Activation and allosteric modulation of a muscarinic acetylcholine receptor (original) (raw)
Related papers
Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist
Nature, 2012
The parasympathetic limb of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G protein coupled receptors (GPCRs) that mediate the response to acetylcholine released from parasympathetic nerves. 1-5 Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M 2 muscarinic acetylcholine receptor (M 2 receptor) is essential for the physiologic control of cardiovascular function through activation of G proteincoupled inwardly-rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we
Muscarinic acetylcholine receptors: novel opportunities for drug development
Nature Reviews Drug Discovery, 2014
The muscarinic acetylcholine receptors (mAChRs) comprise a family of five related G protein-coupled receptors (GPCRs) belonging to the α-branch of class A GPCRs 1 . The mAChR family consists of five distinct subtypes, denoted M 1 to M 5 (and encoded by the genes CHRM1 to CHRM5). Three of these receptor subtypes (M 1 , M 3 and M 5 ) have been shown to couple to G proteins of the G q/11 family, whereas the remaining two subtypes (M 2 and M 4 ) preferentially signal through the G i/o family of G proteins 2 . The mAChRs have a central role in human physiology, regulating heart rate, smooth muscle contraction, glandular secretion and many fundamental functions of the central nervous system (CNS) 3 .
Molecular Pharmacology, 1998
Muscarinic acetylcholine receptors bind allosteric modulators at a site apart from the orthosteric site used by conventional ligands. We tested in cardiac tissue whether modulator binding to ligand-occupied muscarinic M 2 receptors is a preferential event that can be detected using a radioactive allosteric agent. The newly synthesized dimethyl-W84 (N,NЈ-bis[3-(1,3-dihydro-1,3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,NЈ,NЈ-tetramethyl-1,6-hexanediaminium diiodide) has a particular high potency at M 2 receptors occupied by the conventional antagonist N-methylscopolamine (NMS); dissociation of [ 3 H]NMS is halfmaximally retarded at an EC 50,diss value of 3 nM. Using obidoxime as an "allosteric antagonist," evidence was found that dimethyl-W84 interacts with the postulated common allosteric site. Binding of [ 3 H]dimethyl-W84 (0.3 nM; specific activity, 168 Ci/mmol) was measured in porcine heart homogenates (4 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , pH 7.4, 23°) in the presence of 1 M NMS. Homologous competition experiments revealed two components of saturable radioligand binding: one with a high affinity (K D ϭ 2 nM) and small capacity (Ϸ30% of total saturable binding) and the other with a 20,000-fold lower affinity. The B max value of the high affinity sites (68 fmol/mg protein) matched muscarinic receptor density as determined by [ 3 H]NMS (79 fmol/mg). Prototype allosteric agents, alcuronium, W84 (the parent compound of the radioligand), and gallamine, displaced high affinity [ 3 H]dimethyl-W84 binding concentration-dependently (pK i values ϭ 8.62, 7.83, and 6.72, respectively). The binding affinities of the modulators were in excellent correlation with their potencies to allosterically stabilize NMS/ receptor complexes (EC 50,diss ϭ 8.40, 7.72, and 6.74, respectively). We conclude that high affinity binding of [ 3 H]dimethyl-W84 reflects occupation of the common allosteric site of M 2 receptors. This work was supported by the Deutsche Forschungsgemeinschaft und the Fonds der Chemischen Industrie, Germany. H.M.B.C. acknowledges a grant given by the Katholischer Akademischer Austauschdienst.
Structure and activation of muscarinic acetylcholine receptors
Biochemical Society Transactions, 2003
A homology model of the M1 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 h...
Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor
Nature Chemical Biology, 2018
Human muscarinic receptor, M 2 is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype selective ligands against one of the five muscarinic receptors. We report high resolution structures of a thermostabilized mutant M 2 receptor bound to a subtype selective antagonist AF-DX 384 and a non-selective antagonist NMS. The thermostabilizing mutation S110R in M 2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M 2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, that is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular Dynamics simulations reveal that tightening of the ligand-residue contacts in M 2 receptor compared to M 3 receptor leads to subtype selectivity of AF-DX 384.
Muscarinic receptor family interacting proteins: role in receptor function
Journal of neuroscience methods, 2011
a b s t r a c t G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.
Journal of Biological Chemistry, 2004
Antibodies directed against the second extracellular loop of G protein-coupled receptors are known to have functional activities. From a partial agonist monoclonal antibody directed against the M2 muscarinic receptor, we constructed and produced a single chain variable fragment with high affinity for its target epitope. The fragment is able to recognize its receptor on Chinese hamster ovary cells transfected with the M2 muscarinic acetylcholine receptor to block the effect of carbachol on this receptor and to exert an inverse agonist activity on the basal activity of the receptor. The antibody fragment is also able to increase the basal rhythm of cultured neonatal rat cardiomyocytes and to inhibit in a non-competitive manner the negative chronotropic effect of carbachol. This antibody fragment is able to exert its inverse agonist activity in vivo on mouse heart activity. The immunological strategy presented here could be useful to develop specific allosteric inverse agonist reagents for G protein-coupled receptors.
Molecular pharmacology, 2011
Despite the discovery of a diverse range of novel agonists and allosteric modulators of the M(4) muscarinic acetylcholine (ACh) receptor (mAChR), little is known about how such ligands activate the receptor. We used site-directed mutagenesis of conserved residues in transmembrane 3 (TMIII), a key region involved in G protein-coupled receptor activation, to probe the binding and function of prototypical orthosteric mAChR agonists, allosteric modulators, and "atypical" agonists. We found that most mutations did not affect the binding of the allosteric modulators, with the exception of W108(3.28)A and L109(3.29)A (which may contribute directly to the interface between allosteric and orthosteric sites) and mutation D112(3.32)N (which may cause a global disruption of a hydrogen bond network). Although numerous mutations affected signaling, we did not identify amino acids that were important for the functional activity of any one class of agonist (orthosteric, allosteric, or aty...
Antagonist binding profiles of five cloned human muscarinic receptor subtypes
The Journal of pharmacology and experimental therapeutics, 1991
A variety of muscarinic antagonists are currently used as tools to pharmacologically subclassify muscarinic receptors into M1, M2 and M3 subtypes. In the present study, we have determined the affinity profiles of several of these antagonists at five cloned human muscarinic receptors (m1-m5) stably expressed in Chinese hamster ovary cells (CHO-K1). At all five receptors, the (R)-enantiomers of trihexyphenidyl and hexbutinol displayed considerably higher affinities (up to 525-fold) than their corresponding (S)-isomers. The stereoselectivity ratios [inhibition constant(S)/inhibition constant(R)] for both pairs of enantiomers were lowest at m2 receptors, suggesting that less stringent configurational demands are made by this receptor subtype. The "M1-selective" antagonist (R)-trihexyphenidyl displayed high affinities for m1 and m4 receptors. The "M2-selective" antagonists himbacine, (+-)-5,11-dihydro-11- ([(2-[(dipropylamino)methyl]-1- piperidinyl)ethyl)amino]carbony...
Partial functional reconstitution of the cardiac muscarinic cholinergic receptor
Journal of Biological Chemistry
~ Digitonin-solubilized cardiac muscarinic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack high-affinity muscarinic receptors. The number of receptors reconstituted was proportional to the quantity of soluble receptors added to the reconstitution system.