Event:667

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Event Title

iGABAR chloride channel, Binding at picrotoxin site
iGABAR chloride channel, Binding at picrotoxin site

Key Event Overview

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AOPs Including This Key Event

AOP Name Event Type Essentiality
Binding to the picrotoxin site of ionotropic GABA receptors leading to epileptic seizures MIE Strong

Chemical Initiators

The following are chemical initiators that operate directly through this Event:

  1. Picrotoxin
  2. Lindane
  3. Fipronil
  4. RDX
  5. Alpha-endosulfan
  6. Penicillin

Taxonomic Applicability

Name Scientific Name Evidence Links
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI
fruit fly Drosophila melanogaster Strong NCBI
mouse Mus musculus Strong NCBI
dogs Canis sp. Strong NCBI

Level of Biological Organization

Biological Organization
Molecular

How this Key Event works

Figure1.png

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. 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, non-competitive channel blockers (e.g., fipronil, lindane, picrotoxin 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).

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).

Evidence Supporting Taxonomic 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 (GABA-A-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 GABA-A 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).

Evidence for Chemical Initiation of this 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 binding site in the chloride channel lumen lined by five TM2 segments. 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).

References

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.

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.

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.

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, Sieghart W. 2009. GABA A 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.

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.