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Event: 667

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Binding at picrotoxin site, iGABAR chloride channel

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Binding at picrotoxin site, iGABAR chloride channel

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
neuron

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Organ term
brain

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
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

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
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 TFHA/WNT Endorsed

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
fruit fly Drosophila melanogaster High NCBI
mouse Mus musculus High NCBI
dogs Canis lupus familiaris High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
Adult High

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Term Evidence
Unspecific High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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 (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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

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).

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

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.