Native GABA Research Articles

Effects of monoterpenoid insecticides on [ 3H]-TBOB binding in house fly GABA receptor and 36Cl − uptake in American cockroach ventral nerve cord

Monoterpenoids and their derivatives from plant essential oils showed good insecticidal activities in previous studies, but the mechanisms of their action as natural insecticides are not known yet. In the present work, we evaluated the pharmacological action of five monoterpenoids (α-terpineol, carvacrol, linalool, pulegone, and thymol) on native insect GABA receptors from house flies and American cockroaches using radiotracer methods. In the [ 3H]-TBOB binding assay, carvacrol, pulegone, and thymol all enhanced the [ 3H]-TBOB binding to membrane preparation of house fly heads with EC 50 values of 48 μM, 432 μM, and 6 mM, respectively. Moreover, these three monoterpenoids at concentrations of 500 μM and 1 mM also significantly increased the 36Cl − uptake induced by GABA in membrane microsacs prepared from American cockroach ventral nerve cords. These results revealed that carvacrol, pulegone, and thymol are all positive allosteric modulators at insect GABA receptors. The other two monoterpenoids that were tested, α-terpineol and linalool, showed little or no effect in both the [ 3H]-TBOB binding and 36Cl − uptake assays.

Novel 4-(Piperidin-4-yl)-1-hydroxypyrazoles as γ-Aminobutyric AcidA Receptor Ligands: Synthesis, Pharmacology, and Structure−Activity Relationships

A series of substituted 1-hydroxypyrazole analogues of the GABA(A) receptor partial agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL) have been synthesized and pharmacologically characterized. Several of the analogues displayed K(i) in the low nanomolar range at the native GABA(A) receptors and potent antagonism of the alpha(1)beta(2)gamma(2) receptor. It appears that several regions situated in proximity to the core of the orthosteric binding site of the GABA(A) receptor are able to accommodate large hydrophobic substituents.

Inhibitory effects of amiloride on the current mediated by native GABAA receptors in cultured neurons of rat inferior colliculus

1. The diuretic amiloride is known to modulate the activity of several types of ion channels and membrane receptors in addition to its inhibitory effects on many ion transport systems. However, the effects of amiloride on some important ion channels and receptors, such as GABA(A) receptors, in the central nervous system have not been characterized. 2. In the present study, we investigated the functional action of amiloride on native GABA(A) receptors in cultured neurons of rat inferior colliculus using whole-cell patch-clamp recordings. 3. Amiloride reversibly inhibited the amplitude of the GABA-induced current (I(GABA)) in a concentration-dependent manner (IC(50) 454 +/- 24 micromol/L) under conditions of voltage-clamp with a holding potential at -60 mV. The inhibition depended on drug application mode and was independent of membrane potential. Amiloride did not change the reversal potential of I(GABA). Moreover, amiloride induced a parallel right-ward shift in the concentration-response curve for I(GABA) without altering the maximal value and Hill coefficient. 4. The present study shows that amiloride competitively inhibits the current mediated by native GABA(A) receptors in the brain region, probably via a direct action on GABA-binding sites on the receptor. The findings suggest that the functional actions of amiloride on GABA(A) receptors may result in possible side-effects on the central nervous system in the case of direct application of this drug into the cerebrospinal fluid for treatment of diseases such as brain tumours.

New 1,5-benzodiazepine compounds: activity at native GABA A receptors

Various new 1,5-benzodiazepine compounds were synthesized and tested for their biological activity in terms of effects on GABA A receptors of rat cerebellar granules in culture. Their effects were compared to those of a 1,4-benzodiazepine agonist, flunitrazepam and the already known 1,5-benzodiazepine antiepileptic clobazam. The effects were evaluated for the two different GABA A receptor populations present in these neurons, one mediating phasic inhibition and the other one mediating tonic inhibition. Many such compounds display a profile of inverse agonist to both GABA A receptor populations. One of them presents a profile of full agonist at the component mediating phasic inhibition. Interestingly, substitution of just one oxygen atom in that compound with sulphur in a specific position of a morpholine ring resulted in a remarkable change of activity from full agonist to a probable inverse agonist. This indicates such a position as a proton accepting one for the ligand within the benzodiazepine binding pocket of the relevant GABA A receptors. In addition, that position appears to be critical for the pharmacological activity.

GABA A receptor subtype selectivity underlying anxiolytic effect of 6-hydroxyflavone

6-Hydroxyflavone (6HF), a naturally occurring flavonoid, was previously reported to bind to type A γ-aminobutyric acid (GABA A) receptors benzodiazepine (BZ) site with moderate binding affinity. In the present study, we showed that 6HF partially potentiated GABA-induced currents in native GABA A receptors expressed in cortical neurons via BZ site, as the enhancement was blocked by the antagonist flumazenil. Furthermore, in patch clamp studies, 6HF displayed significant preference for α 2- and α 3-containing subtypes, which were thought to mediate anxiolytic effect, compared to α 1- and α 5-containing subtypes expressed in HEK 293T cells. In mice, 6HF exhibited anxiolytic-like effect in the elevated plus-maze test, unaccompanied at anxiolytic doses by the sedative, cognitive impairing, myorelaxant, motor incoordination and anticonvulsant effects commonly associated with classical BZs when tested in the hole-board, step-through passive avoidance, horizontal wire, rotarod, and pentylenetetrazol (PTZ)-induced seizure tests, respectively. The findings therefore identified 6HF as a promising drug candidate for the treatment of anxiety-like disorders.

Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone

The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D(f)) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D(f) motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.

The role of GABA and glutamate receptors in susceptibility and resistance to chloride channel blocker insecticides

Despite a point mutation in the pore-forming segment of the Rdl GABA receptor subunit that is widespread and persistent in insect populations and confers high levels of resistance to dieldrin and other polychlorocycloalkane (PCCA) insecticides, the phenylpyrazole insecticide fipronil, which binds at same site, has proven to be effective in controlling many insects, including dieldrin-resistant populations. Fipronil and its major sulfone metabolite are unique among chloride channel blocking insecticides in that they also potently block GluCls. We present here a patch clamp study of the action of fipronil sulfone on native GABA receptors and GluCl receptors from susceptible and dieldrin-resistant German cockroaches, to provide a better understanding of the effect of the Rdl mutation on the function and insecticide sensitivity of these two targets, and its role in resistance. Dieldrin blocked GABA currents with an IC 50 of 3 nM in wild-type cockroaches, and 383 nM in resistant insects, yielding a resistance ratio of 128. Fipronil sulfone blocked GABA currents with an IC 50 of 0.8 nM in susceptible insects and 12.1 nM, or 15-fold higher, in resistant insects. While both GluClD (desensitizing) and GluClN (non-desensitizing) receptors were found in German cockroach neurons, GluClN receptors were rare and could not be included in this study. GluClD receptors from resistant insects had reduced sensitivity to glutamate and a lower rate of desensitization than those from susceptible insects, but their sensitivity to block by fipronil sulfone was not significantly changed, with an IC 50 of 38.5 ± 2.4 nM ( n = 8) in the susceptible strain and 40.3 ± 1.0 nM ( n = 7) in the resistant strain. Fipronil sulfone also slowed the decay time course of GluClD currents. These results suggest that GluClD receptors contain the Rdl subunit, but their sensitivity to fipronil sulfone is not altered in resistant insects.

(R)-(3-Amino-2-fluoropropyl) Phosphinic Acid (AZD3355), a Novel GABAB Receptor Agonist, Inhibits Transient Lower Esophageal Sphincter Relaxation through a Peripheral Mode of Action

Gastroesophageal reflux disease (GERD) affects >10% of the Western population. Conventionally, GERD is treated by reducing gastric acid secretion, which is effective in most patients but inadequate in a significant minority. We describe a new therapeutic approach for GERD, based on inhibition of transient lower esophageal sphincter relaxation (TLESR) with a proposed peripherally acting GABA(B) receptor agonist, (R)-(3-amino-2-fluoropropyl)phosphinic acid (AZD3355). AZD3355 potently stimulated recombinant human GABA(B) receptors and inhibited TLESR in dogs, with a biphasic dose-response curve. In mice, AZD3355 produced considerably less central side effects than the prototypical GABA(B) receptor agonist baclofen but evoked hypothermia at very high doses (blocked by a GABA(B) receptor antagonist and absent in GABA(B)-/- mice). AZD3355 and baclofen differed markedly in their distribution in rat brain; AZD3355, but not baclofen, was concentrated in circumventricular organs as a result of active uptake (shown by avid intracellular sequestration) and related to binding of AZD3355 to native GABA transporters in rat cerebrocortical membranes. AZD3355 was also shown to be transported by all four recombinant human GABA transporters. AR-H061719 [(R/S)-(3-amino-2-fluoropropyl)phosphinic acid], (the racemate of AZD3355) inhibited the response of ferret mechanoreceptors to gastric distension, further supporting its peripheral site of action on TLESR. In summary, AZD3355 probably inhibits TLESR through stimulation of peripheral GABA(B) receptors and may offer a potential new approach to treatment of GERD.

Brain regional distribution of GABA A receptors exhibiting atypical GABA agonism: Roles of receptor subunits

The major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), has only partial efficacy at certain subtypes of GABA A receptors. To characterize these minor receptor populations in rat and mouse brains, we used autoradiographic imaging of t-butylbicyclophosphoro[ 35S]thionate ([ 35S]TBPS) binding to GABA A receptors in brain sections and compared the displacing capacities of 10 mM GABA and 1 mM 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a competitive GABA-site agonist. Brains from GABA A receptor α1, α4, δ, and α4 + δ subunit knockout (KO) mouse lines were used to understand the contribution of these particular receptor subunits to “GABA-insensitive” (GIS) [ 35S]TBPS binding. THIP displaced more [ 35S]TBPS binding than GABA in several brain regions, indicating that THIP also inhibited GIS-binding. In these regions, GABA prevented the effect of THIP on GIS-binding. GIS-binding was increased in the cerebellar granule cell layer of δ KO and α4 + δ KO mice, being only slightly diminished in that of α1 KO mice. In the thalamus and some other forebrain regions of wild-type mice, a significant amount of GIS-binding was detected. This GIS-binding was higher in α4 KO mice. However, it was fully abolished in α1 KO mice, indicating that the α1 subunit was obligatory for the GIS-binding in the forebrain. Our results suggest that native GABA A receptors in brain sections showing reduced displacing capacity of [ 35S]TBPS binding by GABA (partial agonism) minimally require the assembly of α1 and β subunits in the forebrain and of α6 and β subunits in the cerebellar granule cell layer. These receptors may function as extrasynaptic GABA A receptors.

Expression of GABA A receptor α3-, θ-, and ε-subunit mRNAs during rat CNS development and immunolocalization of the ε subunit in developing postnatal spinal cord

Ionotropic GABA A receptors are heteromeric structures composed of a combination of five from at least 16 different subunits. Subunit genes are expressed in distinct cell types at specific times during development. The most abundant native GABA A receptors consist of α1-, β2-, and γ2-subunits that are co-expressed in numerous brain areas. α3-, θ-, And ε-subunits are clustered on the X chromosome and show striking overlapping expression patterns throughout the adult rat brain. To establish whether these subunits are temporally and spatially co-expressed, we used in situ hybridization to analyze their expression throughout rat development from embryonic stage E14 to postnatal stage P12. Each transcript exhibited a unique or a shared regional and temporal developmental expression profile. The thalamic expression pattern evolved from a restricted expression of ε and θ transcripts before birth, to a θ and α3 expression at birth, and finally to a grouped ε, θ and α3 expression postpartum. However, strong similarities occurred, such as a grouped expression of the three subunits within the hypothalamus, tegmentum and pontine nuclei throughout the developmental process. At early stages of development (E17), ε and θ appeared to have a greater spatial distribution before the dominance of the α3 subunit transcript around birth. We also revealed expression of α3, θ, and ε in the developing spinal cord and identified neurons that express ε in the postnatal dorsal horn, intermediolateral column and motoneurons. Our findings suggest that various combinations of α3-, θ- and ε-subunits may be assembled at a regional and developmental level in the brain.

Differential Drug Responses on Native GABAA Receptors Revealing Heterogeneity in Extrasynaptic Populations in Cultured Hippocampal Neurons

Hippocampal pyramidal neurons potentially express multiple subtypes of GABA(A) receptors at extrasynaptic locations that could therefore respond to different drugs. We activated extrasynaptic GABA(A) receptors in cultured rat hippocampal pyramidal neurons and measured single-channel currents in order to compare the actions of two drugs that potentially target different GABA(A) receptor subtypes. Despite the possible difference in receptor targets of etomidate and diazepam, the two drugs were similar in their actions on native extrasynaptic GABA(A) receptors. Each drug produced three distinct responses that differed significantly in current magnitude, implying heterogeneous GABA(A) receptor populations. In the majority of patches, drug application increased both the single-channel conductance (>40 pS) and the open probability of the channels. By contrast, in the minority of patches, drug application caused an increase in open probability only. In the third group high-conductance channels were observed upon GABA activation and drug application increased their open probability only. The currents potentiated by etomidate or diazepam were substantially larger in patches displaying high-conductance GABA channels compared to those displaying only low-conductance channels. Factors contributing to the large magnitude of these currents were the long mean open time of high-conductance channels and the presence of multiple channels in these patches. In conclusion, we suggest that the local density of extrasynaptic GABA(A) receptors may influence their single-channel properties and may be an additional regulating factor for tonic inhibition and, importantly, differential drug modulation.

Novel, Potent, and Selective GABACAntagonists Inhibit Myopia Development and Facilitate Learning and Memory

This study reports pharmacological and physiological effects of cis- and trans-(3-aminocyclopentanyl)butylphosphinic acid (cis- and trans-3-ACPBPA). These compounds are conformationally restricted analogs of the orally active GABA(B/C) receptor antagonist (3-aminopropyl)-n-butylphosphinic acid (CGP36742 or SGS742). cis-[IC(50)(rho1) = 5.06 microM and IC(50)(rho2) = 11.08 microM; n = 4] and trans-3-ACPMPA [IC(50)(rho1) = 72.58 microM and IC(50)(rho2) = 189.7 microM; n = 4] seem competitive at GABA(C) receptors expressed in Xenopus laevis oocytes, having no effect as agonists (1 mM) but exerting weak antagonist (1 mM) effects on human GABA(A) and GABA(B) receptors. cis-3-ACPBPA was more potent and selective than the trans-compound, being more than 100 times more potent at GABA(C) than GABA(A) or GABA(B) receptors. cis-3-ACPBPA was further evaluated on dissociated rat retinal bipolar cells and dose-dependently inhibited the native GABA(C) receptor (IC(50) = 47 +/- 4.5 microM; n = 6). When applied to the eye as intravitreal injections, cis- and trans-3-ACPBPA prevented experimental myopia development and inhibited the associated vitreous chamber elongation, in a dose-dependent manner in the chick model. Doses only 10 times greater than required to inhibit recombinant GABA(C) receptors caused the antimyopia effects. Using intraperitoneal administration, cis- (30 mg/kg) and trans-3-ACPBPA (100 mg/kg) enhanced learning and memory in male Wistar rats; compared with vehicle there was a significant reduction in time for rats to find the platform in the Morris water maze task (p < 0.05; n = 10). As the physiological effects of cis- and trans-3-ACPBPA are similar to those reported for CGP36742, the memory and refractive effects of CGP36742 may be due in part to its GABA(C) activity.

International Union of Pharmacology. LXX. Subtypes of γ-Aminobutyric AcidAReceptors: Classification on the Basis of Subunit Composition, Pharmacology, and Function. Update

In this review we attempt to summarize experimental evidence on the existence of defined native GABA(A) receptor subtypes and to produce a list of receptors that actually seem to exist according to current knowledge. This will serve to update the most recent classification of GABA(A) receptors (Pharmacol Rev 50:291-313, 1998) approved by the Nomenclature Committee of the International Union of Pharmacology. GABA(A) receptors are chloride channels that mediate the major form of fast inhibitory neurotransmission in the central nervous system. They are members of the Cys-loop pentameric ligand-gated ion channel (LGIC) superfamily and share structural and functional homology with other members of that family. GABA(A) receptors are assembled from a family of 19 homologous subunit gene products and form numerous, mostly hetero-oligomeric, pentamers. Such receptor subtypes with properties that depend on subunit composition vary in topography and ontogeny, in cellular and subcellular localization, in their role in brain circuits and behaviors, in their mechanisms of regulation, and in their pharmacology. We propose several criteria, which can be applied to all the members of the LGIC superfamily, for including a receptor subtype on a list of native hetero-oligomeric subtypes. With these criteria, we develop a working GABA(A) receptor list, which currently includes 26 members, but will undoubtedly be modified and grow as information expands. The list is divided into three categories of native receptor subtypes: "identified," "existence with high probability," and "tentative."

Redox modulation of homomeric ρ1 GABAC receptors

The activity of many receptors and ion channels in the nervous system can be regulated by redox-dependent mechanisms. Native and recombinant GABA(A) receptors are modulated by endogenous and pharmacological redox agents. However, the sensitivity of GABA(C) receptors to redox modulation has not been demonstrated. We studied the actions of different reducing and oxidizing agents on human homomeric GABArho(1) receptors expressed in Xenopus laevis oocytes. The reducing agents dithiothreitol (2 mM) and N-acetyl-L-cysteine (1 mM) potentiated GABA-evoked Cl(-) currents recorded by two-electrode voltage-clamp, while the oxidants 5-5'-dithiobis-2-nitrobenzoic acid (500 microM) and oxidized dithiothreitol (2 mM) caused inhibition. The endogenous antioxidant glutathione (5 mM) also enhanced GABArho(1) receptor-mediated currents while its oxidized form GSSG (3 mM) had inhibitory effects. All the effects were rapid and easily reversible. Redox modulation of GABArho(1) receptors was strongly dependent on the GABA concentration; dose-response curves for GABA were shifted to the left in the presence of reducing agents, whereas oxidizing agents produced the opposite effect, without changes in the maximal response to GABA and in the Hill coefficient. Our results demonstrate that, similarly to GABA(A) receptors and other members of the cys-loop receptor superfamily, GABA(C) receptors are subjected to redox modulation.

The Impact of Sub-Cellular Location and Intracellular Neuronal Proteins on Properties of GABAA Receptors

Most studies of GABA(A) receptor accessory proteins have focused on trafficking, clustering and phosphorylation state of the channel-forming subunits and as a result a number of proteins and mechanisms have been identified that can influence the GABA(A) channel expression and function in the cell plasma membrane. In the light of a growing list of intracellular and transmembrane neuronal proteins shown to affect the fate, function and pharmacology of the GABA(A) receptors in neurons, the concept of what constitutes the native GABA(A) receptor complex may need to be re-examined. It is perhaps more appropriate to consider the associated proteins or some of them to be parts of the receptor channel complex in the capacity of ancillary proteins. Here we highlight some of the effects the intracellular environment has on the GABA-activated channel function and pharmacology. The studies demonstrate the need for co-expression of accessory proteins with the GABA(A) channel-forming subunits in heterologous expression systems in order to obtain the full repertoire of GABA(A) receptors characteristics recorded in the native neuronal environment. Further studies e.g. on gene-modified animal models are needed for most of the accessory proteins to establish their significance in normal physiology and in pathophysiology of neurological and psychiatric diseases. The challenge remains to elucidate the effects that the accessory proteins and processes (e.g. phosphorylation) plus the sub-cellular location have on the "fine-tuning" of the functional and pharmacological properties of the GABA(A) receptor channels.

6,2′-Dihydroxyflavone, a subtype-selective partial inverse agonist of GABA A receptor benzodiazepine site

Neuroactivity of a number of flavonoids is mediated by modulation of type A γ-aminobutyric acid (GABA A) receptor function via benzodiazepine sites, mostly as partial agonists. In the present study, 6,2′-dihydroxyflavone (DHF) was characterized for potential inverse agonistic activity, and its mechanism of action was explored for receptor subtype selectivity. In whole-cell patch clamp studies on neuroblastoma IMR-32 cells expressing native GABA A receptors, DHF decreased GABA-induced currents, to an extent similar to that induced by the partial inverse agonist FG-7142, which could be blocked by flumazenil, a BZ site antagonist. In mouse behavioral models, DHF elicited significant anxiogenic-like effects in the elevated plus-maze test, and enhanced cognitive performance in the step-through passive avoidance test, as expected for an inverse agonist. However, DHF did not exhibit any proconvulsant effects, a typical action of inverse agonists. In electrophysiological studies on subtypes of recombinant GABA A receptors expressed in HEK 293T cells, DHF decreased GABA-induced currents in α 1β 3γ 2, α 2β 3γ 2, or α 5β 3γ 2, but not α 3β 3γ 2 receptors. The results demonstrated DHF as a partial inverse agonist-like modulator of GABA A receptors with selectivity in receptor subtypes as well as behavioral effects. The DHF subtype-selectivity suggested that α 3-containing subtypes could be a mediator of the convulsion activities of GABA A receptor inverse agonists. Moreover, the pharmacological profile displayed in mouse behavioral models supported DHF as a useful lead compound for the development of cognition-enhancing agents devoid of convulsion side effects.

An intracellular motif of P2X3 receptors is required for functional cross‐talk with GABAA receptors in nociceptive DRG neurons

Functional cross-talk between structurally unrelated P2X ATP receptors and members of the 'cys-loop' receptor-channel superfamily represents a recently-discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross-talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross-inhibition between P2X subtypes and GABA(A) receptors, suggesting a P2X subunit-dependent mechanism. We show here that cross-inhibition between neuronal P2X(3) or P2X(2+3) and GABA(A) receptors does not depend on chloride and calcium ions. We identified an intracellular QST(386-388) motif in P2X(3) subunits which is required for the functional coupling with GABA(A) receptors. Moreover the cross-inhibition between native P2X(3) and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABA(A)beta3 subunits. We provide evidence that P2X(3) and GABA(A) receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X(3)-GABA(A) subunit interactions underlie a pre-synaptic cross-talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.

Desensitization and binding properties determine distinct α1β2γ2 and α3β2γ2 GABAA receptor‐channel kinetic behavior

GABAA receptor subtypes comprising the α1 and α3 subunits change with development and have a specific anatomical localization in the adult brain. These receptor subtypes have been previously demonstrated to greatly differ in deactivation kinetics but the underlying gating mechanisms have not been fully elucidated. Therefore, we expressed rat α1β2γ2 and α3β2γ2 receptors in human embryonic kidney 293 cells and recorded current responses to ultrafast GABA applications at macroscopic and single-channel levels. We found that the slow deactivation of α3β2γ2-mediated currents is associated with a relatively small rate and extent of apparent desensitization. In contrast, responses mediated by α1β2γ2 receptors had faster deactivation and stronger desensitization. α3β2γ2 receptors had faster recovery in the paired-pulse agonist applications than α1β2γ2 channels. The onset of currents mediated by α3β2γ2 receptors was slower than that of α1β2γ2 for a wide range of GABA concentrations. Single-channel analysis did not reveal differences in the opening/closing kinetics of α1β2γ2 and α3β2γ2 channels but burst durations were longer in α3β2γ2 receptors. Simulation with a previously reported kinetic model was used to explore the differences in respective rate constants. Reproduction of major kinetic differences required a smaller desensitization rate as well as smaller binding and unbinding rates in α3β2γ2 compared with α1β2γ2 receptors. Our work describes the mechanisms underlying the kinetic differences between two major GABAA receptor subtypes and provides a framework to interpret data from native GABA receptors.

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

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. 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. 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. 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.

Interleukin-1β Enhances GABAA Receptor Cell-surface Expression by a Phosphatidylinositol 3-Kinase/Akt Pathway: RELEVANCE TO SEPSIS-ASSOCIATED ENCEPHALOPATHY

Sepsis-associated encephalopathy (SAE) is a frequent but poorly understood neurological complication in sepsis that negatively influences survival. Here we present clinical and experimental evidence that this brain dysfunction may be related to altered neurotransmission produced by inflammatory mediators. Compared with septic patients, SAE patients had higher interleukin-1beta (IL-1beta) plasma levels; interestingly, these levels decreased once the encephalopathy was resolved. A putative IL-1beta effect on type A gamma-aminobutyric acid receptors (GABA(A)Rs), which mediate fast synaptic transmission in most cerebral inhibitory synapses in mammals, was investigated in cultured hippocampal neurons and in Xenopus oocytes expressing native or foreign rat brain GABA(A)Rs, respectively. Confocal images in both cell types revealed that IL-1beta increases recruitment of GABA(A)Rs to the cell surface. Moreover, brief applications of IL-1beta to voltage-clamped oocytes yielded a delayed potentiation of the GABA-elicited chloride currents (I(GABA)); this effect was suppressed by IL-1ra, the natural IL-1 receptor (IL-1RI) antagonist. Western blot analysis combined with I(GABA) recording and confocal images of GABA(A) Rs in oocytes showed that IL-1beta stimulates the IL-1RI-dependent phosphatidylinositol 3-kinase activation and the consequent facilitation of phospho-Akt-mediated insertion of GABA(A)Rs into the cell surface. The interruption of this signaling pathway by specific phosphatidylinositol 3-kinase or Akt inhibitors suppresses the cytokine-mediated effects on GABA(A)R, whereas activation of the conditionally active form of Akt1 (myr-Akt1.ER*) with 4-hydroxytamoxifen reproduces the effects. These findings point to a previously unrecognized signaling pathway that connects IL-1beta with increased "GABAergic tone." We propose that through this mechanism IL-1beta might alter synaptic strength at central GABAergic synapses and so contribute to the cognitive dysfunction observed in SAE.