Arch Toxicol DOI 10.1007/s00204-017-2089-4

ORGAN TOXICITY AND MECHANISMS

GABAA receptor subtype selectivity of the proconvulsant rodenticide TETS

Brandon Pressly1 · Hai M. Nguyen1 · Heike Wulf1

Received: 16 June 2017 / Accepted: 5 October 2017 © The Author(s) 2017. This article is an open access publication

Abstract The rodenticide tetramethylenedisulfotetramine Abbreviations (TETS) is a potent convulsant (lethal dose in humans AUC Area under the curve 7–10 mg) that is listed as a possible threat agent by the CI Confdence interval United States Department of Homeland Security. TETS has DMSO Dimethylsulfoxide previously been studied in vivo for toxicity and in vitro in EBOB 4 ′-Ethynyl-4-n-propylbicycloorthobenzoate binding assays, with the latter demonstrating it to be a non- EC50 Concentration producing 50% efect competitive antagonist on ­GABAA receptors. To determine Emax Maximal efect whether TETS exhibits subtype selectivity for a particular GABA Gamma-aminobutyric acid ­GABAA receptor combination, we used whole-cell patch- GFP Green fuorescent protein clamp to determine the potency of TETS on the major syn- IC50 Concentration producing 50% inhibition aptic and extrasynaptic ­GABAA receptors associated with nH Hill coefcient convulsant activity. The active component of picrotoxin, PTX Picrotoxinin picrotoxinin, was used as a control. While picrotoxinin did SD Standard deviation not diferentiate well between 13 ­GABAA receptors, TETS TETS Tetramethylenedisulfotetramine exhibited the highest activity on α2β3γ2 (IC­ 50 480 nM, 95% TBPS t-Butylbicyclophosphorothionate CI 320–640 nM) and α6β3γ2 ­(IC50 400 nM, 95% CI 290– 510 nM). Introducing β1 or β2 subunits into these receptor combinations reduced or abolished TETS sensitivity, sug- Introduction gesting that TETS preferentially afects receptors with α2/ β3 or α6/β3 composition. Since α2β3γ2 receptors make up GABAA receptors are heteropentameric ligand-gated chlo- 15–20% of the GABA­ A receptors in the mammalian CNS, ride channels that are activated by gamma-aminobutyric acid we suggest that α2β3γ2 is probably the most important (GABA), the main inhibitory neurotransmitter in the adult ­GABAA receptor for the seizure-inducing activity of TETS. CNS. ­GABAA receptors have a complex and often some- what promiscuous pharmacology with numerous orthosteric Keywords TETS · GABAA receptor · Electrophysiology · and allosteric sites that modulate channel function (Krall Picrotoxinin · Convulsant · Threat agent et al. 2015; Olsen 2015). While ­GABAA receptor agonists and positive allosteric modulators reduce neuronal excit- ability and can be used as anxiolytics and anticonvulsants, compounds that inhibit ­GABAA receptor functions increase * Heike Wulf neuronal fring and promote seizures. TETS (tetramethyl- [email protected] enedisulfotetramine) and picrotoxin are both potent convul-

1 sants (Haskell and Voss 1957; Zolkowska et al. 2012) that Department of Pharmacology, Genome and Biomedical can cause severe tonic–clonic seizures and are, therefore, Sciences Facility Room 3502, 451 Health Sciences Drive, School of Medicine, University of California, Davis, considered threat agents by the United States Department CA 95616, USA of Homeland Security. Both compounds are thought be to

Vol.:(0123456789)1 3 Arch Toxicol work through a similar mechanism; nonselective inhibition a rodenticide in China, where several mass poisonings with of ­GABAA receptors, yet TETS is 30–100-fold more potent malicious intent have occurred (Whitlow et al. 2005; Zhang as a convulsant and lethal toxin in the mouse than picrotoxin et al. 2011). There have also been cases of reported TETS (Lamanna and Hart 1968; Shandra et al. 1996). poisonings in the United States with material sourced from GABAA receptors exhibit a high degree of structural China as an indoor rodenticide (Barrueto et al. 2003; Whit- heterogeneity and exist in multiple subtypes (Olsen and low et al. 2005). The frst pharmacological information about Sieghart 2009), with each subtype being a pentamer assem- TETS was generated by Alfred Haskell and Voss (1957), bled from a pool of 19 possible subunits, α1–α6, β1–β3, who tested TETS on organ preparations from frogs, rats, γ1–γ3, δ, ε, π, θ, and ρ1–ρ3. Based on the fact that TETS cats, and dogs and found that TETS acted exclusively on the and picrotoxin are structurally somewhat similar but dis- brain without exhibiting any activity on peripheral nerves tinct (Fig. 1), we wondered if the two agents would exhibit or muscle. Seizures only terminated with severance below diferent ­GABAA receptor subtype specifcities. Picrotoxin the medulla. The fact that sub-lethal doses of TETS could can be isolated from seeds of the moonseed family and has reverse pentobarbital-induced decreases in blood pressure an extremely bitter taste. It is an equimolar mixture of two and respiration in dogs pointed towards a mechanism poten- tricyclic sesquiterpenes: picrotin and the active component tially involving ­GABAA receptors. TETS was later shown to picrotoxinin (Slater and Wilson 1951). TETS has similar displace ­[35S]t-butylbicyclophosphorothionate ­([35S]TBPS) physicochemical properties, but is easy to synthesize, taste- binding to rat brain membranes with an IC­ 50 of 1 μM (Esser less and odorless, and stable in drinking water for months et al. 1991; Squires et al. 1983), and to prevent 36Cl− uptake (Knaack et al. 2014). These characteristics make TETS a (Ratra et al. 2001) or to inhibit chloride currents (Barnych tangible threat. et al. 2017) through α1β2γ2 GABA­ A receptors expressed in TETS was frst synthesized in 1933 from sulfamide and HEK293 cells with IC­ 50 values of 1.3 or 8 μM, respectively. formaldehyde (Wood and Battye 1933) and then used as Interestingly, recent work from the Casida laboratory (Zhao an anti-mold agent for upholstery. Its severe toxicity frst et al. 2014) showed that “cold”-TETS displaces ­[14C]TETS became apparent in a German furniture factory where work- from rat brain membranes with a much lower IC­ 50 of 80 nM ers accidentally exposed to TETS-impregnated wool suf- than it displaces another radiolabeled caged convulsant, fered from disorientation and seizures (Hagen 1950). TETS 4′-ethynyl-4-n-[3H]propylbicycloorthobenzoate (EBOB), was later resynthesized under laboratory conditions and suggesting that TETS is binding to a site that is only partially found to be exceptionally poisonous with an extremely low overlapping with the EBOB or TBPS site on various GABA­ A parenteral ­LD50 of 0.1–0.3 mg/kg in rodents (Casida et al. receptors or that it might exhibit a specifc ­GABAA recep- 1976; Haskell and Voss 1957). The United States Forestry tor subtype selectivity. We here tested the later possibility Department explored TETS as a rodenticide but eventually using patch-clamp electrophysiology in an attempt to fnd abandoned it due to its extreme toxicity, the lethal dose in an explanation for the discrepancy between the relatively adult humans being 7–10 mg (Guan et al. 1993), the lack of low potency of TETS in inhibiting GABA­ A receptors and its a viable rescue agent, and its persistence in the environment high in vivo toxicity (Lamanna and Hart 1968; Zolkowska (Whitlow et al. 2005). TETS is currently banned world- et al. 2012). wide, but continues to be easily obtainable and popular as

Materials and methods H O O Chemicals

O O O O Picrotoxinin (PTX), fpronil, bicuculline, propofol, sali- N OH S cylidene salicylhydrazide, zinc chloride, GABA, dexameth- H O asone, zeocin, and geneticin were purchased from Sigma N N Aldrich (St. Louis, MO, United States). Diazepam, allopreg- O N N S nanolone, and DS2 (4-chloro- -[2-(2-thienyl)imidazo[1,2-a] pyridin-3-yl]benzamide) were purchased from Tocris Bio- O science (Bristol, United Kingdom). TETS was synthesized in the laboratory of Dr. Bruce Hammock, University of Califor- nia, Davis, CA (Zhao et al. 2014). 10 mM stocks of GABA TETS Picrotoxinin (PTX) were made fresh daily using Ringer solution (see below for composition). 10 mM stocks of PTX and TETS were Fig. 1 Chemical structures of TETS and picrotoxinin prepared in DMSO and diluted down into Ringer solution

1 3 Arch Toxicol immediately before application onto the cell. Both TETS dexamethasone was added to the media to induce α4 and β3 and PTX waste were treated with nitric acid and disposed of expressions (Mortensen et al. 2010). L929 or COS-7 cells using the waste accumulation program at UC Davis. were transfected using FuGENE 6 (ThermoFisher, Grand Island, NY, United States) transfection reagent in Opti- ® Preparation of cells expressing the ­GABAA receptors MEM reduced serum medium (Life Technologies, Benicia, CA, United States) with an equal amount of each of the sub- The human GABA­ A receptors α1, α2, α6, β1, β3, γ2L, and units (1:1:1) in combination with green fuorescent protein δ and the rat ­GABAA receptor β2 cloned into pcDNA3.1 (GFP) expressed from the pEGFP-C1 vector (Invitrogen). expression vectors were a gift from Dr. Robert L. Mac- The ratio of total cDNA to transfection reagent was 2:1. 48 h donald, Vanderbilt University, Nashville, TN. The human post-transfection, and cells were detached by trypsinization, ­GABAA receptor α4 cloned into a pcDNA3.1 expression washed, and plated onto poly-l-lysine-coated glass cover- vector was a gift from Dr. Richard Olson, University of slips. Transfected cells were identifed as GFP-expressing California, Los Angeles, CA. The human GABA­ A receptor cells, using an epifuorescence microscope for electrophysi- γ1 cloned into a pcDNA3.1 expression vector and a Lt-K ological whole-cell voltage-clamp studies. Correct subunit cell line stably expressing α4β3δ (Mortensen et al. 2010) assembly was tested with a battery of GABA­ A receptor posi- were gifts from Dr. Trevor Smart, University College Lon- tive allosteric modulators and inhibitors (Table 1). don, United Kingdom. L929 cells, a mouse fbroblast cell line (CCL-1), were obtained from ATCC (American Type Electrophysiological recordings Culture Collection, Manassas, VA, United States), and were used for expressing all GABA­ A receptors with the excep- Whole-cell voltage-clamp experiments were performed at tion of the receptor combination α6β3δ for which COS-7 room temperature with an EPC-10 HEKA amplifer (HEKA cells (ATCC CRL-1651) were used to increase transfection Elektronik, Lambrecht, Germany). Cells were bathed efcacy and expression. L929, Lt-K, and COS-7 cells were in Ringer solution consisting of 160 mM NaCl, 4.5 mM cultured in Dulbecco’s modifed Eagle’s medium (Lonza, KCl, 1 mM MgCl­ 2, 2 mM CaCl­ 2, 10 mM HEPES, pH 7.4, Basel, Switzerland) supplemented with 10% fetal bovine 311 mOsm. Recording electrodes were pulled from soda serum, 100 U/mL penicillin and 100 mg/mL streptomycin lime glass micro-haematocrit tubes (Kimble Chase, Roch- (Invitrogen, ThermoFisher, Grand Island, NY, United States) ester, NY, United States) and fre-polished to resistances of and maintained in humidifed 95% air and 5% ­CO2 air at 1.8–2.8 MΩ for voltage-clamp. Electrodes were flled with 37 °C. The Lt-K cell line expressing α4β3δ was cultured an internal solution consisting of 154 mM KCl, 2 mM ­CaCl2, with 1 mg/mL geneticin and 0.2 mg/mL of zeocin to main- 1 mM ­MgCl2, 10 mM HEPES, and 10 mM EGTA with pH tain selection pressure. Two days before experiments, 1 μM 7.2 and 305 mOsm. Cells were voltage-clamped at − 80 mV

Table 1 Pharmacological GABAA isoform Sensitivity to positive allosteric modulators Sensitivity to inhibitors signature of expressed GABA­ A receptor isoforms α1β12γL Diazepam+, ­Propofol+, ­Allopregnanolone+ SAS+, ­Bicuculline+, ­Fipronil+ α1β2γ2L Diazepam+, ­Propofol+, ­Allopregnanolone+ Fipronil+ α1β3γ2L Diazepam+, ­Propofol+, ­Allopregnanolone+ Fipronil+ α2β2γ2L Diazepam+, ­Propofol+ Fipronil+ α2β3γ2L Diazepam+, ­Propofol+, ­Allopregnanolone+ SASneg, ­Bicuculline+, ­Fipronil+, ­Zn2+neg α2β3 Diazepamneg, ­Propofol+, Fipronil+ α4β3γ2L Diazepamneg, ­Propofol+, Fipronil+ α4β3δ Diazepamneg, ­Allopregnanolone+, ­DS2+ Fipronil+ α6β1γ2L Diazepamneg SAS+, ­Bicuculline+, ­Fipronil+ α6β2γ2L Diazepamneg, ­Propofol+ Fipronil+ α6β3γ2L Diazepamneg, ­Propofol+, ­Allopregnanolone+ SASneg, ­Bicuculline+, ­Fipronil+, ­Zn2+neg α6β3 ND Zn2+ positive α6β3γ1 Diazepamneg, ­Propofol+ Fipronil+ α6β3δ Diazepamneg, ­DS2+ Fipronil+

Sensitivity to 10 μM diazepam, 250 nM Allopregnanolone, 100 μM Propofol, 50 μM DS2 (4-chloro-N-[2- (2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide), 10 μM Fipronil, 100 μM Bicuculline, 10 μM SAS (sali- cylidene salicylhydrazide) and 10 μM ­ZnCl2 was tested on 3–10 cells per subunit combination ND Not determined

1 3 Arch Toxicol and control currents were recorded under the application et al. 1993). Correct subunit assembly and γ2L or δ incor- of varying GABA concentrations for 5 s followed by a 55 s poration were confrmed with a battery of GABA­ A receptor wash with external Ringer solution using a gravity-fed fast probes consisting of the positive allosteric modulators diaz- perfusion system ­(VC38 system, ALA Scientifc, Farming- epam, propofol, allopregnanolone, and DS2 and the inhibi- dale, NY, United States). tors fbronil, bicuculline, salicylidene salicylhydrazide, and The GABA concentration–response relationships were zinc chloride (Table 1). For both the exemplary synaptic determined by testing increasing concentrations of GABA ­GABAA receptors α1β1γ2L, α1β2γ2L, α1β3γ2L (Fig. 2a), and normalizing the GABA-induced peak currents to the α2β3, α2β2γ2L, and α2β3γ2L (Fig. 2b) and the exemplary peak currents induced by a maximal concentration of GABA. extrasynaptic ­GABAA receptors α4β3δ, α4β3γ2L (Fig. 2c), The normalized current responses were subsequently ftted α6β3δ, α6β3γ1, α6β2γ2L, α6β3γ2L, and α6β1γ2L (Fig. 2d), using the Hill equation to determine ­EC50 and ­EC90. we determined GABA ­EC50 and ­EC90 values to choose the For the TETS and PTX concentration–response curves, most appropriate GABA concentrations for subsequently test solutions were freshly prepared immediately before evaluating TETS and PTX efects on each of these receptors. application onto cells. A near maximal concentration of When comparing our ­EC50 values to the previous work in GABA (GABA EC­ 90) was applied for 5 s and then washed the feld, we found that most of our results are in good agree- out for 55 s. TETS or PTX was then perfused in a volume ment with data obtained by Mortson et al. (2011), who tran- of 3–5 mL directly into the bath through a side port on the siently expressed various synaptic and extrasynaptic GABA­ A perfusion chamber and cells incubated with the inhibitor receptors in HEK cells and reported a very similar overall for 3 min before a 5 s pulse of GABA was applied to the ranking of GABA sensitivity. However, we observed a few patch-clamped cell. The convulsant was then washed out diferences. In our hands, α6 containing combinations were and another control GABA pulse applied. One cell was used generally 2–3-fold less sensitive to GABA than previously per concentration of the convulsant. For analysis of receptor reported. For example, we obtained an EC­ 50 of 0.69 μM for blockade, the area under the current curve ­(AUCMax) was α6β3δ (Fig. 2d), whereas Mortenson et al. reported EC­ 50 of determined for the control (EC­ 90 GABA) and ­AUCEx after 0.17 μM for this same subunit combination (Mortensen et al. exposure: 2011). Despite this diference in sensitivity, we observed

AUCEx the expected pharmacological responses, specifcally, no × 100 =%Blocked. response to diazepam but a pronounced response to DS2 AUCMax (Table 1). For competition experiments, drug was applied for 5 s followed by 55 s of wash with Ringer. The cells were then Importance of the β subunit in TETS activity exposed to GABA or GABA plus the convulsant (TETS or PTX) without any pre-incubation. GABA-induced responses Based on molecular dynamics simulations using a homol- in the presence and absence of convulsant were quantifed ogy model of the pore region of the α1β2γ2 GABA­ A recep- by analyzing the peak response. tor, TETS has been hypothesized to bind in the pore lumen Data analysis was performed using the Excel (Microsoft) by hydrogen bonding with two α1–M2 segments and one and Origin 9.1 (OriginLab Corporation, Northampton, MA, γ2–M2 segment without making any contacts with the β United States) software. Data ftting to the Hill equation to subunit (Zhao et al. 2014). In support of this hypothesis, the obtain ­EC50 or IC50 values were also performed using Ori- β3 homopentamer has been shown to not bind TETS even gin 9.1. Individual data points are presented as mean ± SD at concentrations greater than 10 μM (Ratra et al. 2001). To from 3 to 8 independent recordings. ­EC50 and ­IC50 values determine if the β subunit is really not important for TETS are presented with 95% confdence intervals. activity, as suggested by these studies, we tested all three β subunits, β1, β2, and β3, in combination with α1 and γ2 (Fig. 3a). We started with α1β2γ2, since it is the most abun- dant subunit combination, constituting ~ 60% of GABA­ A Results receptors in the mammalian brain (Rudolph and Knofach 2011; Sur et al. 2001). GABA-induced chloride current GABA concentration responses at GABA EC­ 90 through this subunit exhibited a moderate sensitivity to inhibition by TETS (IC­ 50 3.6 μM). Following Before studying TETS activity, we frst obtained GABA con- pre-incubation with diferent TETS concentrations, block centration–response curves for 13 synaptic and extrasynaptic developed quickly, and TETS inhibition was fully revers- ­GABAA receptor isoforms transiently or stably expressed in ible on washout; however, even concentrations as high as L929 cells, a mouse fbroblast cell line that has been used to 100 μM could not fully block the current and only achieved a express ­GABAA receptors since the early 1990s (Angelotti maximal inhibition of ~ 70%. The remaining GABA-induced

1 3 Arch Toxicol

Fig. 2 GABA concentration–response curves for α1 (a), α2 (b), α4 (c), and α6 (d) containing ­GABAA receptor combinations. Individual data points are presented as mean ± SD from 7–20 independent recordings. EC­ 50 values are presented with 95% confdence intervals chloride current could be blocked by 100 μM of bicuculline both β1 and β3. While α1β1γ2 was not very sensitive to or 10 μM of fpronil (data not shown). To determine the TETS (maximal block < 50% at 100 μM), α1β3γ2 was indis- signifcance of changing the β subunit, we replaced it with tinguishable from the β2 containing subunit combination

1 3 Arch Toxicol

Fig. 3 a Concentration–response curves comparing TETS inhibition are presented with 95% confdence intervals. Example traces show- of diferent β subunits in the α1βXγ2L combination. b Comparison of ing TETS inhibition of α2β3γ2L (c) and α6β3γ2L (d) receptors. EC­ 90 TETS inhibition of α1, α2, α4, and α6 subunits in αXβ3γ2L contain- GABA was applied frst alone as control and then in the presence of 1 ing ­GABAA receptor combinations. Individual data points are pre- or 100 μM of TETS sented as mean ± SD from 3–8 independent recordings. EC­ 50 values in terms of TETS potency and maximal efcacy (Fig. 3a). combination (Fig. 3b). We chose this combination rather We, therefore, concluded that both β2 and β3 subunits can than the αXβ2γ2L combination, because the β3 we are using modulate TETS binding. for this work is human, whereas β2 is a rat clone. We did not investigate α5, because this subunit is not thought to Understanding the signifcance of the α subunit be involved in seizure generation, but rather plays a role in TETS activity on GABA­ A receptors in learning and memory (Collinson et al. 2002; Rudolph and Mohler 2004). Out of the tested subunit combina- We next investigated the role of the α subunit in TETS activ- tions, α2β3γ2L ­(IC50 = 480 nM with a maximal block ity by exchanging α1 for α2, α4, and α6 in the αXβ3γ2L of ~ 80%) and α6β3γ2L ­(IC50 = 400 nM with a maximal

1 3 Arch Toxicol block of ~ 75%) showed the highest sensitivity to TETS Overall, α4 containing receptors are not very sensitive to (Fig. 3b), while α1β3γ2L was roughly tenfold less sensitive TETS (α4β3γ2L: IC­ 50 = 1.82 μM, Emax = 46%, nH = 0.7), (α1β3γ2L: ­IC50 = 3.6 μM with a maximal block of ~ 75%). as previously shown in Fig. 3, but TETS inhibited the δ On the α4β3γ2L combination, even 100 μM of TETS could containing α4β3δ receptor with an increased efficacy not achieve more than 45% of inhibition, making this subunit (IC­ 50 = 2.66 μM, Emax = 85%; Fig. 4c) and a steeper Hill combination the least sensitive. coefcient (nH = 1.2). The same efect was observed in the more TETS-sensitive α6 combination (Fig. 4d). While Does the β subunit afect the TETS sensitivity of α2 α6β3δ was less sensitive ­(IC50 = 3.69 μM) to TETS inhibi- and α6? tion than the α6β3γ2L receptor ­(IC50 = 400 nM, Emax = 64%, nH = 0.7), TETS displayed a greater efcacy (Emax = 85%) We had seen that there was no diference in TETS sensitiv- and a steeper Hill coefcient (nH = 1.3) for α6β3δ (Fig. 4d). ity between β2 and β3 when these subunits were expressed In contrast, exchanging γ2 for γ1 “simply” shifted the TETS with α1 (Fig. 3a). It was important to determine if this was concentration–response curve to the right and reduced also true for the more sensitive α2 and α6 subunits and we, potency, but maintained efcacy (α6β3γ1: ­IC50 = 6.81 μM, therefore, next co-expressed these two subunits with β2 Emax = 60%). We further tested α2β3 to see how removing instead of β3. In both combinations, TETS clearly showed a the γ subunit would change TETS sensitivity and found that preference for β3 containing subunit combinations over β2 the absence of a γ subunit in the receptor pentamer lowered containing combinations (Fig. 4a, b). For the α2βXγ2L com- both the potency and efcacy of TETS (Table 2). bination, replacing β3 with β2 shifted the TETS concentra- tion–response curve signifcantly to the right, and increased Picrotoxinin shows no GABA­ A receptor subtype ­IC50 from 400 nM to 12.2 μM (Fig. 4a). For the α6βXγ2L selectivity combination, the efect was even more dramatic, and in the presence of β2, even 100 μM of TETS no longer achieved As a control, we tested picrotoxinin (PTX), which is a widely more than 40% inhibition of GABA-induced chloride current used ­GABAA receptor inhibitor also classifed as a threat (Fig. 4b). Replacing β3 with β1 further reduced TETS activ- agent on the same receptor subtypes (Table 2 and Fig. 5). ity with 100 μM of TETS only achieving 25% inhibition of As expected based on data in the published literature, PTX current through α6β1γ2L (Fig. 4b). Based on these results, is a relatively nonselective GABA­ A receptor inhibitor that − we conclude that the presence of an α2 or α6 subunit and a blocks GABA-induced Cl­ currents through most GABA­ A β3 subunit is necessary for TETS to inhibit chloride currents receptors with IC­ 50s in 2–8 μM range and Emax of ~ 80% through ­GABAA receptors with high potency and efcacy. current inhibition. Similar to TETS, the presence of a β1 subunit abolishes PTX sensitivity, since α1β1γ2L is insensi- Signifcance of the γ and δ subunits in TETS activity tive to PTX, in contrast to various other β2 (Fig. 5a) or β3 (Fig. 5b) containing GABA­ A receptor combinations, which We next tested if δ could possibly play a role in TETS activ- are all inhibited by PTX (Table 2). ity, and, therefore, determined its potency for blocking cur- rents through the extrasynaptic α4β3δ ­GABAA receptor TETS is a non‑competitive ­GABAA receptor inhibitor stably expressed in Lt-K cells and the α6β3δ receptor tran- siently transfected into COS-7 cells. We used COS-7 cells Finally, to start probing the mechanism of action of TETS on for these experiments, because the α6β3δ combination did the α2β3γ2L receptor, we tested the efect of 2.5 μM TETS, not express well in L929 cells (peak currents < 200 pA). We which corresponds to IC­ 80 on this receptor subtype, on the chose not to try and generate the α2β3δ GABA­ A receptor, GABA concentration–response curve (Fig. 5c). As expected since there seems to be no evidence for the existence of this from the previously performed binding experiments (Zhao subunit combination in the literature (Olsen and Sieghart et al. 2014), TETS depressed the maximum GABA response 2008, 2009). Interestingly, in the two δ containing subunit elicited at 50 or 100 μM GABA and could not be competed combinations, we investigated the presence of a δ subunit of when the GABA concentration was increased to 1 mM, increased the maximal efcacy of TETS inhibition when demonstrating that TETS is a non-competitive inhibitor compared with the γ2L containing GABA­ A receptor com- similar to PTX, which was also tested at ­IC80 for compari- binations, and also changed the Hill coefcient (Fig. 4c, d). son. PTX at 10 μM produced a similar depression of the However, it should be noted here that the two δ containing GABA Emax and was also not able to shift the GABA con- subunit combinations in our study, α4β3δ and α6β3δ, were centration–response curve fully to the right (Fig. 5c). Similar expressed in two diferent cell lines, Lt-K and COS-7, which results were obtained with the other highly TETS-sensitive could have impacted our results. GABA receptor combination, α6β3γ2L (data not shown).

1 3 Arch Toxicol

Fig. 4 a Concentration–response curves for TETS inhibition of cur- for TETS inhibition, while the α4β3δ receptor ­(IC50 3 μM, 95% CI rents evoked by ­EC90 GABA for α2βxγ2L receptors to determine the 1.36–3.96 μM, Emax ~ 85%, nH = 1.2) showed an increased Hill coef- changes induced by exchanging β2 for β3. The concentration response fcient. d In α6 containing receptors (α6β3γ2L: ­IC50 400 nM, 95% CI curve of TETS for α2β2γ2L ­(IC50 12.2 μM, 95% CI 10.5–15.8 μM, 0.29–0.51 nM Emax ~ 70% as previously shown), introduction of a δ Emax ~ 65%) was right-shifted in comparison with α2β3γ2L (IC­ 50 subunit increased Emax and reduced potency (α6β3δ: IC­ 50 3.69 μM, 480 nM, 95% CI 10.5–15.8 nM, Emax ~ 80%). b Both the α6β1γ2L 95% CI 3.18–4.2 μM, Emax ~ 85%), while replacement of γ2L with receptor ­(IC50 > 20 μM, Emax ~ 25%) and the α6β2γ2L receptor ­(IC50 γ1 right-shifted the concentration response curve (α6β3γ1: ­IC50 20 μM, 95% CI 18.3–21.7 μM, Emax ~ 35%) showed a signifcantly 6.81 μM, 95% CI 3.9–9.7 μM, Emax ~ 65%). Individual data points are reduced response to TETS. The α6β3γ2L receptor (IC­ 50 400 nM, presented as mean ± SD from 3–9 independent recordings. ­EC50 val- 95% CI 290–510 nM, Emax ~ 70%) is highly sensitive to TETS as ues are presented with 95% confdence intervals. Please note that the shown previously in Fig. 3. c α4β3γ2L receptor ­(IC50 1.82 μM, 95% α4β3δ receptor combination was stably expressed in Lt-K cells, while CI 1.02–2.62 μM, Emax ~ 46%, nH = 0.7) showed a reduced Emax the α6β3δ combination was transiently expressed in COS-7 cells

Discussion at concentrations between 10 and 100 μM (Bowery et al. 1975), while a group in the former Soviet Union showed The rodenticide TETS has previously been primarily studied in 1989 that 5 μM of TETS increased the excitability of in animal models to assess its convulsant activity and tox- hippocampal slices (Pervukhin et al. 1989). A more recent icity or in binding assays, which demonstrated that TETS paper from our own group describing the synthesis of TETS- acts as a non-competitive ­GABAA receptor inhibitor. How- related haptens for the development of an ELISA assay to ever, so far, only very limited electrophysiological informa- detect TETS (Vasylieva et al. 2017) reported that TETS tion is available for this highly toxic threat agent. In 1975, inhibited chloride currents through α1β2γ2 ­GABAA recep- Bowery et al. demonstrated that TETS could reverse the tors expressed in HEK293 cells with IC­ 50 of 8 μM (Barnych efects of GABA on isolated superior cervical rat ganglions et al. 2017). Since we were somewhat surprised by this

1 3 Arch Toxicol

Table 2 IC50 values for TETS and PTX the α6β2/3γ2 composition constitute less than 5% of the ­GABAA receptors (Mohler et al. 2002; Rudolph and Kno- GABAA isoform TETS (μM) 95% CI PTX (μM) 95% CI fach 2011) and are largely restricted to the cerebellum α1β1γ2L > 20 – > 30 – (Jones et al. 1997). One short coming of our study is that we α1β2γ2L 3.60 2.1–4.1 7.98 6.58–9.38 only used the long splice variant of the γ2 subunit, γ2L, and α1β3γ2L 3.41 1.4–5.4 3.7 2.1–5.3 not the short γ2s subunit, which is known to be expressed α2β2γ2L 12.2 10.5–15.8 11 8.6–13.5 throughout the mammalian CNS in similar proportions and α2β3γ2L 0.48 0.32–0.64 7.5 5.2–9.9 often co-localizes with γ2L in the same receptor complexes α2β3 3.37 2.77–3.97 1.98 0.67–3.29 (Khan et al. 1994). α4β3γ2L 1.82 1.02–2.62 3.0 1.7–4.3 TETS difers from the less selective PTX in showing α4β3δ 2.66 1.36–3.96 ND ND preference for α2 and α6 over α1 and α4 when expressed in α6β1γ2L > 20 – ND ND combination with β3 and γ2. Introducing a β1 subunit into α6β2γ2L 20 18.3–21.7 26.9 20.3–33.2 the ­GABAA receptor heteropentamer dramatically decreases α6β3γ2L 0.40 0.29–0.51 5.8 4.2–7.4 both TETS and PTX potency and efcacy, suggesting that α6β3γ1 6.81 3.9–9.7 ND ND the presence of an α/β1 interface disrupts binding for both α6β3δ 3.69 3.18–4.2 ND ND compounds. In contrast, exchanging β3 for β2 or γ2 for the ND Not determined less commonly found γ1 reduces TETS activity by roughly tenfold (Table 2). However, β3 alone is not sufcient to generate a TETS binding site, since homopentameric β3 relatively low potency in electrophysiological assays con- ­GABAA receptors have been shown to not bind TETS at sidering TETS has a reported ­LD50 of 0.1 mg/kg (Casida concentrations of up to 10 μM, while they bind PTX with et al. 1976; Haskell and Voss 1957), we here investigated an afnity of 32 nM (Ratra et al. 2001). It is interesting that the GABA­ A receptor subtype selectivity of TETS using the presence of a β3 subunit in the preferred α2/β3 or α6/β3 whole-cell patch-clamp electrophysiology. One observa- combination plays such a large role in TETS action on the tion that made us suspect that TETS might exhibit subtype ­GABAA receptor. Of these two β subunits, β2 and β3, that selectivity was a report from the Casida laboratory that are able to participate in TETS binding, the more sensitive unlabeled TETS displaced [­14C]TETS from rat brain mem- β3 subunit is probably more important for the proconvul- branes with ­IC50 of 80 nM (Zhao et al. 2014), which is much sant activity of TETS. Mice lacking the β3 subunit display lower than ­IC50s usually reported for TETS displacement features reminiscent of Angelman syndrome in humans of other radiolabeled ­GABAA receptor antagonists such as including abnormal EEG with interictal spikes and slowing, EBOB (Zhao et al. 2014) or TBPS (Esser et al. 1991; Squires seizures, hyperactivity, impaired learning, and memory and et al. 1983). Another report suggests that the possibility of repetitive behavior (DeLorey et al. 1998; Handforth et al. a higher afnity target was ­Ca2+ dynamics measurements 2005). In contrast, mice lacking β2 do not exhibit spontane- in mouse hippocampal neuronal cultures which develop ous seizures, but are less susceptible to the hypnotic actions spontaneous network activity after about 2 weeks in culture of ethanol and the efects of benzodiazepines (Blednov et al. (Cao et al. 2012). TETS visibly altered ­Ca2+ dynamics in 2003; Sur et al. 2001). these networks at submicromolar concentrations, although While TETS and PTX thus difer in their GABA­ A recep- the reported ­EC50s for altering the frequency and amplitude tor subtype selectivity, with TETS most likely exerting its of the Ca­ 2+-induced fuorescence changes in the assay were major action on α2β3γ2 receptors and while PTX does not the 1–2 μM range (Cao et al. 2012). demonstrate any signifcant receptor selectivity, we hesi- Here, we identifed two GABA­ A receptor subtypes that tate to use this selectivity as an explanation for the greater are sensitive to TETS at submicromolar concentrations: in vivo toxicity of TETS, since the diferences in potency α2β3γ2 with ­IC50 of 480 nM and α6β3γ2 with ­IC50 of could possibly also be pharmacokinetic in nature. When 400 nM. Of these two receptor subtypes, both of which are administered intraperitoneally in mice, TETS induces clonic among the 11 GABA­ A receptors conclusively identifed as seizures with ED­ 50 of 0.14 mg/kg and is lethal at a dose native receptors (Olsen and Sieghart 2008), α2β3γ2 is prob- of 0.3 mg/kg, which is roughly 40 times more potent than ably the more important receptor for the seizure-inducing picrotoxin (Zolkowska et al. 2012). However, when TETS activity of TETS, since α2β3γ2 receptors make up 15–20% and picrotoxin (which in this case contained only 50% of of the ­GABAA receptors in the mammalian CNS (Fritschy the active picrotoxinin) were administered intraventricularly, and Mohler 1995; Pirker et al. 2000; Rudolph and Knofach they were found to be equipotent in their ability to induce 2011) and α2 containing ­GABAA receptors have been shown convulsions (Zolkowska et al. 2012), suggesting that the to signifcantly contribute to the anticonvulsant actions of higher potency of TETS when administered systemically diazepam (Fradley et al. 2007). In contrast, receptors with could be due to brain penetration or brain uptake than PTX.

1 3 Arch Toxicol

Fig. 5 Concentration-response curves for PTX inhibition of β1/β2 centration response curve of the α2β3γ2L receptor. Individual data (a) and β3 (b) containing ­GABAA receptor combinations. c Efect of points are presented as mean ± SD 3–8 independent recordings. ­EC50 TETS and PTX are roughly ­IC80 concentrations on the GABA con- values are presented with 95% confdence intervals

Another possibility is of course that TETS has additional achieve 70–85% of block. It is of course possible that we actions on other receptors than GABA­ A receptors that pro- could have achieved some additional block by applying mil- mote seizures and convulsions. limolar TETS or PTX concentrations, but we chose to not Both TETS and PTX failed to fully block GABA-induced test concentrations higher than 100 μM of TETS on most chloride currents through all tested GABA­ A receptor sub- receptors, since we regard these high concentrations as phys- unit combinations under our experimental conditions in iologically unrealistic. However, it should be mentioned here which we elicited near maximal currents using GABA EC­ 90 that the remaining current could be blocked by bicuculline concentrations for each receptor, and could typically only (100 μM) or fpronil (10 μM). Out of these two compounds,

1 3 Arch Toxicol bicuculline is known to competitively inhibit GABA bind- utilizing a fuorescent substrate/marker gene technique. J Neuro- ing at the orthosteric site, although it also seems to have sci 13(4):1418–1428 Barnych B, Vasylieva N, Joseph T, Hulsizer S, Nguyen H, Cajka T, additional allosteric actions (Johnston 2013), while the phe- Pessah I, Wulf H, Gee S, Hammock B (2017) Development of nylpyrazole fpronil has been shown to bind to the same site tetramethylenedisulfotetramine (TETS) hapten library: synthe- as picrotoxinin and several polychlorocycloalkane insecti- sis, electrophysiological studies and immune response in rabbits. cides in radioligand binding assays on the homopentameric Chemistry. doi:10.1002/chem.201700783 Barrueto F Jr, Furdyna PM, Hofman RS, Hofman RJ, Nelson LS β3 receptor (Chen et al. 2006). This of course raises the (2003) Status epilepticus from an illegally imported Chinese question of the TETS binding site, which based on the non- rodenticide: “tetramine”. J Toxicol Clin Toxicol 41(7):991–994 competitive nature of its inhibition, and the observations that Blednov YA, Jung S, Alva H, Wallace D, Rosahl T, Whiting PJ, TETS displaces pore blockers like ­[35S]TBPS (Esser et al. Harris RA (2003) Deletion of the alpha1 or beta2 subunit of GABAA receptors reduces actions of alcohol and other 1991; Squires et al. 1983) and the cage convulsant EBOB drugs. J Pharmacol Exp Ther 304(1):30–36. doi:10.1124/ (Ratra et al. 2001; Zhao et al. 2014) is most likely located jpet.102.042960 in the pore region of the channel, where TETS has been Bowery NG, Brown DA, Collins JF (1975) Tetramethylenedisulphote- predicted by molecular dynamics simulations to hydrogen tramine: an inhibitor of gamma-aminobutyric acid induced depo- larization of the isolated superior cervical ganglion of the rat. Br bond with two α1–M2 segments and one γ2–M2 segment J Pharmacol 53(3):422–424 in a homology model of the α1β2γ2 ­GABAA receptor. This Cao Z, Hammock BD, McCoy M, Rogawski MA, Lein PJ, Pessah IN model (Zhao et al. 2014), which was based on the crystal (2012) Tetramethylenedisulfotetramine alters Ca­ 2+ dynamics in structure of the homopentameric Caenorhabditis elegans cultured hippocampal neurons: mitigation by NMDA receptor blockade and GABA(A) receptor-positive modulation. Toxicol glutamate-gated chloride channel (Hibbs and Gouaux 2011), Sci 130(2):362–372. doi:10.1093/toxsci/kfs244 and which has never been probed by mutagenesis, showed Casida JE, Eto M, Moscioni AD, Engel JL, Milbrath DS, Verkade TETS making no contacts with the β subunit, thus, appar- JG (1976) Structure-toxicity relationships of 2,6,7-trioxabicy- ently providing an explanation for why the α1β2γ2 GABA­ clo(2.2.2)octanes and related compounds. Toxicol Appl Pharma- A col 36(2):261–279 receptor is sensitive to TETS, while the β3 homopentamer is Chen L, Durkin KA, Casida JE (2006) Structural model for gamma- not (Ratra et al. 2001). This molecular model should be re- aminobutyric acid receptor noncompetitive antagonist binding: examined in light of our fndings that TETS shows the high- widely diverse structures ft the same site. Proc Natl Acad Sci est potency for blocking chloride current through GABA­ USA 103(13):5185–5190. doi:10.1073/pnas.0600370103 A Collinson N, Kuenzi FM, Jarolimek W, Maubach KA, Cothlif R, Sur receptors containing α2 or α6 as well as β3 subunits. Ideally, C, Smith A, Otu FM, Howell O, Atack JR, McKernan RM, Sea- the TETS binding site should be mapped through a combina- brook GR, Dawson GR, Whiting PJ, Rosahl TW (2002) Enhanced tion of site-directed mutagenesis and molecular modeling learning and memory and altered GABAergic synaptic transmis- using new homology models of the α2β3γ2, α2β2γ2 and sion in mice lacking the alpha 5 subunit of the GABAA receptor. J Neurosci 22(13):5572–5580 the α1β2γ2 GABA­ A receptors based on the more recently DeLorey TM, Handforth A, Anagnostaras SG, Homanics GE, Minas- crystallized human β3 homopentamer (Miller and Aricescu sian BA, Asatourian A, Fanselow MS, Delgado-Escueta A, Elli- 2014). son GD, Olsen RW (1998) Mice lacking the beta3 subunit of the GABAA receptor have the epilepsy phenotype and many of the Compliance with ethical standards behavioral characteristics of Angelman syndrome. J Neurosci 18(20):8505–8514 Esser T, Karu AE, Toia RF, Casida JE (1991) Recognition of tetra- Funding This work was supported by the CounterACT Program, methylenedisulfotetramine and related sulfamides by the brain National Institutes of Health Ofce of the Director, and the National GABA-gated chloride channel and a cyclodiene-sensitive mono- Institute of Neurological Disorders and Stroke [U54NS079202]. Bran- clonal antibody. Chem Res Toxicol 4(2):162–167 don Pressly was supported by a National Institute of General Medical Fradley RL, Guscott MR, Bull S, Hallett DJ, Goodacre SC, Waford Sciences funded Pharmacology Training Program [T32GM099608]. KA, Garrett EM, Newman RJ, O’Meara GF, Whiting PJ, Rosahl TW, Dawson GR, Reynolds DS, Atack JR (2007) Diferential Open Access This article is distributed under the terms of the contribution of GABA(A) receptor subtypes to the anticonvul- Creative Commons Attribution 4.0 International License (http://crea- sant efcacy of benzodiazepine site ligands. J Psychopharmacol tivecommons.org/licenses/by/4.0/), which permits unrestricted use, 21(4):384–391. doi:10.1177/0269881106067255 distribution, and reproduction in any medium, provided you give appro- Fritschy JM, Mohler H (1995) GABAA-receptor heterogeneity in priate credit to the original author(s) and the source, provide a link to the adult rat brain: diferential regional and cellular distribu- the Creative Commons license, and indicate if changes were made. tion of seven major subunits. J Comp Neurol 359(1):154–194. doi:10.1002/cne.903590111 Guan FY, Liu YT, Luo Y, Hu XY, Liu F, Li QY, Kang ZW (1993) GC/ MS identifcation of tetramine in samples from human alimentary intoxication and evaluation of artifcial carbonic kidneys for the References treatment of the victims. J Anal Toxicol 17(4):199–201 Hagen J (1950) Schwere Vergiftungen in Liner Polstermöbelfabrik durch einen neuartigen hoch toxischen Giftstof (Tetramethyl- Angelotti TP, Uhler MD, Macdonald RL (1993) Assembly of GABAA endisulfotetramin). Dtsch Med Wochenschr 75:183–184 receptor subunits: analysis of transient single-cell expression

1 3 Arch Toxicol

Handforth A, Delorey TM, Homanics GE, Olsen RW (2005) Phar- Pervukhin G, Sharonova IN, Prokof’ev SE, Fedorov NB, Kotel’nikov macologic evidence for abnormal thalamocortical func- SA (1989) Efect of the gamma-aminobutyric acid antagonist tioning in GABA receptor beta3 subunit-deficient mice, a disulfotetraazaadamantane on the evoked responses of neurons model of Angelman syndrome. Epilepsia 46(12):1860–1870. in hippocampal slices. Neirofziologiia 21(1):66–70 doi:10.1111/j.1528-1167.2005.00287.x Pirker S, Schwarzer C, Wieselthaler A, Sieghart W, Sperk G (2000) Haskell AR, Voss E (1957) The pharmacology of tetramine (tetra- GABA(A) receptors: immunocytochemical distribution of 13 ethylenedisulfotetramine). J Am Pharm Assoc Am Pharm Assoc subunits in the adult rat brain. Neuroscience 101(4):815–850 46(4):239–242 Ratra GS, Kamita SG, Casida JE (2001) Role of human GABA(A) Hibbs RE, Gouaux E (2011) Principles of activation and permeation receptor beta3 subunit in insecticide toxicity. Toxicol Appl Phar- in an anion-selective Cys-loop receptor. Nature 474(7349):54–60. macol 172(3):233–240. doi:10.1006/taap.2001.9154 doi:10.1038/nature10139 Rudolph U, Knofach F (2011) Beyond classical benzodiazepines: Johnston GA (2013) Advantages of an antagonist: bicuculline and other novel therapeutic potential of GABAA receptor subtypes. Nat GABA antagonists. Br J Pharmacol 169(2):328–336. doi:10.1111/ Rev Drug Discov 10(9):685–697. doi:10.1038/nrd3502 bph.12127 Rudolph U, Mohler H (2004) Analysis of GABAA receptor func- Jones A, Korpi ER, McKernan RM, Pelz R, Nusser Z, Makela R, Mel- tion and dissection of the pharmacology of benzodiaz- lor JR, Pollard S, Bahn S, Stephenson FA, Randall AD, Sieghart epines and general anesthetics through mouse genetics. Annu W, Somogyi P, Smith AJ, Wisden W (1997) Ligand-gated ion Rev Pharmacol Toxicol 44:475–498. doi:10.1146/annurev. channel subunit partnerships: GABAA receptor alpha6 subunit pharmtox.44.101802.121429 gene inactivation inhibits delta subunit expression. J Neurosci Shandra AA, Mazarati AM, Godlevsky LS, Vastyanov RS (1996) 17(4):1350–1362 Chemical kindling: implications for antiepileptic drugs—sensi- Khan ZU, Gutiérrez A, De Blas AL (1994) Short and long form gamma tive and resistant epilepsy models. Epilepsia 37(3):269–274 2 subunits of the GABAA/benzodiazepine receptors. J Neurochem Slater SN, Wilson AT (1951) Relationship between picrotin and picro- 63(4):1466–1476 toxinin. Nature 167(4243):324–325 Knaack JS, Hamelin EI, Magnuson M, Silvestri E, Ash D, Johnson Squires RF, Casida JE, Richardson M, Saederup E (1983) [­ 35S]t-butylb- RC (2014) Quantitative analysis and stability of the rodenti- icyclophosphorothionate binds with high afnity to brain-specifc cide TETS (tetramine) in fnished tap water. Anal Methods UK sites coupled to gamma-aminobutyric acid-A and ion recognition 6(8):2780–2784 sites. Mol Pharmacol 23(2):326–336 Krall J, Balle T, Krogsgaard-Larsen N, Sorensen TE, Krogsgaard- Sur C, Wafford KA, Reynolds DS, Hadingham KL, Bromidge F, Larsen P, Kristiansen U, Frolund B (2015) GABAA recep- Macaulay A, Collinson N, O’Meara G, Howell O, Newman R, tor partial agonists and antagonists: structure, binding mode, Myers J, Atack JR, Dawson GR, McKernan RM, Whiting PJ, and pharmacology. Adv Pharmacol 72:201–227. doi:10.1016/ Rosahl TW (2001) Loss of the major GABA(A) receptor subtype bs.apha.2014.10.003 in the brain is not lethal in mice. J Neurosci 21(10):3409–3418 Lamanna C, Hart ER (1968) Relationship of lethal toxic dose to body Vasylieva N, Barnych B, Rand A, Inceoglu B, Gee SJ, Hammock BD weight of the mouse. Toxicol Appl Pharmacol 13(3):307–315 (2017) Sensitive immunoassay for detection and quantifcation Miller PS, Aricescu AR (2014) Crystal structure of a human GABAA of the neurotoxin, tetramethylenedisulfotetramine. Anal Chem receptor. Nature 512(7514):270–275. doi:10.1038/nature13293 89(10):5612–5619. doi:10.1021/acs.analchem.7b00846 Mohler H, Fritschy JM, Rudolph U (2002) A new benzodiazepine phar- Whitlow KS, Belson M, Barrueto F, Nelson L, Henderson AK macology. J Pharmacol Exp Ther 300(1):2–8 (2005) Tetramethylenedisulfotetramine: old agent and new Mortensen M, Ebert B, Waford K, Smart TG (2010) Distinct activi- terror. Ann Emerg Med 45(6):609–613. doi:10.1016/j. ties of GABA agonists at synaptic- and extrasynaptic-type annemergmed.2004.09.009 GABAA receptors. J Physiol 588(Pt 8):1251–1268. doi:10.1113/ Wood FC, Battye AE (1933) The condensation of sulphamide, jphysiol.2009.182444 dimethylsulphamide, and aniline-p-sulphonamide with formal- Mortensen M, Patel B, Smart TG (2011) GABA potency at GABA(A) dehyde. J Soc Chem Ind 56:346–349 receptors found in synaptic and extrasynaptic zones. Front Cell Zhang Y, Su M, Tian DP (2011) Tetramine poisoning: a case report Neurosci 6:1 and review of the literature. Forensic Sci Int 204(1–3):e24–e27. Olsen RW (2015) Allosteric ligands and their binding sites defne doi:10.1016/j.forsciint.2010.06.025 gamma-aminobutyric acid (GABA) type A receptor subtypes. Zhao C, Hwang SH, Buchholz BA, Carpenter TS, Lightstone FC, Adv Pharmacol 73:167–202. doi:10.1016/bs.apha.2014.11.005 Yang J, Hammock BD, Casida JE (2014) GABAA receptor tar- Olsen RW, Sieghart W (2008) International Union of pharmacology. get of tetramethylenedisulfotetramine. Proc Natl Acad Sci USA LXX. Subtypes of gamma-aminobutyric acid(A) receptors: clas- 111(23):8607–8612. doi:10.1073/pnas.1407379111 sifcation on the basis of subunit composition, pharmacology, and Zolkowska D, Banks CN, Dhir A, Inceoglu B, Sanborn JR, McCoy function. Update. Pharmacol Rev 60(3):243–260. doi:10.1124/ MR, Bruun DA, Hammock BD, Lein PJ, Rogawski MA (2012) pr.108.00505 Characterization of seizures induced by acute and repeated expo- Olsen RW, Sieghart W (2009) GABA A receptors: subtypes provide sure to tetramethylenedisulfotetramine. J Pharmacol Exp Ther diversity of function and pharmacology. Neuropharmacology 341(2):435–446. doi:10.1124/jpet.111.190579 56(1):141–148. doi:10.1016/j.neuropharm.2008.07.045

1 3