Key Event Title
|Level of Biological Organization|
Key Event Components
|G-protein coupled acetylcholine receptor binding||muscarinic acetylcholine receptor||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|AChE Inhibition Leading to Neurodegeneration||KeyEvent|
Key Event Description
Muscarinic acetylcholine receptors (mAChRs) are G-protein-coupled receptors (GPCRs) with five different subtypes (M1, M2, M3, M4, and M5). GPCRs are transmembrane receptors that detect extracellular signals and activate internal pathways which modulate a variety of processes such as locomotion, learning and memory, thermoregulation and epileptic seizures (Gainetdinov and Caron, 1999). Subtypes M1, M3, and M5 are Gq- coupled receptors that activate phospholipase C enzyme resulting in two secondary messengers, inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Subtypes M2 and M4 are inhibitory and signal using the Gi pathway (Haga, 2013). Gi protein activation inhibits adenylyl cyclase, and reduces the conversion of ATP to cAMP (Jett and Lein, 2011).
In its resting state, the mAChR G-protein subunits (alpha, beta and gamma) are clustered together and the alpha subunit is bound to GDP. Once a ligand binds to an mAChR, the receptor undergoes a conformation change that allows the alpha subunit to exchange its bound GDP with GTP then the alpha subunit dissociates from the beta and gamma subunits. Once the alpha subunit is free of the beta and gamma subunits, it moves along the cell membrane to affect its target enzyme, which typically sends out secondary messenger signals (Kandel et al., 2013)
How It Is Measured or Detected
Most studies investigating the function of mAChRs involve blocking signaling from these receptors through use of selective antagonists like atropine or scopolamine, or the use of gene targeted knockout specimens (Bymaster et al. 2003; Faria et al. 2017). The distribution and density of mAChRs can be measured using radiolabeled agonists that bind to the mAChR binding site. The receptor activity can be measured by detecting secondary-messengers regulated by the G-protein.
- Use mAChR agonist [3H] quinuclidinyl benzilate (QNB) to label mAChRs (all subtypes; see Fonnum and Sterri (2011) and measure binding levels as described by Fitzgerald and Costa (1993) and Gazit et al. (1979)
- Determination of the relative levels of specific mAChR subtypes in tissues has been found through the use of subtype-specific antisera as described by Dörje et al. (1991)
- Kinetic measurements of DAG production and IP3 release can be obtained through fluorescent reporters as in Falkenburger et al. (2013) and Dickson et al. (2013).
- Changes in the activity and quantity of cAMP and the cAMP-dependent protein kinases can serve as an indicator of the activity of mAChRs bound to Gi-proteins (M2 and M4). cAMP content can be determined using a radioimmunoassay (RIA) kit (Heikkilä et al., 1991).
- Adenylyl cyclase activity can be determined through an assay as described by Salomon et al. (1974) and used by Raheja and Dip Gill (2007).
Domain of Applicability
mAChRs are found in most vertebrates, many of the studies cited are conducted using zebrafish and mice. Zebrafish are frequently used for high-throughput assays as they have well-conserved neurotransmitter structures, including acetylcholine transmitters (Garcia et al., 2016). This can provide valuable data regarding the activation of mAChRs in mammalian systems. Knockout mice also help to elucidate the functions of specific mAChR subtypes (Gainetdinov and Caron, 1999).
mAChRs signal neurons throughout all life stages (Miller and Yeh, 2016). They do not only affect individuals during developmental stages, but there have been some studies conducted specifically on the developmental effects of chemicals that affect acetylcholine signaling (Burke et al., 2017). Most of the whole animal experimental data are from younger specimens, but there have also been experiments on adult individuals (Fitzgerald and Costa, 1993).
mAChRs are found in both males and females, with similar functions (Burke et al., 2017).
Burke, R. D., S. W. Todd, E. Lumsden, R. J. Mullins, J. Mamczarz, W. P. Fawcett, R. P. Gullapalli, W. R. Randall, E. F. R. Pereira and E. X. Albuquerque (2017), "Developmental neurotoxicity of the organophosphorus insecticide chlorpyrifos: from clinical findings to preclinical models and potential mechanisms”, Journal of Neurochemistry 142: 162-177. DOI: 10.1111/jnc.14077.
Dickson, E. J., B. H. Falkenburger and B. Hille (2013), "Quantitative properties and receptor reserve of the IP3 and calcium branch of Gq-coupled receptor signaling”, Journal of General Physiology 141(5): 521-535. DOI: 10.1085/jgp.201210886.
Dörje, F., A. I. Levey and M. R. Brann (1991), "Immunological detection of muscarinic receptor subtype proteins (m1-m5) in rabbit peripheral tissues”, Molecular Pharmacology 40(4): 459-462.
Falkenburger, B. H., E. J. Dickson and B. Hille (2013), "Quantitative properties and receptor reserve of the DAG and PKC branch of Gq-coupled receptor signaling”, The Journal of General Physiology 141(5): 537-555. DOI: 10.1085/jgp.201210887.
Faria, M., Prats, E., Padrós, F., Soares, A. M., & Raldúa, D. (2017). Zebrafish is a predictive model for identifying compounds that protect against brain toxicity in severe acute organophosphorus intoxication. Archives of toxicology, 91(4), 1891-1901.
Fitzgerald, B. B. and L. G. Costa (1993), "Modulation of Muscarinic Receptors and Acetylcholinesterase Activity in Lymphocytes and in Brain Areas Following Repeated Organophosphate Exposure in Rats”, Fundamental and Applied Toxicology 20(2): 210-216. DOI: 10.1006/faat.1993.1028.
Fonnum, F. and S. H. Sterri (2011), “Tolerance Development to Toxicity of Cholinesterase Inhibitors”, in Toxicology of organophosphate and carbamate compounds, R. C. Gupta, Ed., Academic Press: 257-267.
Gainetdinov, R. R. and M. G. Caron (1999), "Delineating muscarinic receptor functions”, Proceedings of the National Academy of Sciences of the United States of America 96(22): 12222-12223. DOI: 10.1073/pnas.96.22.12222.
Garcia, G. R., P. D. Noyes and R. L. Tanguay (2016), "Advancements in zebrafish applications for 21st century toxicology”, Pharmacology and Therapeutics 161: 11-21. DOI: 10.1016/j.pharmthera.2016.03.009.
Gazit, H., I. Silman and Y. Dudai (1979), "Administration of an organophosphate causes a decrease in muscarinic receptor levels in rat brain”, Brain Research 174(2): 351-356. DOI: 10.1016/0006-8993(79)90861-8.
Haga, T. (2013), "Molecular properties of muscarinic acetylcholine receptors”, Proceedings of the Japan Academy Series B: Physical and Biological Sciences 89(6): 226-256. DOI: 10.2183/pjab.89.226.
Heikkilä, J., C. Jansson and K. E. O. Åkerman (1991), "Differential coupling of muscarinic receptors to Ca2+ mobilization and cyclic AMP in SH-SY5Y and IMR 32 neuroblastoma cells”, European Journal of Pharmacology: Molecular Pharmacology 208(1): 9-15. DOI: 10.1016/0922-4106(91)90045-J.
Jett, D. A. and P. J. Lein (2011), “Noncholinesterase Mechanisms of Central and Peripheral Neurotoxicity: Muscarinic Receptors and Other Targets”, in Toxicology of organophosphate and carbamate compounds, R. C. Gupta, Ed., Academic Press: 233-245.
Kandel, E., J. Schwartz, T. Jessell, S. Siegelbaum and A. J. Hudspeth (2013), “Modulation of Synaptic Transmission: Second Messengers”, in Principles of Neural Science, Fifth Edition, Blacklick, United States, McGraw-Hill Publishing: 236-259.
Miller, S. L. and H. H. Yeh (2016), “Neurotransmitters and Neurotransmission in the Developing and Adult Nervous System”, in Conn's Translational Neuroscience: 49-84.
Raheja, G. and K. Dip Gill (2007), "Altered cholinergic metabolism and muscarinic receptor linked second messenger pathways after chronic exposure to dichlorvos in rat brain”, Toxicology and Industrial Health 23(1): 25-37. DOI: 10.1177/0748233707072490.
Salomon, Y., C. Londos and M. Rodbell (1974), "A highly sensitive adenylate cyclase assay”, Anal Biochem 58(2): 541-548. DOI: 10.1016/0003-2697(74)90222-x.