Rat GABAA Receptor Research Articles

Exploring the Orthosteric Binding Site of the γ-Aminobutyric Acid Type A Receptor Using 4-(Piperidin-4-yl)-1-hydroxypyrazoles 3- or 5-Imidazolyl Substituted: Design, Synthesis, and Pharmacological Evaluation

A series of 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP) 3- or 5-imidazolyl substituted analogues have been designed, synthesized, and characterized pharmacologically. All analogues showed binding affinities in the low micro- to low nanomolar range at native rat GABAA receptors and were found to be antagonists at the human α1β2γ2s receptor. The structure-activity relationship of the compound series demonstrates distinct differences in size and architecture of previously discovered cavities in the vicinity of the 4-PHP scaffold in the orthosteric binding site.

The effects of low-frequency electric stimulus on hippocampal of Effects of low-frequency electric stimulus on hippocampal of α5 subunit of extra synapse GABAA receptor in kainic acid-induced epilepsy rats

To explore the effects of low-frequency electric stimulus (LFS) on hippocampal of α5 subunit of extrasynaptic GABAA receptor in kainic acid-induced epilepsy rats and explore the possible mechanism of LFS on hippocampus to treat epilepsy. A total of 32 male Sprague-Dawley rats were divided randomly into 4 groups:epileptic, pseudo-surgery, LFS treatment and control. The expression of α5 subunit of extrasynaptic GABAA receptor and the association with the curative effects of LFS were evaluated by behavioristics, real-time fluorogenic quantitative polymerase chain reaction (PCR) and Western blot. After treatment, the number of seizures in LFS group were significantly lower than that in the epileptic and pseudo-surgery groups (2.88 ± 0.83 vs 8.50 ± 0.93 and 8.88 ± 0.83) (P < 0.01). The results of real-time PCR demonstrated that the expression of mRNA of α5 subunits in LFS treatment group was obviously higher than that in epileptic and pseudo-surgery groups (0.74 ± 0.20 vs 0.30 ± 0.16 and 0.31 ± 0.16) (P < 0.01), but was lower than 1.10 ± 0.23 in control group. Western blot demonstrated that the protein expression of α5 subunits in LFS (0.75 ± 0.09) and control (0.88 ± 0.09) groups were dramatically higher than that in epileptic (0.22 ± 0.08) and pseudo-surgery (0.26 ± 0.08) groups (P < 0.01). Low-frequency electric stimulus on hippocampus is effective in controlling epileptic seizures. And the mechanism may be related with the expression level of α5 subunits of extrasynaptic GABAA receptor.

The effects of scopolamine and the nootropic drug phenotropil on rat brain neurotransmitter receptors during testing of the conditioned passive avoidance task

We studied the effects of administration of the new nootropic drug phenotropil (N-carbamoylmethyl-4-phenyl-2-pyrrolidone) at a dose of 100 mg/kg on the quantitative characteristics of dopamine (DA), serotonin (5-HT), glutamate (NMDA), GABA-A (BDZ), and acetylcholine (nACh) receptors in rats using the conditioned passive avoidance task (PAT) under normal conditions and during scopolamine-induced amnesia ex vivo. We found that the cholinolytic drug scopolamine induced a substantial increase in the density (Bmax) of n-choline receptors in the cortex (by 99% as compared to the control) and NMDA receptors in the hippocampus (by 93%). A single administration of phenotropil (100mg/kg, intraperitoneally) abolished the effect of scopolamine and decreased the number of nACh and NMDA receptors by 46% and 14%, respectively. Phenotropil also abolished the effect of scopolamine on the benzodiazepine receptors and dopamine D1 receptors. Scopolamine decreased the density of D1 receptors by 20% and BDZ receptors by 17%, whereas phenotropil increased the density of receptors by 16% and 25%, respectively. Phenotropil considerably increased the density of dopamine D2 and D3 receptors by 29% and 62%, respectively. Scopolamine also increased the density of D3 receptors by 44% as compared to the control. We did not find any changes in the binding characteristics of 5-HT2 receptors during scopolamine-induced amnesia or during phenotropil treatment. These results demonstrate the role of these receptors in the development of scopolamine-induced amnesia and in neurochemical mechanisms of the anti-amnestic effects of phenotropil.

P03-109 - Involvement of the lateral septal nucleus and GABA a receptors in the antidepressant-like effects of allopregnanolone in wistar rats

IntroductionPharmacological and non-pharmacological antidepressant therapies increase the firing rate of lateral septal nucleus neurons (a brain structure involved in mood state regulation) and reduce immobility in the forced swim test (FST), whereas the neurosteroid allopregnanolone appears to produce antidepressant-like effects through actions at GABAA receptors.ObjectiveTo explore the participation of the lateral septal nucleus and GABAA receptors in the antidepressant-like effects of allopregnanolone in Wistar rats.MethodsFirst, the minimally effective dose of allopregnanolone that produces antidepressant-like effects in the FST was determined. Second, the effect of this minimally effective dose in the FST was evaluated on the firing rate of lateral septal neurons. Third, the antidepressant-like effects of microinjection of allopregnanolone (1.0 μg/rat) into the lateral septal nucleus was evaluated in the FST. Fourth, we explored whether the effects of allopregnanolone on the lateral septal neuron firing rate and FST are blocked by picrotoxin or bicuculline, two GABAA receptor antagonists.ResultsThe minimally effective dose of allopregnanolone with antidepressant-like effects in the FST was 1.0 mg/kg (i.p.) and significantly increased the firing rate of lateral septal neurons. Microinjection of allopregnanolone into the lateral septal nucleus produced antidepressant-like effects in the FST. Pretreatment with picrotoxin and bicuculline blocked the increase in lateral septal neuron firing rate and the antidepressant-like effects of allopregnanolone in the FST.ConclusionThe lateral septal nucleus participates in the antidepressant-like effects of allopregnanolone through actions on GABAA receptors in Wistar rats.

GABAA receptor density is altered by cannabinoid treatment in the hippocampus of adult but not adolescent rats

Cannabinoids are known to induce transient psychotic symptoms and cognitive dysfunction in healthy individuals and contribute to trigger schizophrenia in vulnerable individuals, particularly during adolescence. Converging preclinical evidence suggests important interactions between cannabinoid and GABAergic systems. In the present study, we compared the effects of cannabinoid treatment on GABAA receptor binding in the brain of adolescent and adult rats. Adolescent (5weeks old) and adult (10weeks old) rats were treated with the synthetic cannabinoid HU210 (25, 50 or 100μg/kg/day) or vehicle for 1, 4 or 14days. Rats were sacrificed 24h after the last injection and GABAA receptor density was measured in several brain regions using [35S]TBPS and in vitro autoradiography. Adolescent rats had higher numbers of GABAA receptors compared to adults. A 24% increase of binding in adult rats treated with 100μg/kg HU210 for 14days compared to controls was observed in the CA1 region of the hippocampus (16.1 versus 12.9fmol/mg tissue equivalent, t=2.720, p<0.05). HU210 did not affect GABAA receptors in adolescent rats in any treatment regimen and in adult rats treated with HU210 for 1 or 4days. These data suggest that long-term, high-dose treatment with HU210 increases GABAA receptors in the hippocampus of adult rats, changes that may interfere with associated hippocampal cognitive functions such as learning and memory. In addition, our results suggest that the adolescent brain does not display the same compensatory mechanisms that are activated in the adult brain following cannabinoid treatment.

Identification of a domain in the δ subunit (S238‐V264) of the α4β3δ GABAA receptor that confers high agonist sensitivity

We have expressed the alpha4beta3delta and alpha4beta3gamma2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the alpha4beta3delta receptor (EC50 approximately 1.4 micromol/L) than of the alpha4beta3gamma2L subtype (EC50 approximately 27.6 micromol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, imidazole-4-amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co-expressing chimeric delta/gamma2L subunits with alpha4 and beta3 subunits. A stretch of amino acids in the delta subunit, S238-V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the delta and gamma2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.

Characterization of multimeric complexes formed by the human PTB1 protein on RNA.

Polypyrimidine tract binding protein (PTB or hnRNP I) has several known functions in eukaryotic cells, including exon exclusion during alternative splicing events, mRNA stabilization, and regulation of viral translation and replication. PTB contains four RNA Binding Domains (RBDs, or RRMs), all of which can potentially bind RNA, but their roles in the various biological functions of PTB are not clear. We investigate the properties of the complexes formed by human PTB1 on two target RNAs: the rat GABAA receptor gamma2 subunit pre-mRNA and the Hepatitis C Virus 3' NonTranslated RNA. The GABA RNA contains four polypyrimidine tracts in the intron and exon, while the HCV NTR contains a 75-nt U-rich tract and a highly structured 3'-terminus. Electrophoretic mobility shift assays show that PTB1 protein first binds to both RNAs with nanomolar affinities, but subsequent protein addition leads to formation of higher-order complexes. Stoichiometry experiments show that the ultimate complexes contain up to eight PTB1 proteins per RNA strand. Protein constructs containing two tandem RBDs also bind the two RNAs, but with different affinities and stoichiometries. Nuclease protection assays show that PTB1 protects the polypyrimidine tracts in the GABA RNA, as does a construct consisting of RBD3 and RBD4; however, a construct containing RBD1 and RBD2 enhances cleavage of bound RNA. The binding mechanisms of PTB1 are unique to the full-length protein; these modes appear to include direct association with the RNA as well as weaker intermolecular protein associations.

Recent Progress in the Mechanism of Action of Insecticides: Pyrethroids, Fipronil and Indoxacarb

The mechanisms of action of pyrethroids, fipronil and indoxacarb on neuroreceptors and ion channels are summarized based primarily on our own studies. The major target site of pyrethroids is the sodium channel, and the toxicity is profoundly amplified from channel modulation to hyperactive symptoms in animals. For tetramethrin, only-1% of sodium channel population needs to be modified from brief opening to very prolonged opening to cause hyperactivity. Selective toxicity of pyrethroids between mammals and insects can be largely explained on the basis of differential sodium channel sensitivity to pyrethroids and negative temperature dependence of pyrethroid action. Fipronil blocks rat GABAA receptors, and the effect is exerted in both resting and active states of the receptors. It acts on GABA-receptor site different from the picrotoxinin binding site. Cockroach GABA receptors are more sensitive to fipronil blocking action than rat GABAA receptors. Indoxacarb and its metabolite DCJW have multiple actions on sodium channels, neuronal nicotinic acetylcholine receptors, and GABA receptors in both rat and cockroach. However, modulation of these receptors and channels differs considerably between mammals and insects.

Characterisation of inhibitory and excitatory postsynaptic currents of the rat medial superior olive.

The medial superior olive (MSO) is part of the binaural auditory pathway, receiving excitatory projections from both cochlear nuclei and an inhibitory input from the ipsilateral medial nucleus of the trapezoid body (MNTB). We characterised the excitatory and inhibitory synaptic currents of MSO neurones in 3- to 14-day-old rats using whole-cell patch-clamp methods in a brain slice preparation.A dual component EPSC was mediated by AMPA and NMDA receptors. The AMPA receptor-mediated EPSC decayed with a time constant of 1.99+/-0.16 ms (n = 8). Following blockade of glutamate receptors, a monosynaptic strychnine-sensitive response was evoked on stimulation of the MNTB, indicative of a glycine receptor-mediated IPSC. GABAA receptors contributed to IPSCs in rats under 6 days old (bicuculline blocked 30% of the IPSC). In older rats little or no bicuculline-sensitive component was detectable, except in the presence of flunitrazepam. These glycinergic IPSCs showed a reversal potential that varied with changes in [Cl-]i, as predicted by the Nernst equation. The IPSC exhibited two developmentally relevant changes. (i) At around postnatal day 6, the GABAA receptor-mediated component declined, leaving a predominant glycine-mediated IPSC. The isolated glycinergic IPSC decayed with time constants of 7.8+/-0.3 and 38.3+/-1.7 ms, with the slower component contributing 7.8+/-0.6% of the peak amplitude (n = 121, 3-11 days old, -70 mV, 25 deg C). (ii) After day 11 the IPSC fast decay accelerated to 3.9+/-0.3 ms (n = 12) and the magnitude of the slow component declined to less than 1%. Spontaneous miniature glycinergic IPSCs (mIPSCs) were variable in amplitude and were of large conductance (1.83+/-0.19 nS, n = 8). The amplitude was unchanged on lowering [Ca2+]o. The time course of evoked and spontaneous miniature glycinergic IPSCs were compared. The 10-90% rise times were 0.7 and 0.6 ms, respectively. The evoked IPSC decayed with a fast time constant of 7.2+/-0.7 ms, while the mIPSC decayed with a fast time constant of 5.3+/-0.4 ms in the same seven cells.The glycinergic IPSC decay was voltage dependent with an e-fold change over 118 mV. The temperature dependence of the IPSC decay indicated a Q10 value of 2. Picrotoxin and cyanotriphenylborate had little or no effect on IPSCs from 6- to 14-day-old animals, implying homomeric channels are rare. We conclude that the MSO receives excitatory inputs mediated by AMPA and NMDA receptors and a strong glycinergic IPSC which has a significant contribution from GABAA receptors in neonatal rats. Functionally, the IPSC could increase membrane conductance during the decay of binaural glutamatergic EPSCs, thus refining coincidence detection and interaural timing differences.

Selective Susceptibility to Inhibitors of GABA Synthesis and Antagonists of GABAA Receptor in Rats with Genetic Absence Epilepsy

Thalamocortical spike-and-wave discharges characterize the nonconvulsive absence seizures that occur spontaneously in genetic absence epilepsy rats from Strasbourg (GAERS), a selected strain of Wistar rats. GABA is crucial in the generation of absence seizures. The susceptibility to convulsions induced by threshold doses of various GABA receptor antagonists and inhibitors of GABA synthesis, kainic acid and strychnine, was compared in GAERS and in nonepileptic rats from a selected control strain (NE). The brain structures involved in the drug-elicited convulsive seizures were mapped by c-Fos immunohistochemistry. Injection of various antagonists of the GABAA receptor, bicuculline and picrotoxin, and inverse agonists of the benzodiazepine site (FG 7142 and DMCM) induced myoclonic spike-and-wave discharges followed by clonic or tonic–clonic seizures with high paroxysmal activity on the cortical EEG. The incidence of the convulsions was dose-dependent and was higher in GAERS than in NE rats. Mapping of c-Fos expression showed that the frontoparietal cortex was constantly involved in the convulsive seizures elicited by a threshold convulsant dose, whereas limbic participation was variable. In contrast, GAERS were less susceptible than NE rats to the tonic–clonic convulsions induced by the inhibitors of glutamate decarboxylase, isoniazide and 3-mercaptopropionic acid. The GABAB receptor antagonist CGP 56999 and kainic acid induced a similar incidence of seizures in GAERS and NE rats and predominantly activated the hippocampus. No difference in the tonic seizures elicited by strychnine could be evidenced between the strains. These results suggest that an abnormal cortical GABAergic activity may underlie absence seizures in GAERS.

Bicyclophosphorothionate antagonists exhibiting selectivity for housefly GABA receptors

2,6,7-Trioxa-1-phosphabicyclo[2.2.2]octane 1-sulfides (bicyclophosphorothionates) with various C1–4 alkyl groups at the 3- and 4-positions were synthesized and tested for their ability to compete with [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of γ-aminobutyric acid (GABA) receptors, for specific binding to rat-brain and housefly-head membranes, and for their insecticidal activity against houseflies. Among the 3,4-substituted analogues, 20 compounds were selectively active for housefly GABA receptors versus rat GABA receptors. The 3-alkyl groups of C3 length and the 4-alkyl groups of C4 length were tolerated in housefly receptors, whereas such bulky substituents were deleterious in rat receptors. The 4-isobutyl-3-isopropyl analogue was the most potent in housefly receptors (IC50 = 45.2 nM), and tert-butylbicyclophosphorothionate (TBPS), with the 4-tert-butyl group and no 3-substituent, was the most potent in rat receptors (IC50 = 62.2 nM). Their receptor selectivities (rat IC50/housefly IC50) were 52 and 0.038, respectively. The insecticidal activity (LD50) of 20 active analogues was well correlated with their potency (IC50) in inhibiting [3H]EBOB binding to housefly-head membranes (r = 0.93). The results obtained in the present study indicate that the introduction of appropriate alkyl groups into the 3- and 4-positions of bicyclophosphorothionate leads to non-competitive antagonists with increased affinity and selectivity for housefly ionotropic GABA receptors versus rat GABAA receptors. © 1999 Society of Chemical Industry

EDPC: a novel high affinity ligand for the benzodiazepine site on rat GABAA receptors

Rat recombinant α1β2γ2 γ-aminobutyric acid type A (GABAA) receptors were functionally expressed in Xenopus laevis oocytes and analyzed for the action of EDPC (Ethyl 3-(1,3-dithian-2-yl)-1H-pyrrolo[2,3–c]pyridine-5-carboxylate) using electrophysiological techniques. EDPC inhibited GABA currents at low concentrations (IC50≈2 nM). The inhibition by 100 nM EDPC could be reversed by 1 μM of the benzodiazepine antagonist flumazenil (Ro 15-1788), indicating a negative allosteric modulation via the benzodiazepine binding site. In line with this conclusion are radioactive ligand binding studies. EDPC inhibited the binding of 2 nM [3H]flunitrazepam to membranes from the cerebellum or the cortex with IC50 values of about 8 and 25 nM, respectively.

The Heteroyohimbine Mayumbine Binds with High Affinity to Rat Brain Benzodiazepine Receptors in vitro

Mayumbine, a naturally occurring heteroyohimbine, was isolated from Rauwolfia vomitoria extracts and shown to potently (IC50 = 76 ± 3.5 nM) inhibit the in vitro binding of 3H-diazepam to the benzodiazepine sites within the rat GABAA receptor complex.

Chronic diazepam exposure decreases transcription of the rat GABAA receptor γ2-subunit gene

The rate of transcription of the GABA(A) receptor gamma2-subunit gene in rat cortex has been measured using the nuclear run-off transcriptional assay. Exposure of rats to diazepam (15 mg/kg/day for 14 days) caused a significant reduction in the level of nascent GABA(A) receptor gamma2-subunit transcripts. Therefore, a component of the cellular response to chronic benzodiazepine exposure includes events which take place at the level of transcription of a GABA(A) receptor gene.

GABA and epileptogenesis.

GABA and epileptogenesis.

Development of Stable Cell Lines Expressing Different Subtypes of GabaA Receptors

The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABAA receptors. For this, we developed an original two step selection strategy, in which the first step consisted of transfecting HEK 293 cells with rat GABAA receptor α and β subunits. G 418 resistant colonies isolated at this step were screened for [3H] muscimol binding to select for those that coexpressed α- and β-subunits. The best α and β subunit expressing colony was then supertransfected with a plasmid coding for the γ rat GABAA receptor subunit and a mutant DHFR gene. After a second round of selection, this time in presence of methotrexate, those colonies that coexpressed ternary αβγ GABAA receptor combinations were distinguished using [3H] flumazenil as a probe. This strategy was applied to the isolation of 3 GABAA receptor clones, α1β2γ2S, α1β2γ2S and α1β2γ2S, that expressed relatively high levels of these proteins. These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations. These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABAA receptor subtypes.

Actions of picrodendrin antagonists on dieldrin-sensitive and -resistant Drosophila GABA receptors.

1. A series of terpenoid compounds, recently isolated from Picrodendron baccatum, share a picrotoxane skeleton with picrotoxinin, an antagonist of ionotropic GABA receptors. Referred to as picrodendrins, they inhibit the binding of [35S]-tert-butylbicyclophosphorothionate (TBPS) to rat GABAA receptors. Hitherto, their effects on GABA receptors have not been investigated electrophysiologically. Under two-electrode voltage-clamp, the actions of picrodendrins and related terpenoids have been assayed on homooligomeric GABA receptors formed by the expression of a Drosophila GABA receptor subunit (RDLac) in Xenopus oocytes. 2. All the terpenoids tested, dose-dependently antagonized currents induced by 30 microM (EC50) GABA. 3. Tutin and its analogues (dihydrotutin and isohyenanchin) differ in the structure of their axial C4 substituents. Of these compounds, tutin, which bears an isopropenyl group at this carbon atom, was the most potent antagonist of RDLac homo-oligomers, whereas isohyenanchin, which bears a hydroxyisopropyl group, was the least potent antagonist tested. 4. Picrodendrins differ mainly in the structure of their C9 substituents. The IC50s of picrodendrins ranged from 17 +/- 1.3 nM (picrodendrin-Q) to 1006 +/- 1.3 nM (picrodendrin-O). As such, the most potent picrodendrins (Q, A and B) were approximately equipotent with picrotoxinin as antagonists of RDLac homo-oligomers. 5. Certain picrodendrin compounds effected a use-dependent blockade of RDLac homo-oligomers. Such a biphasic block was not observed with tutin analogues. 6. Picrotoxin-resistant RDLacA3025 homo-oligomers, which have a single amino acid substitution (A302S) in the 2nd transmembrane region, were markedly less sensitive to picrodendrin-O than the wild-type, dieldrin-sensitive, homo-oligomers. 7. The relative potency of tutin analogues demonstrates that the structure-activity relationship of the C4 substituent of picrotoxane-based compounds is conserved in vertebrates and insects. However, the relative order of potency of picrodendrins on RDLac homo-oligomers is distinctly different from that observed in previous radioligand binding studies performed on vertebrate GABAA receptors. As picrodendrin compounds differ in the structure of their C9 substituents, these data suggest that the optimal convulsant pharmacophores of vertebrate GABAA receptors and RDLac homo-oligomers differ with respect to this substituent.

Bicuculline enhances the corticosterone secretion induced by lipopolysaccharide and interleukin-1 alpha in male rats.

Lipopolysaccharide (LPS)-induced inflammatory stress activates the hypothalamus-pituitary-adrenal (HPA) function. Interleukin-I (IL-1) is one of the key factors during this event; however, the mechanisms mediating IL-1 stimulation of HPA axis are still unclear. The present study evaluated the possible involvement of gamma-aminobutyric acid (GABA) in LPS-induced activation of HPA axis in adult male rats. In addition, the possible existence of diurnal changes of LPS-induced HPA axis activity was also investigated. Bicuculline (0.8 mg/kg BW), a GABA-A receptor antagonist and GABA (1 g/kg BW) were intraperitoneally (ip) injected 15 min before LPS (2 mg/kg BW, ip) or recombinant human IL-1 alpha (microgram/rat) administration in intact rats. Control animals received an equivalent volume of 0.9% saline. Rats were sacrificed at 60 min or 90 min after LPS, or IL-1 alpha or saline injection. Plasma corticosterone levels were measured by radioimmunoassay. Results showed that pretreatment with bicuculline enhanced both LPS- and IL-1-induced corticosterone secretion; while pretreatment with GABA significantly reduced the LPS-stimulated corticosterone release (p < 0.05, vs LPS alone). The effect is dependent on the time of sampling and such effect of bicuculline or GABA was not observed when rats were stimulated in the evening. In addition, the maximal changes of plasma corticosterone following LPS administration in the evening were significantly lower than in the morning (p < 0.01). The present study provides evidence that GABA is involved, at least in part, in the neuroendocrine regulation of LPS/interleukin-1a-induced corticosterone secretion via GABA-A receptor in rats. In addition, the response of plasma corticosterone to LPS has a diurnal variation, which corresponds to a diurnal change of GABAergic modulation of the immunoneuroendocrine response.

GABA antagonists differentiate between recombinant GABAA/benzodiazepine receptor subtypes

Seventeen rat GABAA receptor subtypes were transiently expressed in the human embryonic kidney 293 cell line from alpha 1, alpha 2, alpha 3, alpha 5, or alpha 6 variants with any of the three beta subunits and gamma 2S or gamma 3. We obtained fingerprints in the form of subtype characteristic concentration-response curves of 35S-TBPS binding to GABA and the GABAA antagonists SR 95531 and bicuculline. alpha 3 beta 3 gamma 2S/3 and alpha 5 beta 3 gamma 2S/3 containing receptors effectively recognized 35S-TBPS but not when beta 3 was replaced by the beta 1 or beta 2 subunit. This indicates a specific interaction of alpha and beta variants to form high-affinity 35S-TBPS binding sites. At low levels GABA allosterically increased 35S-TBPS binding to all receptors with the concentration and magnitude depending on the subunit combination. Exchange of the beta variant did not alter the concentration-response curves for alpha 1 and alpha 6 containing receptors but did so for alpha 2 containing receptors. alpha 2 beta 3 gamma 3 receptors displayed strong GABA-induced stimulation of 35S-TBPS binding, whereas binding to alpha 2 beta 3 gamma 3 receptors was marginally increased. SR 95531 and bicuculline decreased 35S-TBPS binding to all gamma 3 containing receptors. In addition, bicuculline was effective on alpha 1 beta x gamma 2 receptors. SR 95531 was threefold more potent than bicuculline in reversing GABA-induced modulation of 35S-TBPS binding in most receptor types, but was 30-fold more potent on alpha 2 beta 1 gamma 3 and alpha 6 beta 1 gamma 2S receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of U-97775 as a GABAA receptor ligand of dual functionality in cloned rat GABAA receptor subtypes.

1. U-97775 (tert-butyl 7-chloro-4,5-dihydro-5-[(1-(3,4,5-trimethyl)piperazino)carbonyl]- imidazo[1,5-a])quinoxaline-3-carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells. 2. The drug produced a bell-shaped dose-response profile in the alpha 1 beta 2 gamma 2 receptor subtype as monitored with GABA-induced Cl- currents in the whole cell patch-clamp technique. At low concentrations (< 0.5 microM), U-97775 enhanced the currents with a maximal increase of 120% as normalized to 5 microM GABA response (control). An agonist interaction of U-97775 with the benzodiazepine site is suggested, because Ro 15-1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]-flunitrazepam binding was inhibited by U-97775 with a Ki of 1.2 nM. 3. The enhancement of GABA currents progressively disappeared as the U-97775 concentration was raised above 1 microM, and the current amplitude was reduced to 40% below the control at 10 microM U-97775. The current inhibition by U-97775 (10 microM) was not affected by Ro 15-1788. It appears that U-97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents. 4. U-97775 at low concentrations reduced and at high concentrations enhanced [35S]-TBPS binding. Ro 15-1788 selectively blocked the effect of U-97775 at low concentrations. Analysis of the binding data in the presence of Ro 15-1788 yielded a single low affinity site with an estimated Kd of 407 nM.5. In other alpha beta upsilon receptor subtypes, U-97775 at low concentrations enhanced Cl- currents in the alpha 3 beta 2 upsilon 2,but not in the alpha 6 beta 2 upsilon 2 subtype. On the other hand, U-97775 at high concentrations reduced Cl- currents in all the receptor subtypes we examined, including those of two subunits, alpha 1 beta 2, beta 2 upsilon 2 and alpha 1 upsilon 2 subtypes.6. Therapeutically, U-97775 could be unique among benzodiazepine ligands because of its ability to limit its own agonistic activity such that, at high doses the appearance of agonistic activity would be delayed until occupancy of its second site wanes. This property should make the total agonistic activity of U-97775 relatively constant over a wide range of drug doses, and may minimize its liability to abuse.