Vibrational spectroscopy analysis of ligand efficacy in human M2 muscarinic acetylcholine receptor (M2R).
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ABSTRACT:
SUBMITTER: Katayama K
PROVIDER: S-EPMC8635417 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine (ACh) and mediates various cellular responses in the nervous system. We recently established Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy for ligand binding to M2R reconstituted in lipid membranes, paving the way to understand the mechanism in atomic detail. However, the obtained difference FTIR spectra upon ligand binding contained ligand, protein, lipid, and water signals, so a vibrational assignment was needed for a thorough understanding. In the present study, we compared difference FTIR spectra between unlabeled and 2-13C labeled ACh, and assigned the bands at 1741 and 1246 cm-1 as the C[double bond, length as m-dash]O and C-O stretches of ACh, respectively. The C[double bond, length as m-dash]O stretch of ACh in M2R is close to that in aqueous solution (1736 cm-1), and much lower in frequency than the free C[double bond, length as m-dash]O stretch (1778-1794 cm-1), indicating a strong hydrogen bond, which probably formed with N4046.52. We propose that a water molecule bridges ACh and N4046.52. The other ACh terminal is positively charged, and it interacts with negatively charged D1033.32. The present study revealed that D1033.32 is deprotonated (negatively charged) in both ACh-bound and free states, a suggested mechanism to stabilize the negative charge of D1033.32 in the free M2R.
| S-EPMC8696876 | biostudies-literature
Project description:The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2 and M5 mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.
| S-EPMC6926013 | biostudies-literature
Project description:The M3 muscarinic acetylcholine receptor (CHRM3) is predominantly expressed in the basal epidermal layer where it mediates the effects of the auto/paracrine cytotransmitter acetylcholine. Patients with the autoimmune blistering disease pemphigus develop autoantibodies to CHRM3 and show alterations in keratinocyte adhesion, proliferation and differentiation, suggesting that CHRM3 controls these cellular functions. Chrm3 mice display altered epidermal morphology resembling that seen in patients with pemphigus vulgaris. Here, we characterized the cellular and molecular mechanisms whereby CHRM3 controls epidermal structure and function. We used single cell (sc)RNA-seq to evaluate keratinocyte heterogeneity and identify differentially expressed genes in specific subpopulations of epidermal cells in Chrm3 KO neonatal mice.
2022-05-06 | GSE201885 | GEO
Project description:BACKGROUND: Controlling enzyme activity by ligand binding to a regulatory domain of choice may have many applications e.g. as biosensors and as tools in regulating cellular functions. However, until now only a small number of ligand-binding domains have been successfully linked to enzyme activity. G protein-coupled receptors (GPCR) are capable of recognizing an extraordinary structural variety of extracellular signals including inorganic and organic molecules. Ligand binding to GPCR results in conformational changes involving the transmembrane helices. Here, we assessed whether ligand-induced conformational changes within the GPCR helix bundle can be utilized to control the activity of an integrated enzyme. RESULTS: As a proof of principle, we inserted the luciferase amino acid sequence into the third intracellular loop of the M3 muscarinic acetylcholine receptor. This fusion protein retained both receptor and enzyme function. Receptor blockers slightly but significantly reduced enzyme activity. By successive deletion mutagenesis the enzyme activity was optimally coupled to ligand-induced conformational helix movements. CONCLUSION: Our results demonstrate that in engineered GPCR-enzyme chimeras, intracellular enzyme activity can be directly controlled by a GPCR serving as the extracellular ligand-binding domain.
| S-EPMC2689208 | biostudies-literature
Project description:TBPB and 77-LH-28-1 are selective agonists of the M1 muscarinic acetylcholine receptor (mAChR) that may gain their selectivity through a bitopic mechanism, interacting concomitantly with the orthosteric site and part of an allosteric site. The current study combined site-directed mutagenesis, analytical pharmacology,and molecular modeling to gain further insights into the structural basis underlying binding and signaling by these agonists. Mutations within the orthosteric binding site caused similar reductions in affinity and signaling efficacy for both selective and prototypical orthosteric ligands. In contrast, the mutation of residues within transmembrane helix (TM) 2 and the second extracellular loop (ECL2) discriminated between the different classes of ligand. In particular, ECL2 appears to be involved in the selective binding of bitopic ligands and in coordinating biased agonism between intracellular calcium mobilization and ERK1/2 phosphorylation. Molecular modeling of the interaction between TBPB and the M1 mAChR revealed a binding pose predicted to extend from the orthosteric site up toward a putative allosteric site bordered by TM2, TM3, and TM7, thus consistent with a bitopic mode of binding. Overall, these findings provide valuable structural and mechanistic insights into bitopic ligand actions and receptor activation and support a role for ECL2 in dictating the active states that can be adopted by a G protein-coupled receptor. This may enable greater selective ligand design and development for mAChRs and facilitate improved identification of bitopic ligands.
| S-EPMC4156061 | biostudies-literature
Project description:Nonselective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson's disease and dystonia. Despite their efficacy in these and other central nervous system disorders, antimuscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the antiparkinsonian and antidystonic efficacy observed with the use of nonselective antimuscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of nonselective antimuscarinic therapeutics and may decrease or eliminate the adverse effects associated with these drugs. However, this has not been directly tested due to lack of selective antagonists of M4. Here, we utilize genetic mAChR knockout animals in combination with nonselective mAChR antagonists to confirm that the M4 receptor activation is required for the locomotor-stimulating and antiparkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of the first-in-class selective M4 antagonists VU6013720, VU6021302, and VU6021625 and confirm that these optimized compounds have antiparkinsonian and antidystonic efficacy in pharmacological and genetic models of movement disorders.
| S-EPMC8369681 | biostudies-literature
Project description:Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor control deficits, which is associated with the loss of striatal dopaminergic neurons from the substantia nigra. In parallel to dopaminergic denervation, there is an increase of acetylcholine within the striatum, resulting in a striatal dopaminergic-cholinergic neurotransmission imbalance. Currently, available PD pharmacotherapy (e.g., prodopaminergic drugs) does not reinstate the altered dopaminergic-cholinergic balance. In addition, it can eventually elicit cholinergic-related adverse effects. Here, we investigated the interplay between dopaminergic and cholinergic systems by assessing the physical and functional interaction of dopamine D2 and muscarinic acetylcholine M1 receptors (D2R and M1R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D2R-M1R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET1 and NanoBiT®) assays, performed in transiently transfected HEK293T cells. Subsequently, a D2R-M1R co-distribution in the mouse striatum was observed through double-immunofluorescence staining and AlphaLISA® immunoassay. Finally, we evaluated the functional interplay between both receptors via behavioral studies, by implementing the classical acute reserpine pharmacological animal model of experimental parkinsonism. Reserpinized mice were administered with a D2R-selective agonist (sumanirole) and/or an M1R-selective antagonist (VU0255035), and alterations in PD-related behavioral tasks (i.e., locomotor activity) were evaluated. Importantly, VU0255035 (10 mg/kg) potentiated the antiparkinsonian-like effects (i.e., increased locomotor activity and decreased catalepsy) of an ineffective sumanirole dose (3 mg/kg). Altogether, our data suggest the existence of putative striatal D2R/M1R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects.
| S-EPMC7083216 | biostudies-literature
Project description:The gene encoding the human muscarinic receptor, type 1 (CHRM1), was genotyped from 245 samples of the Coriell Collection (Coriell Institute for Medical Research, Camden, NJ). Fifteen single nucleotide polymorphisms (SNPs) were discovered, 9 of which are located in the coding region of the receptor. Of these, 8 represent synonymous SNPs, indicating that CHRM1 is highly conserved in humans. Only a single allele was found to contain a nonsynonymous SNP, which encodes an amino acid change of Cys to Arg at position 417. This may have functional consequences because a C417S point mutation in rat M1 was previously shown to affect receptor binding and coupling. Furthermore, 0 of 4 SNPs within CHRM1 previously deduced from sequencing of the human genome were found in this study despite a prediction that a majority of such inferred SNPs are accurate. The consensus sequence of CHRM1 obtained in our study differs from the deposited reference sequence (AC NM_000738) in 2 adjacent nucleotides, leading to a V173M change, suggesting a sequencing error in the reference sequence. The extraordinary sequence conservation of the CHRM1 gene-coding region was unexpected as M1-knockout mice show only minimal functional impairments.
| S-EPMC2751220 | biostudies-literature
Project description:1. Muscarinic acetylcholine receptors in a plasma-membrane fraction derived from mouse neuroblastoma clone NIE-115 bind [3-3H]quinuclidinyl benzilate according to the Law of Mass Action (Kdissociation 40 pM, h0.96). 2. Antagonist and agonist binding to the receptor was studied by displacement of [3-3H]quinuclidinyl benzilate with non-radioactive ligands. The data show good agreement with similar data obtained on rat brain and ideal smooth muscle [Birdsall & Hulme (1976) J. Neurochem. 27, 7-16] indicating that the receptor is very similar in these three tissues.
| S-EPMC1185723 | biostudies-other
Project description:The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. 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 M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.
| S-EPMC3345277 | biostudies-literature