Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α 2 ß 3 γ 2 GABAA Receptor.

Tetramethylenedisulfotetramine (TETS) is a so-called "caged" convulsant that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABA receptor type A (GABAA) antagonist picrotoxinin, TETS has been proposed to bind to the noncompetitive antagonist (NCA) site in the pore of the receptor channel. However, the TETS binding site has never been experimentally mapped, and we here set out to gain atomistic level insights into how TETS inhibits the human α 2 ß 3 γ 2 GABAA receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α 2 ß 3 γ 2 receptor in the open, desensitized, and closed/resting state. Three different ligand-docking algorithms (RosettaLigand, Glide, and Swissdock) identified two possible TETS binding sites in the channel pore. Using a combination of site-directed mutagenesis, electrophysiology, and modeling to probe both sites, we demonstrate that TETS binds at the T6' ring in the closed/resting-state model, in which it shows perfect space complementarity and forms hydrogen bonds or makes hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6' in either the α 2 or ß 3 subunit reduces the IC50 of TETS by ∼700-fold in whole-cell patch-clamp experiments. TETS is thus interacting at the NCA site in the pore of the GABAA receptor at a location that is overlapping but not identical to the picrotoxinin binding site. SIGNIFICANCE STATEMENT: Our study identifies the binding site of the highly toxic convulsant tetramethylenedisulfotetramine (TETS), which is classified as a threat agent by the World Health Organization. Using a combination of homology protein modeling, ligand docking, site-directed mutagenesis, and electrophysiology, we show that TETS is binding in the pore of the α2ß3γ2 GABA receptor type A receptor at the so-called T6' ring, wherein five threonine residues line the permeation pathway of the pentameric receptor channel.

Pharmacological characterization of the neurotrophic sesquiterpene jiadifenolide reveals a non-convulsant signature and potential for progression in neurodegenerative disease studies.

The 'neurotrophic sesquiterpenes' refer to a group of molecules derived from the Illicium genus of flowering plant. They display neurotrophic effects in cultured neuron preparations and have been suggested to be cognitive enhancers and potential therapeutics for neurodegenerative disorders and dementias. Recent synthetic advances generated sufficient quantities of jiadifenolide for in vivo investigation into its biological effects. Jiadifenolide did not induce convulsions in mice nor did it enhance or diminish convulsions induced by pentylenetetrazole. Other negative allosteric modulators of GABAA receptors, picrotoxin, tetramethylenedisulfotetramine (TETS), and bilobalide all induced convulsions. Either i.p. or i.c.v. dosing generated micromolar plasma and brain levels of jiadifenolide but only small effects on locomotion of mice. However, jiadifenolide decreased d-amphetamine-induced hyperlocomotion in mice, an antipsychotic-like drug effect. Jiadifenolide did not significantly alter body temperature or behavior in the forced-swim test in mice. Molecular simulation data suggested a potential site in the pore/M2 helix region that is at an overlapping, yet lower position than those observed for other 'cage convulsant' compounds such as TETS and picrotoxin. We hypothesize that a position nearer to the entrance of the pore channel may allow for easier displacement of jiadifenolide from its blocking location leading to lower potency and lower side-effect liability. Like jiadifenolide, memantine (Namenda), one of the few drugs used in the symptomatic treatment of dementias, occupies a unique site on the NMDA receptor complex that creates low binding affinity that is associated with its reduced side-effect profile. Given the potential therapeutic applications of jiadifenolide and its relatively inert effects on overt behavior, the possibility of clinical utility for jiadifenolide and related compounds becomes intriguing.

Oxazolidinone-based allosteric modulators of mGluR5: Defining molecular switches to create a pharmacological tool box.

Herein we describe the structure activity relationships uncovered in the pursuit of an mGluR5 positive allosteric modulator (PAM) for the treatment of schizophrenia. It was discovered that certain modifications of an oxazolidinone-based chemotype afforded predictable changes in the pharmacological profile to give analogs with a wide range of functional activities. The discovery of potent silent allosteric modulators (SAMs) allowed interrogation of the mechanism-based liabilities associated with mGluR5 activation and drove our medicinal chemistry effort toward the discovery of low efficacy (fold shift) PAMs devoid of agonist activity. This work resulted in the identification of dipyridyl 22 (BMS-952048), a compound with a favorable free fraction, efficacy in a rodent-based cognition model, and low potential for convulsions in mouse.

Effect of ring-constrained phenylpropyloxyethylamines on sigma receptors.

A series of ring-constrained phenylpropyloxyethylamines, partial opioid structure analogs and derivatives of a previously studied sigma (σ) receptor ligand, was synthesized and evaluated at σ and opioid receptors for receptor selectivity. The results of this study identified several compounds with nanomolar affinity at both σ receptor subtypes. Compounds 6 and 9 had the highest selectivity for both σ receptor subtypes, compared to µ opioid receptors. In addition, compounds 6 and 9 significantly reduced the convulsive effects of cocaine in mice, which would be consistent with antagonism of σ receptors.

A pro-convulsive carbamazepine metabolite: quinolinic acid in drug resistant epileptic human brain.

Drugs and their metabolites often produce undesirable effects. These may be due to a number of mechanisms, including biotransformation by P450 enzymes which are not exclusively expressed by hepatocytes but also by endothelial cells in brain from epileptics. The possibility thus exists that the potency of systemically administered central nervous system therapeutics can be modulated by a metabolic blood-brain barrier (BBB). Surgical brain specimens and blood samples (ex vivo) were obtained from drug-resistant epileptic subjects receiving the antiepileptic drug carbamazepine prior to temporal lobectomies. An in vitro blood-brain barrier model was then established using primary cell culture derived from the same brain specimens. The pattern of carbamazepine (CBZ) metabolism was evaluated in vitro and ex vivo using high performance liquid chromatography-mass spectroscopy. Accelerated mass spectroscopy was used to identify (14)C metabolites deriving from the parent (14)C-carbamazepine. Under our experimental conditions carbamazepine levels could not be detected in drug resistant epileptic brain ex situ; low levels of carbamazepine were detected in the brain side of the in vitro BBB established with endothelial cells derived from the same patients. Four carbamazepine-derived fractions were detected in brain samples in vitro and ex vivo. HPLC-accelerated mass spectroscopy confirmed that these signals derived from (14)C-carbamazepine administered as parental drug. Carbamazepine 10, 11 epoxide (CBZ-EPO) and 10, 11-dihydro-10, 11-dihydrooxy-carbamazepine (DiOH-CBZ) were also detected in the fractions analyzed. (14)C-enriched fractions were subsequently analyzed by mass spectrometry to reveal micromolar concentrations of quinolinic acid (QA). Remarkably, the disappearance of carbamazepine-epoxide (at a rate of 5% per hour) was comparable to the rate of quinolinic acid production (3% per hour). This suggested that quinolinic acid may be a result of carbamazepine metabolism. Quinolinic acid was not detected in the brain of patients who received antiepileptic drugs other than carbamazepine prior to surgery or in brain endothelial cultures obtained from a control patient. Our data suggest that a drug resistant BBB not only impedes drug access to the brain but may also allow the formation of neurotoxic metabolites.

Isolation, amino acid sequence and biological activities of novel long-chain polyamine-associated peptide toxins from the sponge Axinyssa aculeata.

A novel family of functionalized peptide toxins, aculeines (ACUs), was isolated from the marine sponge Axinyssa aculeate. ACUs are polypeptides with N-terminal residues that are modified by the addition of long-chain polyamines (LCPA). Aculeines were present in the sponge extract as a complex mixture with differing polyamine chain lengths and peptide structures. ACU-A and B, which were purified in this study, share a common polypeptide chain but differ in their N-terminal residue modifications. The amino acid sequence of the polypeptide portion of ACU-A and B was deduced from 3' and 5' RACE, and supported by Edman degradation and mass spectral analysis of peptide fragments. ACU induced convulsions upon intracerebroventricular (i.c.v.) injection in mice, and disrupted neuronal membrane integrity in electrophysiological assays. ACU also lysed erythrocytes with a potency that differed between animal species. Here we describe the isolation, amino acid sequence, and biological activity of this new group of cytotoxic sponge peptides.

Actions of two GABAA receptor benzodiazepine-site ligands that are mediated via non-γ2-dependent modulation.

The potent sedative-hypnotic zolpidem and the convulsant methyl-6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate (DMCM) act primarily by binding to the benzodiazepine site of the main inhibitory neurotransmitter receptor, the pentameric γ-aminobutyric acid type A receptor (GABA(A)). This binding depends critically on the wild-type F77 residue of the GABA(A) receptor γ2 subunit. Mice with γ2 subunit F77I point mutation (γ2I77 mouse line) lose the high-affinity nanomolar binding of these ligands as well as their most robust behavioral actions at low doses. Interestingly, the γ2I77 mice offer a tool to study the actions of these substances mediated via other possible binding sites of the GABA(A) receptor. In ligand autoradiographic experiments, we discovered in γ2I77 mouse brain sections a significant amount of residual non-γ2 subunit-dependent benzodiazepine site binding enriched to the striatum and septum. Zolpidem only weakly affected this residual binding at micromolar concentrations, and only a high zolpidem dose (≥ 40 mg/kg) caused sedation and deficits in motor coordination in γ2I77 mice. DMCM had an agonistic action through a secondary, low-affinity non-benzodiazepine binding site of the GABA(A) receptor in the forebrain of γ2I77 mice, and this drug also fully displaced the residual benzodiazepine-site labeling. In behavioral tests, a high dose (20mg/kg) of DMCM was sedative and modulated fear learning. DMCM, but not zolpidem, acted as an agonist in recombinant GABA(A) α1/6ß3 receptors studied using ligand binding and electrophysiological assays. Our results highlight the less well-known actions of high doses of DMCM and zolpidem that are not mediated via the γ2 subunit-containing benzodiazepine site of the GABA(A) receptor.

Neurobehavioral effects of acute exposure to aromatic hydrocarbons.

This article reports the results of neurobehavioral tests on representative aromatic constituents, specifically C(9) to C(11) species. The testing evaluated effects in several domains including clinical effects, motor activity, functional observations, and visual discrimination performance. Exposures ranging from 600 to 5000 mg/m(3), depending on the molecular weights of the specific aromatic constituents, produced minor, reversible effects on the central nervous system (CNS), particularly in the domains of gait and visual discrimination. There was little evidence of effects at lower exposure levels. There was some evidence of respiratory effects at 5000 mg/m(3) in 1 study, and there were also minor changes in body weight and temperature. The CNS effects became less pronounced with repeated exposures, corresponding to lower concentrations in the brain of 1 representative substance, 1,2,4-trimethyl benzene (TMB). At high exposure levels, the alkyl benzenes apparently induced their own metabolism, increasing elimination rates.

Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of diisopropyl fluorophosphatase (DFPase) with (1)H- (31)P HSQC NMR spectroscopy.

The enzyme diisopropyl fluorophosphatase (DFPase, EC 3.1.8.2) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Until now, determination of kinetic data has been achieved by use of techniques such as pH-stat titration, ion-selective electrodes, and a recently introduced method based on in situ Fourier-transform infrared (FTIR) spectroscopy. We report the use of 1D (1)H-(31)P HSQC NMR spectroscopy as a new method for real-time quantification of the hydrolysis of toxic organophosphonates by DFPase. The method is demonstrated for the agents sarin (GB), soman (GD), and cyclosarin (GD) but can also be used for V-type nerve agents, for example VX. Besides buffered aqueous solutions the method was used to determine enzymatic activities in a biodiesel-based bicontinuous microemulsion that serves as an example of complex decontamination media, for which other established techniques often fail. The method is non-invasive and requires only limited manual handling of small volumes of liquid (700 microL), which adds to work safety when handling highly toxic organophosphorus compounds. Limits of detection are slightly below 100 micromol L(-1) on a 400 MHz spectrometer with 16 FIDs added for a single time frame. The method is not restricted to DFPase but can be used with other phosphotriesterases, for example paraxonase (PON), and even reactive chemicals, for example oximes and other nucleophiles, as long as the reaction components are compatible with the NMR experiment.

Effect of Brewer's yeast-induced pyrexia on aminophylline-elicited convulsions in mice.

Theophylline-associated convulsions have been observed most frequently in children with fever, but the mechanism is not fully understood. In this study, we investigated the basic mechanism of aminophylline [theophylline-2-ethylenediamine]-induced convulsions and the effects of Brewer's yeast-induced pyrexia in mice. Diazepam (5-10mg/kg, i.p.), a benzodiazepine receptor agonist, significantly prolonged the onset and significantly decreased the incidence of convulsions induced by aminophylline (350 mg/kg, i.p.). However, the gamma aminobutyric acid (GABA)A receptor agonist muscimol (1-4 mg/kg, i.p.), the GABAB receptor agonist baclofen (2-4 mg/kg, i.p.) and the N-methyl-D-aspartic acid receptor antagonist dizocilpine (0.1-0.3 mg/kg, i.p.) failed to protect against the convulsions. 20% Brewer's yeast (0.02 ml/g, s.c.) increased body temperature by 1.03, and also significantly shortened the onset and significantly increased the incidence of convulsions induced by aminophylline. The anticonvulsant action of diazepam (2.5-10mg/kg, i.p.) on the convulsions induced by aminophylline was reduced by Brewer's yeast-induced pyrexia. The proconvulsant actions of the GABAA receptor antagonists picrotoxin (3-4 mg/kg, i.p.) and pentylenetetrazol (40-60 mg/kg, i.p.) were enhanced by Brewer's yeast. These results suggest that the anticonvulsant action of diazepam against aminophylline is reduced by Brewer's yeast-induced pyrexia, and that GABAA receptors are involved in the aggravation of the convulsions by Brewer's yeast in mice.

[Model of Huntington's disease induced with 3-nitropropionic acid]. / Modelo de enfermedad de Huntington inducido con ácido 3-nitropropiónico.

INTRODUCTION: Huntington's disease is an autosomal dominant hereditary disorder. This neurodegenerative illness is characterized by mutation of the huntingtin protein gene, causing the formation of intracellular protein aggregates. DEVELOPMENT: Intensive research efforts have been made to investigate the molecular mechanism involved. For this reason, the development of animal and cellular models of Huntington's disease has offered alternative approaches to study of this disease. The alteration of succinate dehydrogenase activity has been linked to Huntington's disease. 3-nitropropionic acid is an inhibitor of this enzyme, prompting oxidative stress and death neuronal, mimic some aspects of Huntington's disease as anatomical, physiological and chemical changes. CONCLUSION: This model is a useful tool to study the mechanisms involved in this disease and to evaluate new therapeutic strategies.

Expression and possible role of creatine transporter in the brain and at the blood-cerebrospinal fluid barrier as a transporting protein of guanidinoacetate, an endogenous convulsant.

Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a K(m) value of 269 microM/412 microM which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that of d-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB.

Neuroprotective effect of Ro5-4864 following brain injury.

The 18 kDa translocator protein (TSPO) is a protein complex located at the outer mitochondrial membrane and interacting with the mitochondrial permeability transition pore (mPTP), indicating its involvement in the control of mPTP opening. We intended to explore the effect of TSPO ligands, PK 11195 and Ro5-4864 on apoptosis in a rat model of cortical injury. Sprague-Dawley rats received a daily intraperitoneal injection of dimethylsulfoxide (vehicle), PK 11195, or Ro5-4864, starting 2 days prior the injury and a third injection after the injury. At 6 weeks, immunohistochemistry analysis showed that Ro5-4864 resulted in a significant increase in the number of surviving neurons and in the density of the neurofilament network in the perilesional cortex in comparison with animals of the vehicle and PK 11195 groups. In tissue samples dissected from the injured area, Ro5-4864 caused a significant reduction in activation of caspases 3 and 9 but not of caspase 8 in comparison with the vehicle and PK 11195 groups. In addition, measurements of transmembrane mitochondrial potential of mitochondria (Deltapsi(M)) isolated from normal rat brain showed that loss of Deltapsi(M) induced by recombinant Bax could be significantly reduced by Ro5-4864 in a concentration-dependent manner. Our findings indicate that the neuroprotective effect shown by Ro5-4864 in the present model of brain injury involves the mitochondrial pathway of apoptosis modulation of mPTP.

mGlu5 receptor deletion does not confer seizure protection to mice.

Metabotropic glutamate mGlu5 receptors have been implicated in the regulation of seizures and have been suggested as a target against which discovery of novel anticonvulsants may be possible. However, the experimental literature is not consistent in reporting anticonvulsant efficacy of mGlu5 receptor antagonists. Additional assessment of this target was approached in the present study by comparing convulsions in wild-type (WT) and mGlu5 receptor null (knockout or KO) mice. Chemically induced seizures induced by a variety of mechanisms including pentylenetetrazole, N-methyl-d-aspartic acid (NMDA), cocaine, kainic acid, aminophylline, 4-aminopyridine, strychnine, and nicotine did not differentially increase clonic, clonic/tonic, or lethality in WT vs. mGlu5 receptor KO mice. The mGlu5 receptor antagonist 3-[(2-Methyl-1,3-thiazol-4-yl) ethynyl]-pyridine (MTEP) did not significantly prevent seizures induced by NMDA; in contrast, the uncompetitive NMDA receptor antagonist, dizocilpine, significantly prevented NMDA-induced seizures and lethality in both WT and KO mice. The present findings do not support the idea that mGlu5 receptors play as important a role in seizure control as previously speculated.

The pentylenetetrazole-induced activity in the hippocampus can be inhibited by the conversion of L-kynurenine to kynurenic acid: an in vitro study.

The kynurenine pathway converts tryptophan into various compounds, including L-kynurenine, which in turn can be converted into the excitatory amino acid receptor antagonist kynurenic acid. The ionotropic glutamate receptors have been considered to be attractive targets for new anticonvulsants in neurological disorders such as epileptic seizure. This study was designed to examine the conversion of L-kynurenine to kynurenic acid and to investigate the effects of kynurenic acid on pentylenetetrazole-treated rat brain slices, and in parallel to draw attention to the fact that a well-designed in vitro model has many advantages in pharmacological screening. Schaffer collateral stimulation-evoked field EPSPs were recorded from area CA1 of rat hippocampal slices in vitro; drugs were bath-applied. Pretreatment with the kynurenic acid precursor L-kynurenine led to the elimination of the effect of pentylenetetrazole on hippocampal slices in vitro. N-Omega-nitro-L-arginine, which inhibits kynurenine aminotransferase I and II, abolished this neuroprotective effect. This study has furnished the first in vitro electrophysiological evidence that rat brain slices have the enzymatic capacity to convert exogenously administered L-kynurenine (16 microM) to kynurenic acid in an amount sufficient to protect them against pentylenetetrazole (1 mM)-induced hyperexcitability.

Normalization of GABAA receptor specific binding in the substantia nigra reticulata and the prevention of L-dopa-induced dyskinesias in MPTP parkinsonian monkeys.

L-Dopa therapy in Parkinson's disease (PD) is counfounded by the development of involuntary movements such as L-Dopa-induced dyskinesias (LIDs). In this study GABA(A) receptor autoradiography was assessed using [(3)H]flunitrazepam binding to the benzodiazepine site of the GABA(A) receptor and [(35)S]t-butylbicyclophosphorothionate (TBPS) binding to the chloride channel of GABA(A) receptors in the substantia nigra reticulata (SNr) and subthalamic nucleus (STN). L-Dopa-treated parkinsonian monkeys experiencing LIDs were compared to animals in which LIDs was prevented by adjunct treatments with CI-1041, a selective antagonist of the NR1A/2B subtype of NMDA receptor, or low doses of the dopamine D2 receptor agonist, cabergoline. Our results demonstrated a decrease of GABA(A) receptor specific binding in the posterior part of the SNr in dyskinetic monkeys compared to nondyskinetic animals, while no modulation has been observed in the STN. These results provide evidence for the first time that pharmacological treatments preventing LIDs in nonhuman primate model of PD are associated with normalization of GABA(A) receptor-mediated signalling in the SNr.

Gender difference in the influence of antioxidants on the blood-brain barrier permeability during pentylenetetrazol-induced seizures in hyperthermic rat pups.

Our purpose in this study was to investigate the protective effects of selenium and vitamin E on the blood-brain barrier (BBB) permeability in rats with convulsion under hyperthermic conditions. To eliminate the effect of sex on BBB, we performed our study on 4- to 5-week-old prepubertal rat pups. Evans-blue was used as a BBB tracer. Convulsions were induced by administration of i.p. pentylenetetrazol. In the selenium group, 4 ppm selenium was added to the drinking water for 4-5 weeks. Vitamin E was administered at 700 mg/kg ip. It was shown that the convulsions, both under normothermic and hyperthermic conditions, caused widespread increase in the BBB permeability (p < 0.05). In addition, a significant difference was observed among female and male rats (f [1, 102] = 6.387, p < 0.05). In convulsions under normothermic conditions, there was a further increase in the BBB permeability (F[3, 102] = 43.534, p < 0.001) and a greater increase of permeability in males compared to females (F[1, 102] = 6.387, p < 0.05). Selenium and vitamin E significantly decreased the BBB destruction caused by convulsions under hyperthermic conditions in males (p < 0.05). Treatment with selenium or vitamin E has beneficial effects on the BBB breakdown during convulsions. But gender differences are very important in BBB permeability under pathological conditions and antioxidant treatments.

Differential expression of cerebellar metabotropic glutamate receptors mGLUR2/3 and mGLUR4a after the administration of a convulsant drug and the adenosine analogue cyclopentyladenosine.

Metabotropic glutamate receptors (mGluR) play a role in synaptic transmission, neuronal modulation and plasticity but their action in epileptic activity is still controversial. On the other hand adenosine acts as a neuromodulator with endogenous anticonvulsive properties. Since cerebellum from epileptic patients has shown neuronal damage, sometimes associated with Purkinje cells loss, we have explored the effect of repetitive seizures on two types of mGluR in the cerebellum. Seizures were induced by the convulsant drug 3-mercaptopropionic acid (MP) and the effect of the adenosine analogue cyclopentyladenosine (CPA) alone or before MP administration (CPA+MP) were also evaluated. The expression of the receptors subtypes 2/3 (mGluR2/3) and 4a (mGluR4a) was assessed by immunocitochemistry. Granular cell layer was labeled with mGluR2/3 antibody and increased immunoreactivity was observed after MP (60%), CPA (53%) and CPA + MP (85%) treatments. Control cerebellum slices showed mGluR4a reactivity around Purkinje cells, while MP, CPA and CPA+MP treatment decreased this immunostaining. Repetitive administration of MP and CPA induces an increased cerebellar mGluR2/3 and a decreased mGluR4a immunostaining, suggesting a distinct participation of both receptors that may be related to the type of cell involved. A protective action and /or an apoptotic effect may not be discarded. CPA repetitive administration although increase seizure latency, cannot prevent seizure activity.

Characterization of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain.

BACKGROUND AND PURPOSE: The aim of the present study was to determine whether binding of [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) to the convulsant binding site of GABA(A) receptors in human postmortem brain samples can be used as an in vitro index of the functional activation of these receptors. EXPERIMENTAL APPROACH: Postmortem stability of [(35)S]TBPS binding was assessed in rat brain samples harvested at various times after death and the binding properties of [(35)S]TBPS binding (K(D) and B(max)) were determined in human postmortem brain using radioligand binding studies. In addition, the ability of human brain [(35)S]TBPS binding to be allosterically modulated by compounds that bind at recognition sites distinct from the convulsant binding site was measured. KEY RESULTS: Whereas binding of [(3)H]Ro 15-1788 to the benzodiazepine binding site and [(3)H]muscimol to the agonist (GABA) binding site were retained over a 20 h postmortem interval, there was a significant decrease in the affinity and number of [(35)S]TBPS binding sites. Nevertheless, [(35)S]TBPS binding in human brain could be inhibited by TBPS, picrotoxin, loreclezole and pentobarbital and modulated by GABA with potencies comparable to those observed in rats. In addition, the GABA-induced reduction in human brain [(35)S]TBPS binding could be modulated by benzodiazepine site ligands in a manner that reflected their intrinsic efficacies. CONCLUSIONS AND IMPLICATIONS: These results suggest that allosteric coupling between the [(35)S]TBPS, GABA and benzodiazepine binding sites is preserved in postmortem human brain and that [(35)S]TBPS binding in this tissue may be used to study functional characteristics of native human GABA(A) receptors.

Mapping convulsants' binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites.

Gamma-aminobutyric acid (GABA) type A receptors play a key role in brain inhibitory neurotransmission, and are ligand-activated chloride channels blocked by numerous convulsant ligands. Here we summarize data on binding of picrotoxin, tetrazoles, beta-lactams, bicyclophosphates, butyrolactones and neurotoxic pesticides to GABA-A ionophore, and discuss functional and structural overlapping of their binding sites. The paper reviews data on convulsants' binding sensitivity to different point mutations in ionophore-lining second trans-membrane domains of GABA-A subunits, and maps possible location of convulsants' sites within the chloride ionophore. We also discuss data on inhibition of glycine, glutamate, serotonin (5-HT3) and N-acetylcholine receptors by GABA-A channel blockers, and examine the applicability of this model to other homologous ionotropic receptors. Positioning various convulsant-binding sites within ionophore of GABA-A receptors, this model enables a better understanding of complex architectonics of ionotropic receptors, and may be used for developing new channel-modulating drugs.