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The crystal structures of antagonist and agonist complexes of isolated β(2) and β(1) adrenoceptors have recently been supplemented by antagonist structures of M(2) and M(3) muscarinic acetylcholine receptors. Importantly, a structure of an agonist-ligated β(2) adrenoceptor complexed with its cognate G protein has provided the first view of a ternary complex representing the transition state in agonist-mediated G protein activation. This review interprets these G-protein-coupled receptor (GPCR) structures through the focus provided by extensive mutagenesis studies on muscarinic receptors, revealing an activation mechanism that is both modular and dynamic. Specific motifs, based around highly conserved residues, functionalise the seven-transmembrane architecture of these receptors. While exploiting conserved motifs, the ligand binding and signal transduction pathways work around and through water-containing cavities, an emerging feature of GPCR structures. These cavities may have undergone evolutionary selection to adapt GPCRs to particular signalling niches, and may provide targeting opportunities to enhance drug selectivity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We have used alanine-scanning mutagenesis followed by functional expression and molecular modeling to analyze the roles of the 14 residues, Asn422 to Cys435, C-terminal to transmembrane (TM) helix 7 of the M(1) muscarinic acetylcholine receptor. The results suggest that they form an eighth (H8) helix, associated with the cytoplasmic surface of the cell membrane in the active state of the receptor. We suggest that the amide side chain of Asn422 may act as a cap to the C terminus of TM7, stabilizing its junction with H8, whereas the side chain of Phe429 may restrict the relative movements of H8 and the C terminus of TM7 in the inactive ground state of the receptor. We have identified four residues, Phe425, Arg426, Thr428, and Leu432, which are important for G protein binding and signaling. These may form a docking site for the C-terminal helix of the G protein α subunit, and collaborate with G protein recognition residues elsewhere in the cytoplasmic domain of the receptor to form a coherent surface for G protein binding in the activated state of the receptor.

The focus of this review paper is factors affecting data interpretation in ligand binding assays under equilibrium conditions. Protocols for determining K(d) (the equilibrium dissociation constant) and K(dA) (the equilibrium inhibitor constant) for receptor ligands are discussed. The basic theory describing the interaction of a radiotracer and an unlabelled competitor ligand with a receptor is developed. Inappropriate experimental design may result in ligand depletion and non-attainment of equilibrium, distorting the calculation of K(d) and K(dA) . Strategies, both theoretical and practical, will be given to avoid and correct such errors, thus leading to the determination of reliable values for these constants. In determining K(dA) from competition binding studies, two additional concepts are discussed. First, the necessity to measure an adequate specific binding signal from the bound radiotracer ligand limits the range of affinity constants that can be measured: a particular set of assay conditions may lead to an upper limit on the apparent affinity of unlabelled ligands. Second, an extension of the basic assay methodology can indicate whether the interaction between the tracer and a test ligand is mediated by a competitive or an allosteric mechanism. Finally, the review ends with a discussion of two factors that are often overlooked: buffer composition and the temperature at which the assay is conducted, and the impact these can have on affinity measurements and the understanding of drug interactions., (© 2010 The Authors. Journal compilation © 2010 The British Pharmacological Society.)

Point mutations and molecular modeling have been used to study the activation of the M(1) muscarinic acetylcholine receptor (mAChR) by the functionally selective agonists 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42), and 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1), comparing them with N-desmethylclozapine (NDMC) and acetylcholine (ACh). Unlike NDMC and ACh, the activities of AC-42 and 77-LH-28-1 were undiminished by mutations of Tyr404 and Cys407 (transmembrane helix 7), although they were reduced by mutations of Tyr408. Signaling by AC-42, 77-LH-28-1, and NDMC was reduced by L102A and abolished by D105E, suggesting that all three may interact with transmembrane helix 3 at or near the binding site Asp105 to activate the M(1) mAChR. In striking contrast to NDMC and ACh, the affinities of AC-42 and 77-LH-28-1 were increased 100-fold by W101A, and their signaling activities were abolished by Y82A. Tyr82 and Leu102 contact the indole ring of Trp101 in a structural model of the M(1) mAChR. We suggest the hypothesis that the side chain of Trp101 undergoes conformational isomerization, opening a novel binding site for the aromatic side chain of the AC-42 analogs. This may allow the positively charged piperidine nitrogen of the ligands to access the neighboring Asp105 carboxylate to activate signaling following a vector within the binding site that is distinct from that of acetylcholine. NDMC does not seem to use this mechanism. Subtype-specific differences in the free energy of rotation of the side chain and indole ring of Trp101 might underlie the M(1) selectivity of the AC-42 analogs. Tryptophan conformational isomerization may open up new avenues in selective muscarinic receptor drug design.

Alanine substitution mutagenesis has been used to investigate residues that make up the roof and floor of the muscarinic binding pocket and regulate ligand access. We mutated the amino acids in the second extracellular loop of the M1 muscarinic acetylcholine receptor that are homologous to the cis-retinal contact residues in rhodopsin, the disulfide-bonded Cys178 and Cys98 that anchor the loop to transmembrane helix 3, the adjoining acidic residue Asp99, and the conserved aromatic residues Phe197 and Trp378 in the transmembrane domain. The effects on ligand binding, kinetics, and receptor function suggest that the second extracellular loop does not provide primary contacts for orthosteric ligands, including acetylcholine, but that it does contribute to microdomains that are important for the conformational changes that accompany receptor activation. Kinetic studies suggest that the disulfide bond between Cys98 and Cys178 may contribute to structures that regulate the access of positively charged ligands such as N-methyl scopolamine to the binding pocket. Asp99 may act as a gatekeeper residue to this channel. In contrast, the bulkier lipophilic ligand 3-quinuclidinyl benzilate may require breathing motions of the receptor to access the binding site. Trp378 is a key residue for receptor activation as well as binding, whereas Phe197 represents the floor of the N-methyl scopolamine binding pocket but does not interact with acetylcholine or 3-quinuclidinyl benzilate. Differences between the binding modes of N-methyl scopolamine, 3-quinuclidinyl benzilate, and acetylcholine have been modeled. Although the head groups of these ligands occupy overlapping volumes within the binding site, their side chains may follow significantly different directions.

Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M1 muscarinic acetylcholine receptor, and of the highly conserved Asn43 in TM1. The mutation of Asn43, Asn61, and Leu64 caused an enhanced ACh affinity phenotype. Interpreted using a rhodopsin-based homology model, these results suggest the presence of a network of specific contacts between this group of residues and Pro415 and Tyr418 in the highly conserved NPXXY motif in TM7 that exhibit a similar mutagenic phenotype. These contacts may be rearranged or broken when ACh binds. D71A, like N414A, was devoid of signaling activity. We suggest that formation of a direct hydrogen bond between the highly conserved side chains of Asp71 and Asn414 may be a critical feature stabilizing the activated state of the M1 receptor. Mutation of Leu67, Ala70, and Ile74 also reduced the signaling efficacy of the ACh-receptor complex. The side chains of these residues are modeled as an extended surface that may help to orient and insulate the proposed hydrogen bond between Asp71 and Asn414. Mutation of Leu72, Gly75, and Met79 in the outer half of TM2 primarily reduced the expression of functional receptor binding sites. These residues may mediate contacts with TM1 and TM7 that are preserved throughout the receptor activation cycle. Thermal inactivation measurements confirmed that a reduction in structural stability followed the mutation of Met79 as well as Asp71.

GPCRs (G-protein-coupled receptors) such as the M(1) muscarinic receptor have so far proved recalcitrant to direct structure determination. Nevertheless systematic mutagenesis, particularly alanine scanning, has advanced our understanding of their structure-function relationships. GPCRs exhibit multiple conformational states with different affinities for and abilities to activate their cognate G-proteins. Ligand binding alters these conformational equilibria, thus promoting or inhibiting signalling. Alanine-scanning mutagenesis probes the relative contributions of a particular amino acid side chain to the stability of the ground and activated states of the receptor and its complexes. These determine the phenotype of the mutant receptor. Classification of the phenotypes suggests functional roles for particular amino acid side chains, allowing us to group them accordingly. From a rhodopsin-based homology model of the M(1) mAChR, a coherent view emerges of how these clusters of residues function in ligand anchoring, transduction of binding energy, global structural stabilization and selective stabilization of the ground state or the activated state of the receptor. We can identify differences in ligand-binding modes, and suggest inter- and intra-molecular interactions that are weakened or broken, or formed or intensified during acetylcholine-induced activation. In due course, we may be able to extend these insights to activation by unconventional agonists.

Neutron diffraction augmented with hydrogen isotope substitution has been used to examine the water structure around the acetylcholine molecular ion in aqueous solution. It is shown that the nearest-neighbor water molecules in the region around the trimethylammonium headgroup are located either in a ring around the central nitrogen atom or between the carbon atoms, forming a sheath around the onium group. Moreover the water molecules in this cavity do not bond to the onium group but rather form hydrogen bonds with water molecules in the surrounding aqueous environment. Given that in the bound state the onium headgroup must be completely desolvated, the absence of bonding between the onium headgroup and the surrounding water solvent may be selectively favorable to acetylcholine-binding in the receptor site. Away from the headgroup, pronounced hydrogen-bonding of water to the carbonyl oxygen is observed, but not to the ether oxygen in the acetylcholine chain.

A homology model of the M(1) muscarinic acetylcholine receptor, based on the X-ray structure of bovine rhodopsin, has been used to interpret the results of scanning and point mutagenesis studies on the receptor's transmembrane (TM) domain. Potential intramolecular interactions that are important for the stability of the protein fold have been identified. The residues contributing to the binding site for the antagonist, N -methyl scopolamine, and the agonist, acetylcholine, have been mapped. The positively charged headgroups of these ligands probably bind in a charge-stabilized aromatic cage formed by amino acid side chains in TM helices TM3, TM6 and TM7, while residues in TM4 may participate as part of a peripheral docking site. Closure of the cage around the headgroup of acetylcholine may be part of the mechanism for transducing binding energy into receptor activation, probably by disrupting a set of Van der Waals interactions between residues lying beneath the binding site that help to constrain the receptor to the inactive state, in the absence of agonist. This may trigger the reorganization of a hydrogen-bonding network between highly conserved residues in the core of the receptor, whose integrity is crucial for achievement of the activated state.

Following the solution of the structure of bovine rhodopsin by X-ray crystallography, it has been possible to build an improved homology model of the M(1) muscarinic acetylcholine receptor. This has been used to interpret the outcome of an extensive series of scanning and point mutagenesis studies on the transmembrane domain of the receptor. Potential intramolecular interactions enhancing the stability of the protein fold have been identified. The residues contributing to the binding site for the antagonist, N-methylscopolamine, and the agonist, acetylcholine have been mapped. The positively charged headgroups of these ligands appear to bind in a charge-stabilized aromatic cage formed by amino acid side chains in transmembrane (TM) helices 3, 6, and 7, while residues in TM 4 may participate in a peripheral docking site. Closure of the cage around the headgroup of acetylcholine may help to transduce binding energy into receptor activation, possibly disrupting a set of Van der Waals interactions between a set of residues underlying the binding site which help to constrain the receptor to the inactive state, in the absence of agonist. This may trigger the reorganization of a hydrogen bonding network between highly conserved residues in the core of the receptor, whose integrity is crucial for activation.

The X-ray structure of the photoreceptor rhodopsin has provided the first atomic-resolution structure of a seven-transmembrane (7-TM) G-protein-coupled receptor. This has provided an improved template for interpreting the huge body of structure--activity, mutagenesis and affinity labelling data available for related 7-TM receptors, such as muscarinic acetylcholine receptors. Ligand contacts, and the intramolecular interactions that stabilize the ground state structure, can be identified with some degree of confidence. We now have a firm basis for attempts to predict the structure of the receptor--G-protein complex, and understand the mechanism by which the agonist--receptor complex activates the G protein.

Activation of the muscarinic acetylcholine receptors requires agonist binding followed by a conformational change, but the ligand binding and conformation-switching residues have not been completely identified. Systematic alanine-scanning mutagenesis has been used to assess residues 142-164 in transmembrane helix 4 and 402-421 in transmembrane helix 7 of the M(1) muscarinic acetylcholine receptor. Several inward-facing amino acid side chains in the exofacial parts of transmembrane helices 4 and 7 contribute to acetylcholine binding. Alanine substitution of the aromatic residues in this group reduced signaling efficacy, suggesting that they may form part of a charge-stabilized aromatic cage, which triggers rotation and movement of the transmembrane helices. The mutation of adjacent residues modulated receptor activation, either reducing signaling or causing constitutive activation. In the buried endofacial section of transmembrane helix 7, alanine substitution mutants of the conserved NSXXNPXXY motif displayed strongly reduced signaling efficacy, despite having increased or unchanged acetylcholine affinity. These residues may have dual functions, forming intramolecular contacts that stabilize the receptor in the inactive ground state, but that are broken, allowing them to form new intramolecular bonds in the activated state. This conformational rearrangement is critical to produce a G protein binding site and may represent a key mechanism of receptor activation.

The recently-determined structure of rhodopsin has provided a suitable basis for modeling the three-dimensional structure of the M1 muscarinic acetylcholine receptor. Using this as a framework for interpreting mutagenesis studies, we have been able to suggest most of the contacts which the receptor makes with acetylcholine and many of the intramolecular contacts which are important for the ground-state structure of the receptor. It is possible to outline a mechanism of G-protein interaction.

The exofacial part of transmembrane domain 5 (TMD 5) of the cationic amine-binding subclass of 7-transmembrane receptors is thought to be important in binding the side chain of the agonist. Residues Ile-188 through Ala-196 in TMD 5 of the M(1) muscarinic acetylcholine receptor (mAChR) have been studied by Cys- and Ala-scanning mutagenesis. The results are consistent with a helical conformation for this sequence. The positively charged sulfhydryl reagent N-trimethyl-2-aminoethyl methanethiosulfonate reacted selectively with Phe-190 --> Cys, Thr-192 --> Cys, and Ala-193 --> Cys, indicating that the face of TMD 5 accessible from the binding site crevice is consistent with a recent model by Baldwin and colleagues of the transmembrane domain of the 7-transmembrane receptors. In contrast, the acetylcholine derivative bromoacetylcholine reacted selectively with Thr-192 --> Cys, which forms the focus of a group of amino acids (Ile-188, Thr-189, Thr-192, Ala-196) whose mutation decreased the binding affinity of the transmitter ACh itself. The center of this patch of residues is offset to one side of the binding pocket, suggesting that a rotation of TMD 5, relative to that implied by the Baldwin model, may be necessary to optimize the anchoring of acetylcholine within the binding site of the M(1) mAChR. An induced rotation of TMD 5 could contribute to the formation of the activated state of the receptor.

Activation of the rhodopsin-like 7-transmembrane (7-TM) receptors requires switching interhelical constraints that stabilize the inactive state to a new set of contacts in the activated state, which binds the cognate G-protein. The free energy to drive this is provided by agonist binding, which has higher affinity to the active than to the inactive conformation. We have sought specific interhelical constraint contacts, using the M(1) muscarinic acetylcholine receptor as a model. Histidine substitutions of particular groups of amino acids, in transmembrane domains 3, 6, and 7, created high-affinity Zn(2+) binding sites, demonstrating the close proximity of their side chains in the inactive state. Alanine point substitutions have shown the effect of weakening the individual intramolecular contacts. In each case, the acetylcholine affinity was increased, implying promotion of the activated state. These amino acids are highly conserved throughout the 7-TM receptor superfamily. We propose that they form an important part of a network of conserved interhelical contacts that defines the off-state of a general transmembrane switch mechanism.

Muscarinic acetylcholine receptors (mAChRs) are known to be involved in learning and memory, but the molecular basis of their involvement is not well understood. The availability of new and specific biochemical tools has revealed a crucial role for the mitogen-activated protein kinase (MAPK) family in learning and memory. Here, we examine the link between mAChRs and MAPK in neurons. Using the MAPK kinase (MEK)-specific inhibitor PD98059, we first demonstrate a necessary role for active ERKI/II in long-term potentiation in vivo. Using phospho-specific antibodies that recognize the activated form of ERKI/II, we find that the level of ERKI/II activation in brain is regulated by mAChRs. Carbachol, a muscarinic agonist, induces prolonged activation of ERKI/II, without effect on the related kinase SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal protein kinase) in primary cortical cultures. ERKI/II activation is Src-dependent and partially phosphoinositide-3 kinase- and Ca(2+)-dependent but is PKC-independent. M1-M4 mAChR subtypes expressed in COS-7 cells can all induce ERKI/II activation using a signal transduction pathway similar to that operating in neurons. The nature of the signal transduction suggests that ERKI/II can serve as a convergence site for mAChR activation and other neurotransmitter receptors.

Transmembrane domain 6 of the muscarinic acetylcholine (ACh) receptors is important in ligand binding and in the conformational transitions of the receptor but the roles of individual residues are poorly understood. We have carried out a systematic alanine-scanning mutagenesis study on residues Tyr381 to Val387 within the binding domain of the M(1) muscarinic ACh receptor. The seven mutations were then analyzed to define the effects on receptor expression, agonist and antagonist binding, and signaling efficacy. Tyr381Ala produced a 40-fold reduction in ACh affinity and a 50-fold reduction in ACh-signaling efficacy. Leu386Ala had similar but smaller effects. Asn382Ala caused the largest inhibition of antagonist binding. The roles of the hydroxyl group and benzene ring of Tyr381 were probed further by comparative analysis of the Tyr381Phe and Tyr381Ala mutants using three series of ligands: ACh analogs, azanorbornane- and quinuclidine-based ligands, and atropine analogs. These data suggested that the hydroxyl group of Tyr381 is primarily involved in forming hydrogen bond interactions with the oxygen atoms present in the side chain of ACh. We propose that this interaction is established in the ground state and preserved in the activated state of the receptor. In contrast, the Tyr381 benzene ring may form a cation-pi interaction with the positively charged head group of ACh that contributes to the activated state of the receptor but not the ground state. However, the hydroxyl group and benzene ring of Tyr381 both participate in interactions with azanorbornane- and quinuclidine-based ligands and atropine analogs in the ground state as well as the activated state of the receptor.

The rhodopsin-like superfamily of 7-transmembrane receptors is the largest class of signalling molecules in the mammalian genome. Recently, a combination of mutagenesis, biophysical and modelling studies have suggested a credible model for the alpha-carbon backbone in the transmembrane region of the 7-transmembrane receptors, and have started to reveal the structural basis of the conformational switch from the inactive to the active state. A key feature may be the replacement of a network of radial constraints, centred on transmembrane helix three, which stabilise the inactive ground state of the receptor by a new set of axial interactions which help to stabilise the activated state. Transmembrane helix three may act as a rotary switch in the activation mechanism.

Alanine-scanning mutagenesis has been applied to residues 100-121 in transmembrane domain 3 of the M1 muscarinic acetylcholine receptor. This study complements a previous investigation of the triad Asp122-Arg123-Tyr124 (Lu, Z-L., Curtis, C. A., Jones, P. G., Pavia, J., and Hulme, E. C. (1997) Mol. Pharmacol. 51, 234-241). The results demonstrate the alpha-helical secondary structure of the domain and suggest its orientation with respect to the other transmembrane domains. The C-terminal part of the helix appears to be largely buried within the receptor structure. On its surface, there is a patch of three residues, Val113, Leu116, and Ser120, which may form intramolecular contacts that help to stabilize the inactive ground state of the receptor. Mutagenic disruption of these increased agonist affinity and signaling efficacy. In two cases (L116A and S120A), this led to constitutive activation of the receptor. Parallel to the helix axis and spanning the whole transmembrane region, a distinct strip of residues on one face of transmembrane domain 3 forms intermolecular (acetylcholine-receptor, receptor-G protein) or intrareceptor bonds that contribute to the activated state. The binding of acetylcholine may destabilize the first set of contacts while favoring the formation of the second.

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor has revealed a highly-differentiated alpha-helical structure. Lipid-facing residues are distinguished from a patch of residues which selectively stabilise the ground state of the receptor, and from a band of amino acids extending the full length of the helix, which contribute to the active agonist-receptor-G protein complex. The most important residues are strongly conserved in the GPCR superfamily.

An Asp-Arg-Tyr triad occurs in a majority of rhodopsin-like G protein-coupled receptors. The fully conserved Arg is critical for G protein activation, but the function of the flanking residues is not well understood. We expressed in COS-7 cells m1 muscarinic receptors that were mutated at Asp122 and Tyr124. Most mutations at either position strongly attenuated or prevented the expression of binding sites for the antagonist [3H]N-methylscopolamine. However, sites that were expressed displayed unaltered affinity for the antagonist. Receptor protein, visualized with a carboxyl-terminally directed antibody, was reduced but never completely abolished. The effects of these mutations were partially reversed by the deletion of 129 amino acids from the third intracellular loop of the receptor. In several cases, comparison of immunocytochemistry with binding measurements suggested the presence of substantial amounts of inactive, presumably misfolded, receptor protein. Some of the variants that bound [3H]N-methylscopolamine underwent small changes in their affinities for acetylcholine. All retained nearly normal abilities to mediate an acetylcholine-induced phosphoinositide response. We propose that Asp122 and Tyr124 make intramolecular contacts whose integrity is important for efficient receptor folding but that they do not participate directly in signaling. The role of these residues is completely distinct from that of Arg123, whose mutation abolishes signaling without diminishing receptor expression.

Mutation of the Asp in transmembrane domain three of the muscarinic receptors to Asn (M1) or Glu (M1 and M2) strongly reduced the high-affinity component of agonist binding, and the M1 phosphoinositide response. Formation of the acetylcholine-receptor binary complex was also strongly inhibited. In contrast, binary complex formation by other agonists, as well as the antagonist (-)-N-methylscopolamine, was less affected. Ionic bonding between the carboxylate oxyanion and the positively-charged headgroup probably anchors acetylcholine when it is bound in its active conformation, but alternative, non-productive, binding modes, promoted by non-polar forces, may contribute to binary complex formation by other ligands.

Arg123 in the rat muscarinic M1 receptor is part of the highly conserved triad Asp-Arg-Tyr found at the junction of transmembrane helix 3 with the second intracellular loop. Mutation of Arg123 to Lys, Ala, Leu, Glu and Gln had no effect on levels of receptor expression in COS-7 cells, or on affinities for the antagonist N-methylscopolamine. Acetylcholine stimulation of the Lys123 receptor evoked the same maximum phosphoinositide response as the wild type, although the potency was reduced six-fold, but mutation to other residues strongly disrupted receptor function. Mutation of Arg123 always decreased the ratio of the high affinity to the low affinity agonist binding constant, but the absolute effect on the latter varied from a 4-fold increase for the Lys123 to a small decrease for the Leu123 mutation. These results suggest that a positive charge at position 123 is central to the activation of G-proteins by the muscarinic M1 receptor.

The m3 muscarinic acetylcholine receptor from rat heterologously produced in insect cells after infection with a recombinant baculovirus has an apparent molecular mass of approximately 75 kDa. Polyclonal antibodies raised against a carboxy-terminal nonapeptide that is unique to the m3 subtype can detect the receptors produced in the insect cells by Western blot and can also immunoprecipitate solubilized receptor. Immunofluorescence microscopy as well as electron microscopy revealed that the receptor was located intracellularly, visualized as a ring around the nucleus of the infected insect cells. Solubilization of the receptor was accomplished with digitonin which was added in increments (over 10 min) to a final concentration of 0.8% (mass/vol). The solubilized receptor is unstable when the ligand-binding site is not protected by a ligand. Here the low-affinity ligand propylbenzilylcholine (approximately 10 nM) has demonstrable protective ability during solubilization, but the usefulness of this ligand is limited by a very slow off rate. From the behaviour of the solubilized receptor during DEAE-Sephacel chromatography and lectin-affinity chromatography it can be deduced that the receptor produced in insect cells is heterogeneously glycosylated in the producing insect cells.

Site-directed mutagenesis has been used to evaluate the roles of the key aspartate and arginine residues in transmembrane domain three of the muscarinic receptors. The results suggest that the formation of an ionic bond between the Asp carboxylate group and the onium headgroup is essential to anchor acetylcholine in its active, bound conformation in both binary agonist-receptor and ternary agonist-receptor-G-protein complexes, but that secondary, non-productive binding modes, promoted by non-polar forces, may contribute to binary complex formation by other ligands. The positive charge of the arginyl side-chain is central to the recognition, and subsequent activation of G-proteins by the agonist-M1 mAChR complex.

Acetylcholine mustard (AChM) is an analogue of acetylcholine (ACh) in which the onium headgroup is replaced by a chemically reactive aziridinium moiety. AChM aziridinium has agonist activity, but, having bound, reacts with and blocks the muscarinic receptor (mAChR) binding site. Purified mAChRs from rat forebrain have been specifically labeled with [3H]AChM. The linkage formed is cleaved by hydroxylamine, is found within cyanogen bromide (CNBr) peptides with molecular masses of approximately 2.4 and 3.9 kDa, and is close to a disulfide-bonded cysteine. Edman degradation reveals a site of label attachment 26 residues C-terminal to a CNBr cleavage site. As in the case of the alkylating antagonist analogue [3H]propylbenzilylcholine mustard, these findings indicate that a conserved aspartic acid residue in transmembrane helix 3 of the mAChRs, corresponding to Asp-105 (m1 sequence), is the site of label attachment.

1. Membranes from rat cerebral cortex, myocardium and extraorbital lacrimal gland were used as sources of M1, M2 and M3 muscarinic acetylcholine receptors respectively and the affinities of seven antagonists for the three subtypes were examined under different experimental conditions. 2. The affinities for the membrane-bound receptors were measured at different ionic strengths and temperatures and compared with those determined on the receptor solubilised in the neutral detergent digitonin or the zwitterionic detergent, CHAPSO. 3. The range of measured affinity constants of a given antagonist for a specific subtype varied from 2 (atropine at M1 receptors) to 1000 (AF-DX 116 at M2 receptors). 4. As a consequence of these changes in affinity, which were dependent on the drug, the subtype and the experimental conditions, both the structure-binding relationships of a given subtype can be markedly changed as well as the selectivity of a drug for the different subtypes. For example it is possible to change the relative affinities of AF-DX 116 and gallamine at membrane-bound M1 receptors from 50:1 to 1:60. 5. Experimental conditions for the observation of high selectivity of pirenzepine, AF-DX 116, gallamine and hexahydrosiladiphenidol for the three subtypes are given. 6. When the receptors are removed from their membrane environment by solubilisation in detergent, antagonist affinities are changed but the subtypes still retain different structure-binding relationships. 7. In general, AF-DX 116 and the allosteric antagonist, gallamine, behave differently from the other antagonists, suggesting that they bind in different ways to muscarinic receptors. Careful attention should therefore be paid to the experimental conditions in binding assays used to assess the affinities and selectivities of new muscarinic antagonists in order to avoid misleading results. 9. The ability to produce enhanced or attenuated affinities and selectivities of antagonists, resulting from the induction of different conformations of the receptor by a variety of physical, chemical or molecular biological perturbations may lead to a better understanding of the structural basis of drug receptor interactions.

Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [3H]propylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]N-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. m1 mAChRs were purified from CHO-K1 cells stably expressing the cloned receptor sequence and labeled with [3H]PrBCM. The labeled receptors were cleaved with the lysine-specific protease Lys-C and, after fractionation of the products, subcleaved with cyanogen bromide. Two major CNBr cleavage products were found with a molecular mass of approximately 3.9 and approximately 2.4 kDa, labeled either by [3H]PrBCM or [3H]NEM. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned m1 mAChR. Edman degradation was applied to the CNBr peptides. The results were compatible with the attachment of the [3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, m1 sequence) and of [3H]NEM to a conserved cysteine residue (corresponding to Cys-98, m1 sequence). These results support the hypothesis that the cysteine residue participates in a disulfide bond on the extracellular surface of the mAChRs and related G-protein-coupled receptors, while the aspartic acid residue is involved in binding the positively charged headgroup of muscarinic antagonists.

The muscarinic acetylcholine receptors (mAChRs) are members of the class of G-protein coupled receptors. 5 mAChR genes have been cloned and sequenced. The receptor subtypes have a characteristic tissue distribution which correlates with their pharmacological and functional properties. The mAChRs appear to be monomeric in their active state. They conform to a common generic structure. The receptor polypeptide chain is believed to traverse the cell-surface membrane 7 times, forming a helical cluster which contains the ligand binding site. Amino-acid residues important in ligand binding have been identified both by affinity-labelling and peptide sequencing, and by site-directed mutagenesis and expression studies. The cytoplasmic domain of the receptor is composed of 3 cytoplasmic loops, and the C-terminal tail of the receptor polypeptide chain. The cytoplasmic domain binds, recognises and activates specific hetero-trimeric GTP-binding proteins (G-proteins) in an agonist-dependent manner. The activated, GTP-liganded G-proteins then mediate a wide variety of events, which include, in the case of mAChRs, the inhibition of adenylyl cyclase, the breakdown of polyphosphoinositides, the opening and closing of potassium channels, and the production of arachidonic acid. Mutational analysis shows that the N-terminal portion of the third intracellular loop of the mAChR sequences is important in conveying specificity for G-protein recognition. The receptor sequences contain a number of additional features which are functionally important. These include potential substrate sites for receptor-specific kinases, and a cysteine residue which is likely to be palmitoylated.

Three muscarinic acetylcholine receptor subtypes may be defined on the basis of functional and binding studies using selective antagonists. The subtypes may be solubilized in a stable form in digitonin. In solution, the subclasses still exhibit different structure-binding relationships but these have been perturbed by solubilization. The binding of the selective antagonist, pirenzepine, to the purified cortical receptor is complex and similar to that found in membranes. The muscarinic receptor subclasses thus appear to be different molecular entities. Possible explanations for the molecular heterogeneity are discussed. It has also been possible to solubilize receptor-GTP binding protein complexes which have higher sedimentation coefficients (13.4 S) than the apparently monomeric receptor (11.6 S).

Levels of muscarinic receptors were measured in the nucleus accumbens of rat following 0.8 microgram 6-hydroxydopamine or vehicle injections (0.2 microliter into the ventral tegmental area to investigate whether the dopaminergic terminals destroyed by this procedure bear muscarinic receptors. Dopamine levels in the nucleus accumbens ipsilateral to the injection of 6-hydroxydopamine were substantially reduced by 83% as compared to the unlesioned side after 7 days. Significant decreases in the specific binding of [3H]N-methylscopolamine of 9 and 15% were also seen in the nucleus accumbens ipsilateral to the lesion after 7 and 14 days respectively. The class of muscarinic receptor depleted by the lesion was further investigated using [3H]oxotremorine-M to label the 'super high' affinity binding sites. The percentage occupancy of total muscarinic receptors by [3H]oxotremorine-M was significantly decreased by lesion e.g. 23% after 7 days indicting a selective loss of 'super high' affinity binding sites. The lesion caused no change in the affinity constant for the muscarinic antagonist, propylbenzilylcholine. Studies of the binding of the agonist carbachol and oxotremorine-M by competition with [3H] propylbenzilylcholine showed little change in the concentrations or affinity constants of the 'high' and 'low' affinity binding sites with the 6-hydroxydopamine lesion.

Muscarinic receptors from rat forebrain have been solubilized by Lubrol PX, lysophosphatidylcholine (LPC), digitonin and cholate/1 M sodium chloride. The overall level of solubilization was characterized using receptors prelabelled with an irreversible antagonist. The recovery of nondenatured soluble binding activity was estimated using reversible tritiated antagonists. All these detergents solubilized 60-85% of the total binding sites. In Lubrol PX most of the receptors were recovered in a denatured form. In the other detergents 30-90% of the solubilized receptors were stable and capable of binding reversible [3H]-antagonists with high affinity. The hydrodynamic properties of the soluble receptors have been examined by gel filtration and sucrose gradient centrifugation in H2O and D2O. The soluble receptors in Lubrol PX, lysophosphatidylcholine and cholate were, in general, heterogeneous as regards their molecular size. Estimates of the molecular weight after correction for bound detergent, varied from 82,000 to 134,000. Conditions were identified under which the receptor was largely monodisperse, and the estimates of molecular weight agreed with values (ca. 83,000) from sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis. The amount of bound detergent could not be calculated for the digitonin-muscarinic receptor complex which had an estimated overall median molecular weight of about 290,000. It is concluded that a subpopulation of muscarinic receptors from the rat forebrain is capable of existing in a monomeric soluble form and binding ligands. There is also evidence that complexes with other proteins can exist, but their specificity and functional relevance are not known.

McN-A-343 behaves as a competitive agonist in binding to muscarinic receptors in the cerebral cortex. In its interaction with myocardial muscarinic receptors it is not competitive but it retains features of agonist binding.

1 Muscarinic receptors from the brain of rat, guinea-pig and frog have been labelled with tritiated propylbenzilylcholine mustard ([3H]-PrBCM). 2 After solubilisation and sodium dodecyl sulphate (SDS)-polycarylamide gel electrophoresis a single radiolabelled peak was seen which was completely suppressed when labelling was carried out in the presence of 10(-6) M atropine. The results suggest the presence of a single polypeptide chain with a molecular weight of similar to or approximately 80,000. 3 In the case of intestinal smooth muscle multiple labelled peaks were found. Precautions against proteolysis led to the identification of a major peak with a molecular weight in the region of 80,000.

Structural and binding studies on the purified rat forebrain muscarinic acetylcholine receptor (mAChR) are consistent with the idea that the receptor is structurally analogous to rhodopsin. We propose that it consists of a short, glycosylated N-terminus, followed by seven transmembrane helices, joined by extra-membranous loops. The transmembrane helices make up a substantial proportion of the molecule. Two peptides, one of which is glycosylated, and therefore close to the N-terminal part of the molecule are labelled on acidic residues by the irreversible antagonist, [3H]-PrBCM, and may thus form part of the ligand binding site. The conformational flexibility of the mAChR, and hence its ability to bind rigid selective ligands such as pirenzepine is strongly affected by the oxidation state of key cysteine residues. The molecule is proposed to terminate in a cytoplasmic C-terminal tail, which participates, together with the cytoplasmic loops, in the recognition of GTP-binding proteins. The C-terminus is susceptible to proteolytic attack, and shows evidence of sequence homology to the C-terminus of the beta-adrenergic receptor.

Stable, soluble preparations of rat brain muscarinic receptors can be prepared by extracting membranes with digitonin, or with combinations of sodium cholate and sodium chloride. The stability of the cholate/NaCl extract is enhanced by the addition of egg phosphatidylcholine, which, at the same time, suppresses the considerable dispersity apparent in the hydrodynamic behaviour of the solubilised receptor. The Stokes radius of the brain muscarinic receptor in cholate/NaCl/lecithin extracts is 6.7 nm, with very similar values in other detergents, including digitonin and sodium dodecyl sulphate. Its sedimentation coefficient is 3.78s, and its molecular weight approximately 110,000 after correction for detergent binding. The isoelectric point of the digitonin - solubilised receptor is approximately 4.5.

Pirenzepine has been shown to evoke a variety of antimuscarinic effects in animals. However, in contrast to classical antimuscarinic drugs, pirenzepine inhibits gastric acid secretion at much lower doses than those required to block smooth muscle action and to cause tachycardia. To investigate this novel behaviour we have compared the binding properties of pirenzepine to muscarinic receptors with those of a classical muscarinic antagonist, N-methyl-scopolamine. Binding has been determined in subcellular preparations of the sublingual, submandibular, parotid and lacrimal glands, smooth muscle of ileum and stomach, heart and several brain regions of the rat. Whereas N-methyl-scopolamine binds to an apparently uniform population of muscarinic receptors with little regional variation in affinity constant, the binding of pirenzepine in peripheral tissues is characterised by its low affinity in smooth muscle and the heart and considerably higher affinities to muscarinic receptors in exocrine glands and fundus mucosa, in agreement with the pharmacological profile of the drug. Furthermore, within the central nervous system and some exocrine glands, pirenzepine distinguishes between subclasses of muscarinic receptors which are not detected by classical antagonists. This evidence suggests that the unusual receptor binding properties of pirenzepine may make its selective antimuscarinic action possible.

1 There are no selective effects of Na+, K+, Ca2+, Mg2+ or Cl- on the binding of antagonists or agonists to muscarinic receptors in rat brain. A decrease in affinity related to ionic strength is found for all these ions. 2 Larger effects were produced by T1+, La3+, and some transition metal ions.

The ontogeny of muscarinic acetylcholine receptors in the rat brain has been examined using the radioligands, [3H]N-methylscopolamine, [3H]propylbenzilylcholine and [3H]oxotremorine-M. In the 3 regions of the brain selected for study, the cerebral cortex, the diencephalon and the medulla-pons, the receptors develop at different rates. The most rapid development takes place in the medulla with considerably slower maturation in the diencephalon and cerebral cortex. In the cortex, the agonist binding properties of the muscarinic receptors vary during development. There appears to be a 6--7 day lag in the appearance of high affinity sites following formation of low affinity sites.