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Key Event Title
Binding at picrotoxin site, iGABAR chloride channel
|Level of Biological Organization|
Key Event Components
|GABA receptor binding||gamma-aminobutyric acid receptor subunit alpha-1||increased|
|GABA receptor binding||gamma-aminobutyric acid receptor subunit alpha-5||increased|
|GABA receptor binding||gamma-aminobutyric acid receptor subunit alpha-6||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Blocking iGABA receptor ion channel leading to seizures||MolecularInitiatingEvent||Ping Gong (send email)||Open for citation & comment||WPHA/WNT Endorsed|
Key Event Description
Figure 1. Structure of ionotropic GABA receptors based on the consensus in multiple literature reviews (Source: Gong et al. 2015). Shown is a common subtype α1β2γ2 of GABAA receptors found in the mammalian CNS. (A) Five subunits from three subunit subfamilies assemble to form a heteropentameric chloride permeable channel. (B) Stoichiometry and subunit arrangement of the GABAA receptor. Also shown are the binding sites for GABA and BZ. (C) Receptor subunits consist of four hydrophobic transmembrane domains (TM1-4), where TM2 is believed to line the pore of the channel. The large extracellular N-terminus is the site for ligand binding as well as the site of action of various drugs (e.g., picrotoxin and dieldrin). Each receptor subunit also contains a large intracellular domain between TM3 and TM4, which is the site for various protein–protein interactions as well as the site for post-translational modifications that modulate receptor activity. BZ: Benzodiazepines; CNS: Central nervous system; TM: Transmembrane.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- As shown in Figure 1, binding of GABA opens up the chloride channel in the iGABAR complex, leading to neuronal inhibition (Olsen et al. 2004). Non-competitive channel blockers (e.g., fipronil, lindane, picrotoxin, penicillin and alpha-endosulfan) indirectly modulate the iGABAR activity (i.e., alter the response of the receptor to agonist) by noncompetitively binding at or near the central pore of the receptor complex (e.g., the picrotoxin site), an allosteric site distinct from that of the orthosteric agonist binding site, and inducing a conformational change within the receptor (Ernst et al. 2005; Johnston 2005).
Mounting evidence indicates that iGABAR chloride channel blockers bind to overlapping but not identical sites (Kalueff 2007). Based on the mapping of convulsants' binding domains to the iGABAR chloride ionophore (Olsen 2006), Kalueff (2007) proposed a "big picrotoxin binding pocket" model for the ligand-binding area of ionophore. For instance, bicyclophosphates, butyrolactones, pentylenetetrazole and penicillin act on the N-terminus of TM2 (‘‘main’’ picrotoxin binding site), whereas picrotoxin may act on the "main" as well as a hypothetical second ‘‘allosteric’’ picrotoxin site located on the C-terminus of TM2 (Kalueff 2007; Olsen 2006). In spite of minor differences, these blockers share similarity in channel blockage characteristics (e.g., voltage-dependency, binding to closed channel, reversible, and non-competitive) and effects of channel state (open/closed frequency and duration) (Kalueff 2007). Nevertheless, the 3D structure of iGABAR ionophore is unavailable. More homology modeling work (similar to the electron microscopic structure of nicotinic receptor channel (Unwin 2005)) may allow us to gain in-depth understanding of the picrotoxin site's accessibility for and interactions with channel blockers (i.e., convulsants), clarify the biological functions of the binding sites, and verify the impact of mutations in TM2 on ligand binding activity (Kalueff 2007).
How It Is Measured or Detected
Binding to a specific site on iGABAR can be determined using a variety of methods including mutagenesis, pore structure studies, ligand binding, and molecular modeling (more details on methods can be found in Chen et al. 2006). One should choose a method in accordance with specific goal and also on the basis of available laboratory facilities. For example, Atack et al. (2007) chose the radioligand [35S]t-butylbicyclophosphorothionate (TBPS) binding assay to determine the binding properties (i.e., inhibition by TBPS, picrotoxin, loreclezole and pentobarbital and modulation by GABA) at the convulsant binding site.
Domain of Applicability
Theoretically, this MIE is applicable to any organisms that possess ionotropic GABA receptors (iGABARs) in their central and/or peripheral nervous systems. Many reviews (e.g., Hoisie et al. 1997; Buckingham et al. 2005; Michels and Moss 2007; Olsen and Sieghart 2009) have summarized evidence of ubiquitous existence of iGABARs (GABAA-R in vertebrates including the humans) in species spanning from invertebrates to human. For instance, an ionotropic GABA receptor gene (GABA-receptor subunit-encoding Rdl gene) was isolated from a naturally occurring dieldrin-resistant strain of D. melanogaster (Ffrench-Constant et al., 1991,1993; Ffrench-Constant and Rocheleau, 1993). Nineteen GABAA receptor genes have been identified in the human genome (Simon et al. 2004). Direct evidence is mostly derived from in silico molecular modeling that docks ligands to the binding pockets of iGABARs in human (Carpenter et al. 2013; Chen et al. 2006; Sander et al. 2011), fruitfly and zebrafish (Zheng et al. 2014).
It should be noted that this MIE is limited to adult brains because of the age- or developmental stage-dependent nature of iGABAR where GABA acts as an excitatory (instead of inhibitory) neurotransmitter due to a higher intracellular chloride concentration in immature or developing neurons (Taketo and Yoshioka 2000; Galanopoulou 2008; Ben-Ari 2006).
This MIE is not applicable to GABAB receptors because, as metabotropic G-protein-coupled receptors, they mediate slow and sustained inhibitory responses and are involved in absence epilepsy (Han et al. 2012).
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Chemicals non-competitively bind at or near the central pore of the receptor complex (e.g., the picrotoxin site) and directly block chloride conductance through the ion channel (Kalueff 2007). It has been postulated that they fit a single "big picrotoxin binding pocket" in the chloride channel lumen lined by five TM2 segments (Kalueff 2007; Olsen 2006). This hypothesis was examined with the β3 homopentamer by mutagenesis, pore structure studies, ligand binding, and molecular modeling (Chen et al. 2006). Results suggest that they fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. More computational evidence can be found in Sander et al. (2011), Carpenter et al. (2013) and Zheng et al. (2014).
Atack JR, Ohashi Y, McKernan RM. 2007. Characterization of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain. Br J Pharmacol. 150(8):1066-74.
Buckingham SD, Biggin PC, Sattelle BM, Brown LA, Sattelle DB. 2005. Insect GABA receptors: splicing, editing, and targeting by antiparasitics and insecticides. Mol Pharmacol 68(4):942-951.
Ben-Ari Y. 2006. Seizures beget seizures: the quest for GABA as a key player. Crit Rev Neurobiol 18(1-2): 135-44.
Carpenter TS, Lau EY, Lightstone FC. 2013. Identification of a possible secondary picrotoxin-binding site on the GABAA receptor. Chem Res Toxicol. 26(10):1444-54.
Chen L, Durkin KA, Casida JE. 2006. Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same site. Proc Natl Acad Sci USA 103(13):5185-5190.
Ernst M, Bruckner S, Boresch S, Sieghart W. 2005. Comparative models of GABAA receptor extracellular and transmembrane domains: important insights in pharmacology and function. Mol Pharmacol 68(5):1291-1300.
Ffrench-Constant RH, Mortlock DP, Shaffer CD, MacIntyre RJ, Roush RT. 1991. Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate gamma-aminobutyric acid subtype A receptor locus. Proc Natl Acad Sci USA 88:7209–7213.
Ffrench-Constant RH and Rocheleau TA. 1993 Drosophila gamma-aminobutyric acid receptor gene Rdl shows extensive alternative splicing. J Neurochem 60:2323–2326.
Ffrench-Constant RH, Steichen JC, Rocheleau TA, Aronstein K, and Roush RT. 1993. A single-amino acid substitution in a gamma-aminobutyric acid subtype A receptor locus is associated with cyclodiene insecticide resistance in Drosophila populations. Proc Natl Acad Sci USA 90:1957–1961.
Galanopoulou AS. 2008. GABAA Receptors in Normal Development and Seizures: Friends or Foes? Curr Neuropharmacol. 6(1):1-20.
Gong P, Hong H, Perkins EJ. 2015. Ionotropic GABA receptor antagonism-induced adverse outcome pathways for potential neurotoxicity biomarkers. Biomarkers in Medicine 9(11):1225-39.
Han HA, Cortez MA, Snead OC III. 2012. GABAB Receptor and Absence Epilepsy. In: Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th edition. Bethesda (MD): National Center for Biotechnology Information (US); 2012. Available from: https://www.ncbi.nlm.nih.gov/books/NBK98192/.
Hosie AM, Aronstein K, Sattelle DB, Ffrench-Constant RH. 1997. Molecular biology of insect neuronal GABA receptors. Trends Neurosci 20(12):578-583.
Johnston GA. 2005. GABA(A) receptor channel pharmacology. Curr Pharm Des 11(15):1867-1885.
Kalueff AV. 2007. Mapping convulsants' binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites. Neurochem Int 50(1): 61-68.
Michels G, Moss SJ. 2007. GABAA receptors: properties and trafficking. Crit Rev Biochem Mol Biol 42(1):3-14.
Olsen RW. 2006. Picrotoxin-like channel blockers of GABAA receptors. Proc Natl Acad Sci USA. 103:6081-82.
Olsen RW, Chang C-SS, Li G, Hanchar HJ, Wallner M. 2004. Fishing for allosteric sites on GABAa receptors. Biochem Pharmacol. 68:1675-84.
Olsen RW, Sieghart W. 2009. GABAA receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology. 56(1):141-8.
Sander T, Frolund B, Bruun AT, Ivanov I, McCammon JA, Balle T. 2011. New insights into the GABAA receptor structure and orthosteric ligand binding: receptor modeling guided by experimental data. Proteins. 79(5):1458-77.
Simon J, Wakimoto H, Fujita N, Lalande M, Barnard EA. 2004. Analysis of the set of GABA(A) receptor genes in the human genome. J. Biol. Chem. 279(40), 41422–41435.
Taketo M, Yoshioka T. 2000. Developmental change of GABAA receptor-mediated current in rat hippocampus. Neuroscience 96(3):507-514.
Unwin N. 2005. Refined structure of the nicotinic acetylcholine receptor at 4 A˚ resolution. J Mol Biol. 346:967-89.
Zheng N, Cheng J, Zhang W, Li W, Shao X, Xu Z, Xu X, Li Z. 2014. Binding difference of fipronil with GABAARs in fruitfly and zebrafish: insights from homology modeling, docking, and molecular dynamics simulation studies. J Agric Food Chem 62(44):10646-53.