     

Guanine nucleotides differentiate agonist and antagonist interactions with opiate receptors.

Opiate receptor binding is regulated by guanine nucleotides differentially for agonists and antagonists. Guanosine-5′-triphosphate (GTP), its stable analogue guanyl-5′-yl-imidodiphosphate (Gpp(NH)p) and GDP inhibit binding of the ³H-agonists dihydromorphine, etorphine and enkephalins but not the ³H-antagonists naloxone or diprenorphine. GMP, ATP, ADP and AMP fail to alter either agonist or antagonist binding. Effects are more pronounced in the presence than in the absence of sodium.     

Corticosteroid modulation of muscarinic receptors in rat myocardial membranes

Rat ventricular myocardial membanes contain muscarinic acetylcholine receptors which can be identified by binding of the muscarinic antagonist (-)-[³H]quinuclidinyl benzilate. Scatchard analysis of saturation binding data revealed binding to a single class of non-cooperative sites (0.693 pmol/mg protein) with high affinity (i.e. with an equilibrium dissociation constant of 0.24 nM). Competition binding curves of the agonist carbamylholine were shallow (with a Hill coefficient, n_H of 0.71) for membranes of untreated rats, suggesting the presence of two receptor subpopulations with different agonist affinity. These curves were steeper (n_H = 0.86) for adrenalectomized animals and more shallow (n_H = 0.62) for hydrocortisone-treated animals. In contrast, both treatments did not affect the total receptor number. This suggests that corticosteroids are required for the myocardial muscarinic receptors to adopt high agonist affinity. However, the inhibition of adenylate cyclase by muscarinic agonists disappeared after both corticosteroid treatment and adrenalectomy. But agonist receptor binding could still be modulated by guanine nucleotides. This indicates that both high and low affinity froms of muscarinic receptors induced by altered corticosteroid states retain functional coupling with the inhibitory nucleotide binding site, but are uncoupled from the adenylate cyclase catalytic subunit, C.     

Hippocampal and Cerebellar β-Adrenergic Receptors and Adenylate Cyclase Are Differentially Altered by Chronic Ethanol Ingestion

Chronic ethanol ingestion by mice resulted in the loss of high-affinity beta-adrenergic agonist binding sites and a significant decrease in activation of adenylate cyclase by guanine nucleotides and beta-adrenergic agonists in the hippocampus, although no significant change was noted in the total number of beta-adrenergic receptors, as defined by the binding of the antagonist [125]iodocyanopindolol. In cerebellum, chronic ethanol ingestion resulted in a 16% decrease in the total concentration of beta-adrenergic receptors and in a decrease in the affinity for agonist of the high-affinity beta-adrenergic agonist binding sites. However, neither the amount of the high-affinity agonist binding sites nor the activation of adenylate cyclase by agonist was affected. The different responses to ethanol in hippocampus and cerebellum may result from quantitative differences in distribution of beta 1- and beta 2-adrenergic receptors in the tested brain areas and/or differential effects of ethanol on stimulatory guanine nucleotide binding protein in these brain areas.     

A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor

The unique properties of agonist binding to the frog erythrocyte beta-adrenergic receptor include the existence of two affinity forms of the receptor. The proportion and relative affinity of these two states of the receptor for ligands varies with the intrinsic activity of the agonist and the presence of guanine nucleotides. The simplest model for hormone-receptor interactions which can explain and reproduce the experimental data involves the interaction of the receptor R with an additional membrane component X, leading to the agonist-promoted formation of a high affinity ternary complex HRX. Computer modeling of agonist binding data with a ternary complex model indicates that the model can fit the data with high accuracy under conditions where the ligand used is either a full or a partial agonist and where the system is altered by the addition of guanine nucleotide or after treatment with group-specific reagents, e.g. p-hydroxymercuribenzoate. The parameter estimates obtained indicate that the intrinsic activity of the agonist is correlated significantly with the affinity constant L of the component X for the binary complex HR. The major effect of adding guanine nucleotides is to destabilize the ternary complex HRX from which both the hormone H and the component X can dissociate. The modulatory role of nucleotides on the affinity of agonists for the receptor is consistent with the assumption that the component X is the guanine nucleotide binding site. The ternary complex model was also applied successfully to the turkey erythrocyte receptor system. The model provides a general scheme for the activation by agonists of adenylate cyclase-coupled receptor systems and also of other systems where the effector might be different.     

Regulation of the solubilized bovine cerebral cortex muscarinic receptor by GTP and Na+

The muscarinic acetylcholine receptor was solubilized, in a sensitive form for GTP and Na+, from bovine cerebral cortex using a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The solubilized muscarinic receptor displayed characteristics as follows: (1) high affinity to nanomolar concentration of Z-[3H]quinuclidinyl benzilate; (2) muscarinic agonists and antagonists had similar inhibitory potencies as on the membrane-bound receptor; (3) without Na+, GTP did not significantly alter the binding affinity of muscarinic agonists and antagonists; (4) GTP in the presence of Na+, selectively decreased the affinity of muscarinic agonists, carbamylcholine and oxotremoline, but not the antagonist binding affinity; (5) Na+ in the absence or presence of GTP, reduced both muscarinic agonist and antagonist affinities.