Upstream eventBinding at picrotoxin site, iGABAR chloride channel
Reduction, Ionotropic GABA receptor chloride channel conductance
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Binding to the picrotoxin site of ionotropic GABA receptors leading to epileptic seizures in adult brain||adjacent||High||High|
|Drosophila melanogaster||Drosophila melanogaster||High||NCBI|
Life Stage Applicability
Key Event Relationship Description
Acting as the major inhibitory neurotransmitter receptors, the ionotropic GABA receptors (iGABARs) are ligand-gated ion channels (LGICs) (Carpenter et al. 2013). Upon binding of an agonist (e.g., GABA), the iGABAR opens and increases the intraneuronal concentration of chloride ions, thus hyperpolarizing the cell and inhibiting the transmission of the nerve action potential. iGABARs also contain many other modulatory binding pockets that differ from the agonist-binding site. The picrotoxin-binding site is a noncompetitive channel blocker site located at the cytoplasmic end of the transmembrane channel (Olsen 2015). Binding to this pocket blocks GABA-induced chloride current, hence reduces chloride conductance.
Evidence Supporting this KER
The mechanisms for noncompetitive picrotoxin site binding-induced reduction in chloride conductance have been investigated intensively for several decades. The consensus has been reached with ample support of computational and experimental evidence. Noncompetitive channel blockers fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6' and L9' alkyl substituents (Chen et al. 2006), which is the primary binding site in the chloride channel lumen lined by five TM2 segments, thereby blocking the channel. Recent evidence suggests that there also exists a secondary modulatory pocket at the interface between the ligand-binding domain and the transmembrane domain of the iGABAR (Carpenter et al. 2013). It is believed that the two mechanisms mediate the blockage of chloride conductance (Yoon et al. 1993; Carpenter et al. 2013).
Numerous pharmacological and computational studies have lent strong support for this relationship. For instance, picrotoxin, applied intracellularly, was capable of blocking GABA-activated chloride current (Akaike et al. 1985). Recently, a computational study using homology modeling, docking and molecular dynamics simulation methods revealed that difference in binding affinity of fipronil with different iGABARs may lead to differential toxicity (potency) (Zheng et al. 2014).
Uncertainties and Inconsistencies
As a heteropentameric receptor, the iGABAR consists of five protein subunits arranged around a central pore that form an ion channel through the membrane. The subunits are drawn from a pool of 19 distinct gene products, including six alpha, three beta, and three gamma subunits. The high diversity of subunit genes, in combination with alternative splicing and editing, leads to an enormous variety and, consequently, variability in function and sensitivity. This constitutes the main source of uncertainties.
Quantitative Understanding of the Linkage
Is it known how much change in the first event is needed to impact the second? There is no study that quantitatively measured both receptor binding affinity and inhibition of chloride flux.
Are there known modulators of the response-response relationships? There is no known modulator that acts in between receptor binding and channel blocking, even though there are many binding sites other than the picrotoxin-binding sites that may affect chloride conductance.
Are there models or extrapolation approaches that help describe those relationships? No, however, there exist computational models based on 3D structure modeling that have been used to predict the binding affinity of ligands/chemicals at specific pockets of the ion channel (Yoon et al. 1993; Zheng et al. 2014).
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Due to the universal existence of iGABARs in the animal kingdom, it would be a very long list of studies that provide supporting evidence with regard to taxonomic applicability of this key event relationship. The following are two examples: Williams et al. (2011) determined the binding affinity of RDX to the picrotoxin-binding site and the blockage of GABAA receptor-mediated currents in the rat amygdala; Grolleau and Sattelle (2000) reported a complete blocking of inward current by 100 μM picrotoxin in the wild-type RDL (iGABAR) of Drosophila melanogaster.
Akaike N. Hattori K, Oomura Y, Carpenter D0. 1985. Bicuculline and picrotoxin block γ-aminobutyric acid-gated Cl-conductance by different mechanisms. Experientia 41:70-71.
Carpenter TS, Lau EY, Lightstone FC. 2013. Identification of a possible secondary picrotoxin-binding site on the GABA(A) receptor. Chem Res Toxicol. 26(10):1444-54.
Chen L, Durkin KA, Casida J. 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.
Grolleau F, Sattelle DB. 2000. Single channel analysis of the blocking actions of BIDN and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a Drosophila cell line. Br J Pharmacol. 130(8):1833-42.
Olsen RW. 2015. Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes. Adv Pharmacol. 73:167-202.
Williams LR, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon D I et al. 2011. RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures. Environ Health Perspect. 119(3):357-363.
Yoon KW, Covey DF, Rothman SM. 1993. Multiple mechanisms of picrotoxin block of GABA-induced currents in rat hippocampal neurons. J Physiol. 464:423-39.
Zheng N, Cheng J, Zhang W, Li W, Shao X, Xu Z, Xu X, Li Z. 2014. Binding difference of fipronil with GABA(A)Rs in fruitfly and zebrafish: Insights from homology modeling, docking, and molecular dynamics simulation studies. J Agric Food Chem. 62:10646-53.