Binding to the picrotoxin site of ionotropic GABA receptors leading to epileptic seizures in adult brain

Ping Gong, Edward J. Perkins, US Army Engineer Research and Development Center

Email: ping.gong@usace.army.mil or edward.j.perkins@usace.army.mil

Point of contact for this AOP entry: Dr. Ping Gong

This AOP begins with a molecular initiating event (MIE) where a chemical binds to the picrotoxin binding site at or near the central pore of the ionotropic GABA receptor complex causing blockage of the ion channel. As a result, the first key event (KE) is a decrease in inward chloride conductance through the ligand-gated ion channel. This leads to the second KE, a reduction in postsynaptic inhibition, reflected as reduced frequency and amplitude of spontaneous inhibitory postsynaptic current (sIPSC) or abolishment of GABA-induced firing action in GABAergic neuronal membranes. Consequently, the resistance of excitatory neurons to fire is decreased, resulting in the generation of a large excitatory postsynaptic potential (EPSP), i.e., the third KE. The large EPSP is reflected as a spike (rise) of intracellular Ca²⁺ observed in the affected region, where a large group of excitatory neurons begin firing in an abnormal, excessive, and synchronized manner. Such a giant Ca²⁺-mediated excitatory firing (depolarization) causes voltage-gated Na⁺ to open, which results in action potentials. The depolarization is followed by a period of hyper-polarization mediated by Ca²⁺-dependent K⁺ channels or GABA-activated Cl⁻ influx. During seizure development, the post-depolarization hyperpolarization becomes smaller, gradually disappears, and is replaced by a depolarization. This characteristic depolarization-shrinking hyperpolarization sequence of events represents the fourth KE known as “paroxysmal depolarizing shift” (PDS), which forms a “seizure focus”. A PDS is, essentially, an indication of epilepsy at the cellular level, which serves as the foci to initiate the adverse outcome at the organismal level of epileptic seizure. The severity of symptoms is often dose- and duration- dependent, while the toxicological symptoms are associated with the type and location of affected iGABARs. Mortality can occur if the individual sustains a prolonged or pronounced convulsion or seizure. Neurotoxicity, of which seizures are an end point, is a regulated outcome for chemicals. This AOP allows for screening chemicals for the potential to cause neurotoxicity through the use of in vitro assays that demonstrate binding to the picrotoxin site, electrophysiological assays demonstrating depolarization of neuronal membranes, or electroencephalography that records electrical activity of the adult brain.

Ionotropic GABA receptors (iGABARs) are ligand-gated ion channels which play important functional roles in the nervous system. As the major player in inhibitory neurotransmission, iGABARs are widely distributed in both vertebrates and invertebrates (McGonigle and Lummis 2010; Garcia-Reyero et al. 2011). In vertebrates, the iGABAR includes two subclasses of fast-responding ion channels, GABA_A receptor (GABA_A-R) and GABA_C receptor (GABA_C-R). Invertebrate iGABARs do not readily fit the vertebrate GABA_A/GABA_C receptor categories (Sieghart 1995). The majority of insect iGABARs are distinguished from vertebrate GABA_A receptors by their insensitivity to bicuculline and differ from GABA_C-Rs in that they are subject to allosteric modulation, albeit weakly, by benzodiazepines and barbiturates (Hosie et al. 1997).

Chemical interactions with iGABARs can cause a variety of pharmacological and neurotoxicological effects depending on the location of the active or allosteric site affected. Three distinct types of interactions at binding sites on iGABARs can antagonize the postsynaptic inhibitory functions of GABA and lead to epileptic seizures and death. These three types of interactions correspond to three AOPs (Gong et al. 2015). One of the three types of interaction is non-competitive channel blocking at the picrotoxin convulsant site located inside of the iGABAR pore that spans neuronal cell membranes, which is the MIE described in the current AOP. The other two types of interactions are negative modulation at allosteric sites and competitive binding at the active orthosteric sites (MIEs to be developed in the future).

It is worth noting that there exist another class of GABA receptors called metabotropic G-protein-coupled receptors (mGABAR) or GABA_B receptors. This AOP is not applicable to GABA_B receptors because they mediate slow and sustained inhibitory responses to GABA and are involved in absence epilepsy (Han et al. 2012).

Biological plausibility

The biological mechanisms underlying epilepsy (defined as a disorder of the central nervous system characterized by recurrent seizures unprovoked by an acute systemic or neurologic insult) have been investigated for more than six decades and are well understood except for a few intermediate details (Bromfield et al. 2006; Lomen-Hoerth and Messing 2010). As one of the cellular mechanisms of action, blocking postsynaptic GABA-mediated inhibition can lead to epileptic seizure (Dichter and Ayala 1987; Gong et al. 2015). It has been extensively documented that non-competitive ion channel blockers such as picrotoxin, lindane, α-endosulfan and fipronil act through binding to iGABARs (Chen et al. 2006; Casida and Durkin 2015). Despite large structural diversity, it has been postulated that these blockers fit a single binding site in the chloride channel lumen lined by five TM2 (TransMembrane domain 2) segments, which was supported in the β3 homopentamer by mutagenesis, pore structure studies, ligand binding, and molecular modeling (Chen et al. 2006; Casida and Durkin 2015). The downstream cascading key events of this AOP have also been reviewed in multiple publications (e.g., Dichter and Ayala 1987; Bromfield et al. 2006; Lomen-Hoerth and Messing 2010). Based on the extensive evidence supporting the MIE, KEs and the AO, there is a high likelihood and certainty that such GABA antagonists as non-competitive channel blockers produce seizures in both invertebrates and vertebrates that possess GABAergic inhibitory neurotransmission in central nervous systems (Treiman 2001; Raymond-Delpech et al. 2005).

Concordance of dose-response relationships

Numerous pharmacological studies have reported quantitative dose-response relationships between the dose of non-competitive antagonists and the recorded electrophysiological response of epileptic seizures. See examples for picrotoxin (Newland and Cull-Candy 1992; Ikeda 1998; Stilwell et al. 2006), RDX (Williams et al. 2011) and dieldrin (Babot et al. 2007; Ikeda 1998).

Temporal concordance among the key events and the adverse outcome

Given that the basic mechanism of neuronal excitability is the action potential, a hyperexcitable state can result from many causes including decreased inhibitory neurotransmission (KE2). Moreover, action potentials occur due to depolarization of the neuronal membrane, with membrane depolarization propagating down the axon to induce neurotransmitter release at the axon terminal. The action potential occurs in an all-or-none fashion as a result of local changes in membrane potential brought about by net positive inward ion fluxes. Membrane potential thus varies with the activation of ligand-gated channels, whose conductance is affected by binding to neurotransmitters. For instance, the conductance is decreased (KE1) due to the binding at allosteric sites in the chloride channel of iGABAR by non-competitive blockers (MIE).

Seizure initiation: The hypersynchronous discharges that occur during a seizure may begin in a very discrete region of the cortex and then spread to neighboring regions. Seizure initiation is characterized by two concurrent events: 1) high-frequency bursts of action potentials, and 2) hypersynchronization of a neuronal population. The synchronized bursts from a sufficient number of neurons result in a so-called spike discharge on the EEG (electroencephalogram), i.e., amplified excitatory postsynaptic potential (KE3). At the level of single neurons, epileptiform activity consists of sustained neuronal depolarization resulting in a burst of action potentials, a plateau-like depolarization associated with completion of the action potential burst, and then a rapid repolarization followed by hyperpolarization. This sequence is called the paroxysmal depolarizing shift (KE4).

Seizure propagation (AO), the process by which a partial seizure spreads within the brain, occurs when there is sufficient activation to recruit surrounding neurons. This leads to a loss of surround inhibition and spread of seizure activity into contiguous areas via local cortical connections, and to more distant areas via long association pathways such as the corpus callosum. The propagation of bursting activity is normally prevented by intact hyperpolarization and a region of surrounding inhibition created by inhibitory neurons. With sufficient activation there is a recruitment of surrounding neurons via a number of mechanisms. The above description is excerpted and summarized from Bromfield et al. (2006).

Strength, consistency, and specificity of association of adverse effect and initiating event

Drug- or chemical-induced focal or generalized seizures are not limited to any specific group of chemical structures, neuroreceptors or taxonomy. This AOP addresses a specific group of chemicals that are capable of binding to the picrotoxin convulsant site of iGABARs, leading to epileptic seizures. Literature evidence strongly and consistently supports such a forward association, i.e., binding to the picrotoxin site leads to epileptic seizures (see reviews Gong et al. 2015; Bromfield et al. 2006; Raymond-Delpech et al. 2005; Treiman 2001; Dichter and Ayala 1987). For instance, dose- and time-dependent correlations between picrotoxin site binding by chlorinated pesticides and convulsions were observed in mice (Cole and Casida 1986), whereas poisoning with the organochlorine insecticide endosulfan caused seizure, status epilepticus, or refractory status epilepticus in humans (Durukan et al. 2009; Moon et al. 2017; Moses and Peter 2010; Parbhu et al. 2009; Roberts et al. 2004), and eventually led to the death of a farmer (Roberts et al. 2004) and a toddler (Parbhu et al. 2009).

Uncertainties, inconsistencies, and data gaps

No inconsistencies have been reported so far, though some uncertainties and data gaps do exist. For instance, the process by which seizures typically end, usually after seconds or minutes, and what underlies the failure of this spontaneous seizure termination in the life-threatening condition known as status epilepticus are less well understood (Bromfield et al. 2006). The spread of epileptic activity throughout the brain, the development of primary generalized epilepsy, the existence of “gating", mechanisms in specific anatomic locations, and the extrapolation of hypotheses derived from simple models of focal epilepsy to explain more complex forms of epilepsies observed in human and other animals, all are also not yet fully understood (Dichter and Ayala 1987). The remarkable plasticity of GABAergic neurons and iGABARs (e.g., iGABAR regulation by phosphorylation, and expression of potassium chloride cotransporters (KCCs) or sodium dependent anion exchangers (NDAE)) in response to insults and injury constitutes additional complexity and creates another layer of uncertainties in the emergence of epileptic seizures (Ben-Ari 2006; Galanopoulou 2008; Scharfman and Brooks-Kayal 2014).

This AOP can be used to establish the mode of neurotoxicological actions for chemicals capable of binding to the picrotoxin convulsant site of iGABARs. It can also be applied to risk assessment where AOP can assist in predictive modeling of chemical toxicity. Chemicals acting through this AOP can be distinguished from neurotoxicants acting on other types of iGABAR sites (e.g., orthosteric or allosteric binding sites) or other types of neuroreceptors (e.g., ardrenergic, dopaminergic, glutaminergic, cholinergic and serotonergic receptors). More information relevant to this topic can be found in Gong et al. (2015).

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