Modulation of the structure-binding relationships of antagonists for muscarinic acetylcholine receptor subtypes.

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. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation.

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.

Binding and hydrodynamic properties of muscarinic receptor subtypes solubilized in 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate.

The muscarinic receptor from the cerebral cortex, heart, and lacrimal gland can be solubilized in the zwitterionic detergent 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate (CHAPSO) with retention of high affinity [3H]N-methyls-copolamine binding. However, in this detergent there are significant differences in the binding properties of the receptors, compared with those observed in membranes and digitonin solution. Some agents retain a degree of selectivity. In the heart and cortex, agonists can bind with high affinity to a receptor-GTP-binding protein complex. A second, lower affinity, agonist binding state is also present, which resembles a class of sites seen in membranes but not in digitonin solution. The high affinity agonist binding state has been resolved from the lower affinity state on sucrose density gradient centrifugation. Hydrodynamic analysis suggests that the high affinity state is approximately 110,000 Da larger than the lower affinity state. The binding properties of the receptor in CHAPSO can be altered to those seen in digitonin by exchanging detergents after CHAPSO solubilization.

Propylbenzilylcholine mustard labels an acidic residue in transmembrane helix 3 of the muscarinic receptor.

Muscarinic acetylcholine receptors were purified from rat forebrain and labeled with [3H]N-(2-chloroethyl)N-(2',3'-[3H2]propyl)-2-aminoethylbenzilate. Cleavage of the labeled muscarinic acetylcholine receptors with a lysine-specific protease yielded labeled, glycosylated peptides about 130 and 200 residues in length, which came from different receptor sequences. The probable cleavage sites are in the second intracellular loop and in the second extracellular or third intracellular loop. The N-terminal 130 residues are disulfide-bonded to another part of the receptor structure, supporting the presence of a link between the second and third extracellular loops. The [3H]propylbenzilylcholine mustard-receptor link is cleaved by nucleophiles, acids, and bases under denaturing conditions, suggesting modification of an acidic residue. Cyanogen bromide cleavage points to transmembrane helix 3 as the site of label attachment.

Muscarinic receptor subtypes and the selectivity of agonists and antagonists.

Receptors may be grouped into superfamilies, according to their mechanism. Muscarinic receptors belong to one such superfamily, that of the G-protein coupled receptors. Subtypes of muscarinic receptors may be defined according to their primary amino acid sequence, their mechanism and their pharmacology. However, there is as yet no consistent scheme to explain the pharmacology and function of each molecular subtype. The pharmacological tools which distinguish between muscarinic receptor subtypes are described and the emerging evidence for the location of the ligand binding site is discussed.

Muscarinic acetylcholine receptors: structure, function subtypes and therapeutic perspectives.

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.

Soluble and membrane-bound muscarinic acetylcholine receptors.

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).