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The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABA A receptors (GABA A -Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to “tonic” inhibition that can control levels of excitability and network activity. The GABA A -R α4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABA A -R-mediated tonic inhibition. Mice were engineered to lack the α4 subunit by targeted disruption of the Gabra4 gene. α4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABA A -Rs containing the α4 subunit and that gaboxadol achieves its effects via the activation of this GABA A -R subtype.

Although it is now more than two decades since it was first reported that the imidazobenzodiazepine Ro15-4513 reverses behavioral alcohol effects, the molecular target(s) of Ro15-4513 and the mechanism of alcohol antagonism remain elusive. Here, we show that Ro15-4513 blocks the alcohol enhancement on recombinant “extrasynaptic” α4/6β3δ GABA A receptors at doses that do not reduce the GABA-induced Cl − current. At low ethanol concentrations (≤30 mM), the Ro15-4513 antagonism is complete. However, at higher ethanol concentrations (≥100 mM), there is a Ro15-4513-insensitive ethanol enhancement that is abolished in receptors containing a point mutation in the second transmembrane region of the β3 subunit (β3N265M). Therefore, α4/6β3δ GABA receptors have two distinct alcohol modulation sites: ( i ) a low-dose ethanol site present in α4/6β3δ receptors that is antagonized by the behavioral alcohol antagonist Ro15-4513 and ( ii ) a site activated at high (anesthetic) alcohol doses, defined by mutations in membrane-spanning regions. Receptors composed of α4β3N265Mδ subunits that lack the high-dose alcohol site show a saturable ethanol dose-response curve with a half-maximal enhancement at 16 mM, close to the legal blood alcohol driving limit in most U.S. states (17.4 mM). Like in behavioral experiments, the alcohol antagonist effect of Ro15-4513 on recombinant α4β3δ receptors is blocked by flumazenil and β-carboline-ethyl ester (β-CCE). Our findings suggest that ethanol/Ro15-4513-sensitive GABA A receptors are important mediators of behavioral alcohol effects.

GABAAreceptors (GABARs) are heteromultimeric proteins composed of five subunits. The specific subunit composition determines critical properties of a GABAR such as pharmacological sensitivities and whether the receptor contributes to synaptic or extrasynaptic forms of inhibition. Classically, synaptic but not extrasynaptic GABARs are thought to respond to benzodiazepines, whereas the reverse has been suggested for ethanol. To examine the effects of subunit composition on GABAR functionin situ, we took advantage of two naturally occurring alleles of the rat gene for GABAR subunit α6 (Gabra6100RandGabra6100Q). Depending on their subunit partners, these two variants of α6 can lead to differential sensitivities to benzodiazepines and ethanol. An examination of synaptic and extrasynaptic GABA-mediated currents in cerebellar granule cells fromGabra6100R/100RandGabra6100Q/100Qrats uncovered marked allele-dependent differences in benzodiazepine sensitivity. Unexpectedly, we found that the benzodiazepines flunitrazepam and diazepam enhanced extrasynaptic inhibition mediated by δ subunit-containing GABARs inGabra6100Q/100Qrats. Complementary experiments on recombinant GABARs confirmed that, at subsaturating [GABA], flunitrazepam potentiates α6/δ subunit-containing GABARs. Based on data and a simple theoretical analysis, we estimate that the average extrasynaptic [GABA] is ∼160 nmin perfused slices. These results (1) demonstrate contributions of α6 subunits to both synaptic and extrasynaptic GABA responses, (2) establish that δ subunit-containing GABARs are benzodiazepine sensitive at subsaturating [GABA] and, (3) provide an empirical estimate of extrasynaptic [GABA] in slices.

Gamma-aminobutyric acid-type A receptors (GABA(A)-Rs) have been proposed as a target for many general anesthetics. We recently created knockin (KI) mice harboring a point mutation (serine 270 to histidine) in the GABA(A)-R alpha1 subunit. This mutation abolishes sensitivity of recombinant GABA(A)-Rs to isoflurane while maintaining normal sensitivity to halothane and increasing the potency of GABA. KI mice showed abnormalities in the EEG baseline, including occasional spike-wave activity and spindle-like bursts. When anesthetized with isoflurane, the KI mice but not the control mice revealed repetitive 4-5 Hz slow wave discharges in the cortical EEG. KI mice did not differ from controls in response to isoflurane or halothane in the standard tail clamp/withdrawal and loss of righting reflex assays. We recorded miniature inhibitory postsynaptic currents (mIPSCs) from hippocampal interneurons and pyramidal cells in brain slices. mIPSCs in neurons from KI mice were of normal amplitude, but decayed more slowly than controls. Hippocampal mIPSCs in control mice were significantly prolonged by 0.4 and 0.9 MAC isoflurane, and by 0.5 MAC halothane. In KI mice, the effect of isoflurane on mIPSC decay was dramatically reduced, while halothane prolonged mIPSCs as for controls. We conclude that the kinetic and pharmacological properties of hippocampal GABA(A)-Rs in the KI mouse recapitulate many features of mutant alpha1beta2gamma2 GABA(A)-Rs observed in vitro. GABA(A)-Rs containing alpha1 subunits do not appear to contribute to the actions of isoflurane in the spinal cord, but both EEG and synaptic recordings provide evidence for effects of isoflurane on these GABA(A)-R isoforms in cortical structures.

Summary: Purpose:γ-Aminobutyric acid receptor (GABAAr) subunit β3–deficient mice model Angelman syndrome by displaying impaired learning, abnormal EEG with interictal spikes and slowing, myoclonus, and convulsions. The β3-subunit deficiency causes a failure of intrathalamic reticular nucleus inhibition, leading to abnormally synchronized thalamocortical oscillations. We postulated that this pathophysiology underlies the abnormal cortical EEG and triggers interictal spikes and seizures, but extrathalamic regions also contribute to interictal spikes and seizures, so that the EEG slowing should reveal an absence-like response profile, whereas spikes and seizures have dual responsiveness to absence and partial-seizure drugs. Methods: Recording electrodes were implanted over the parietal cortices of wild-type, heterozygotes, and homozygous null mice. In each experiment, EEG was recorded for 45 min, either drug or vehicle administered, and EEG recorded for another 3 h. Each EEG was scored for slow-wave activity, interictal spikes, and seizures by a reader blinded to treatments. Results: Interictal spiking and percentage of time in EEG slowing in heterozygotes were increased by the proabsence drug baclofen (GABAB-receptor agonist), whereas CGP 35348 (GABAB-receptor antagonist) had the opposite effect. The antiabsence drug ethosuximide markedly suppressed EEG slowing and interictal spiking in heterozygote and null mice. Broad-spectrum clonazepam and valproate were more effective on interictal spiking than on EEG slowing, and fosphenytoin suppressed only interictal spiking. Conclusions: The results suggest that this model of Angelman syndrome, although not expressing typical absence seizures, is characterized by hypersynchronous thalamocortical oscillations that possess absence-like pharmacologic responsiveness and promote EEG slowing, interictal spikes, and convulsive seizures.

Enhancement of GABAA receptor-mediated inhibition is the major candidate mechanism for the molecular action of general anesthetics. The GABAA receptors are members of the cys-loop superfamily of ligand-gated ion channel receptors whose structures are being elucidated. Benzodiazepines, barbiturates and non-barbiturate intravenous anesthetics, ethanol and other alcohols, volatile anesthetics, and neuroactive steroids modulate GABAA receptor function, via allosteric sites on the receptor protein. The benzodiazepine binding sites are located in the extracellular domain at subunit interfaces, similar to agonist binding pockets; these ‘agonist sites’ may be sensitive to other modulators. We showed that modulation by intravenous anesthetics requires residues in the membrane-spanning domain, TM1, while others found that volatile anesthetic modulation requires residues in TM2 and TM3. Affinity labeling of nicotinic acetylcholine receptors with anesthetic ligands by Cohen and associates reveals attachment to these membrane-spanning domains as well as residues in the agonist binding pockets and the pre-TM1 region. We purified bovine brain GABAA receptor protein to homogeneity in mg quantities using refined benzodiazepine affinity chromatography in mild detergent, and photolabeled with [3H]azi-etomidate. A single band on SDS–PAGE corresponding to the GABAA receptor β2/3 subunit was obtained in amounts suitable for microsequencing to identify the binding site.

The knockout mouse for the beta3 subunit of the GABAA receptor exhibits spontaneous epilepsy and hyperactivity, and has been proposed as a model for the severe developmental disorder, Angelman's syndrome, which is known to be of genetic origin. We have used this mutant to test an approach of therapeutic intervention prior to seizure onset by daily injection with diazepam during either the first or second postnatal week. Results showed differences between postnatal week 1 and week 2 injections both acutely, with respect to sedative effects, and in long-term outcome, with respect to EEG and behavioral tests measured at 12-14 weeks of age. The EEG of control mice remained unaffected under all conditions, but the EEG of beta3 (-/-) injected with diazepam in week 1 was worsened, showing increased oscillatory activity at 5-6Hz, and more myoclonic jerks, particularly among males. For beta3 (-/-) injected with diazepam in week 2, the EEG was normalized in half the mice but worsened similarly to week 1 in the other half. Neonatal diazepam injection had a long-term normalizing effect on behavior of beta3 (-/-) mice injected in week 1, but diazepam treatment in week 2 did not affect the hyperactive and circling behavior characteristic of the beta3 knockout mouse. Diazepam treatment in postnatal week 2 significantly decreased anxiety in the adult beta3 group. Diazepam treatment in both postnatal weeks 1 and 2 improved the motor coordination of beta3 (-/-) on the rotarod, although performance of control mice injected with diazepam in postnatal week 2 was significantly impaired. The observed long-term outcome of neonatal diazepam injections may result from interference with developmental processes, and shows that enhancing GABAergic activity with diazepam during the period where GABA can be excitatory can produce narrow stage-related effects on brain development.

GABA(A) receptor-associated protein (GABARAP) was isolated previously in a yeast two-hybrid screen using the intracellular loop of the gamma2 subunit of the GABA(A) receptor as bait. GABARAP has been shown to participate in the membrane-clustering and intracellular-trafficking of GABA(A) receptors, including a stimulation of the surface expression of GABA(A) receptors. To assess this quantitatively, we used Xenopus laevis oocytes expressing alpha1beta2gamma2S-containing GABA(A) receptors to demonstrate that coexpression of GABARAP increased net surface levels of GABA(A) receptors as shown by both increased GABA currents and surface-expressed protein. This GABARAP stimulation of GABA currents required the receptor gamma2 subunit and full-length GABARAP: deletion of the microtubule-binding domain (amino acids 1-22) or disrupting the polymerization of microtubules abolished the enhancement, indicating that the effect of GABARAP was derived from the interaction with microtubules. GABARAP coexpression did not alter the general properties of GABA(A) receptors such as sensitivity to GABA or benzodiazepines, but it increased surface levels of receptor protein in oocytes. Rather, it seems to supplement inadequate amounts of endogenous GABARAP to support optimum trafficking and/or stabilization of surface GABA(A) receptors.

The trafficking of GABAAreceptors is an important component of the pathway that regulates plasticity of inhibitory synapses. The 17 kDa GABAAreceptor-associated protein (GABARAP) has been implicated in the trafficking of GABAAreceptors because of its ability to interact not only with the γ2 subunit of the receptor but also with microtubules and theN-ethylmaleimide-sensitive factor (NSF). To elucidate the role of GABARAP in the trafficking of GABAAreceptors, we have constructed a yellow fluorescent protein (YFP) fusion protein of GABARAP and expressed it in neurons using adenovirus, so that its function may be examined. YFP-GABARAP colocalized with γ2 subunit-containing GABAAreceptors and NSF to the perinuclear cytoplasm in cultured hippocampal neurons and to the proximal regions of dendrites that are making synaptic contact. Expression of YFP-GABARAP in Cos7 cells and cultured hippocampal neurons was able to increase the level of GABAAreceptors detected at the plasma membrane, even at low levels of YFP-GABARAP expression. This effect is specific to the function of GABARAP on GABAAreceptor trafficking, because point mutations in the γ2-binding domain of YFP-GABARAP interfered with the ability of YFP-GABARAP to increase GABAAreceptor surface levels. These mutations also disrupted the colocalization of YFP-GABARAP with the γ2 subunit and with NSF in hippocampal neurons. The results of this study show for the first time that GABARAP has a functional effect on the trafficking of GABAAreceptors and provide decisive evidence for the role of GABARAP in transporting GABAAreceptors to the plasma membrane in neurons.

The administration of ethanol on a chronic intermittent regimen (CIE) involving multiple withdrawal episodes is a model for ethanol dependence. After CIE, rats exhibited reduced seizure threshold, increased anxiety, tolerance to GABAergic sedative-hypnotic drugs, and changes in GABA(A) receptor function and subunit composition in hippocampus. Previous studies have shown that acute and chronic ethanol may induce changes in the levels of the neurosteroid 3alpha-hydroxysteroid-5alpha-pregnan-20-one (3alpha, 5alpha-THP) in the brain. Therefore, the current study analyses the correlation between chronic intermittent ethanol effects on the level of 3alpha, 5alpha-THP in hippocampus of CIE rats and the behavioral changes in sensitivity to neurosteroids. After CIE, the levels for 3alpha, 5alpha-THP were significantly reduced in hippocampus of rats. The mRNA levels for the enzymes 5alpha-reductase and 3alpha-HSD in hippocampus were also reduced. In vivo, (in contrast to a tolerance to the hypnotic effect of steroids), CIE rats showed increased sensitivity to the anticonvulsant and to the anxiolytic effect of the steroid alphaxalone. Perhaps, this is a response to lowered levels of endogenous neuroactive steroids. CIE rats also showed impairment of hippocampus-dependent memory function. These results suggest that changes in neurosteroids level and in vivo sensitivity to these compounds are involved in the development of ethanol dependence in the CIE model.

γ-Aminobutyric acid type A receptors (GABARs) have long been implicated in mediating ethanol (EtOH) actions, but so far most of the reported recombinant GABAR combinations have shown EtOH responses only at fairly high concentrations (≥60 mM). We show that GABARs containing the δ-subunit, which are highly sensitive to γ-aminobutyric acid, slowly inactivating, and thought to be located outside of synapses, are enhanced by EtOH at concentrations that are reached with moderate, social EtOH consumption. Reproducible ethanol enhancements occur at 3 mM, a concentration six times lower than the legal blood-alcohol intoxication (driving) limit in most states (0.08% wt/vol or 17.4 mM). GABARs responsive to these low EtOH concentrations require the GABAR δ-subunit, which is thought to be associated exclusively with α 4 - and α 6 -subunits in vivo , and the β 3 -subunit, which has recently been shown to be essential for the in vivo anesthetic actions of etomidate and propofol. GABARs containing β 2 -instead of β 3 -subunits in α 4 βδ- and α 6 βδ-receptor combinations are almost 10 times less sensitive to EtOH, with threshold enhancement at 30 mM. GABARs containing γ 2 -instead of δ-subunits with α 4 β and α 6 β are three times less sensitive to EtOH, with threshold responses at 100 mM, a concentration not usually reached with social EtOH consumption. These combined findings suggest that “extrasynaptic” δ-subunit-containing GABARs, but not their “synaptic” γ-subunit-containing counterparts, are primary targets for EtOH.

The δ subunit of the γ-aminobutyric acid (A) receptor (GABAAR) is expressed postnatally mostly in the cerebellum, thalamus, and dentate gyrus. Previous studies in mice with a targeted disruption of the δ subunit revealed a considerable attenuation of behavioral responses to neuroactive steroids but not to other neuromodulatory drugs. Here we show that δ subunit loss leads to a concomitant reduction in hippocampal α4 subunit levels. These changes were accompanied by faster decay of evoked inhibitory postsynaptic potentials (IPSPs) in dentate granule neurons of –/– mutants (decay τ = 25 ms) compared with +/+ controls (τ = 50 ms). Furthermore, the GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs) also decayed faster in δ-mutants (τ = 6.3 ms) than controls (τ = 7.2 ms) and had decreased frequency (controls, 10.5 Hz; mutants, 6.6 Hz). Prolongation of mIPSCs by the neuroactive steroid anesthetic, alphaxalone (1–10 μM), was smaller in δ-mutants (at 10 μM, 65% increase) compared with +/+ littermates (308% increase). In competition binding experiments, alphaxalone (0.03–1 μM) modulation of [35S]t-butylbicyclophosphorothionate binding was reduced in δ-mutant brain homogenates, indicating that the decreased alphaxalone effects on mIPSCs were due to changes in the GABAAR protein. Faster decay of evoked IPSPs and mIPSCs in δ-mutants suggests presence of the δ subunit at both synaptic and extrasynaptic GABAARs. Decreased synaptic and extrasynaptic inhibition likely contributes to the pro-epileptic phenotype of δ-mutants. Reduced neurosteroid sensitivity might also contribute to seizure susceptibility. While the simplest explanation is that δ subunit-containing GABAARs represent the actual target of neurosteroids, it is possible that the behavioral and physiological sensitivity to neuroactive steroids is indirectly altered in the δ –/– mice.

To locate general anesthetic binding sites on ligand-gated ion channels, a diazirine derivative of the potent intravenous anesthetic, R-(+)-etomidate (2-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate), has been synthesized and characterized. R-(+)-Azietomidate [2-(3-methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate] anesthetizes tadpoles with an EC(50) of 2.2 microM, identical to that of R-(+)-etomidate. At this concentration both agents equally enhanced GABA-induced currents and decreased binding of the caged-convulsant [(35)S]TBPS to GABA(A) receptors. In all of the above actions R-(+)-azietomidate is about an order of magnitude more potent than S-(-)-azietomidate, an enantioselectivity comparable to etomidate's. R-(+)-Azietomidate also inhibits acetylcholine-induced currents in nicotinic acetylcholine receptors, with about twice the potency of the parent compound. [(3)H]Azietomidate photoincorporated into Torpedo nicotinic acetylcholine receptor-rich membranes. Desensitization decreased photoincorporation into the delta-subunit and increased that into the alpha-subunit. The latter increase was confined to a proteolytic fragment containing the first three transmembrane segments. Thus, R-(+)-azietomidate is a potent stereoselective general anesthetic and an effective photolabel.

The majority of fast inhibitory neurotransmission in the CNS is mediated by the GABA type-A (GABAA) receptor, a ligand-gated chloride channel. Of the approximately 20 different subunits composing the hetero-pentameric GABAA receptor, the γ2 subunit in particular seems to be important in several aspects of GABAA receptor function, including clustering of the receptor at synapses. In this study, we report that the intracellular loop of the γ2 subunit interacts with itself as well as with γ1, γ3 and β1–3 subunits, but not with the α subunits. We further show that γ2 subunits interact with photolabeled pentameric GABAA receptors composed of α1, β2/3 and γ2 subunits, and calculate the dissociation constant to be in the micromolar range. By using deletion constructs of the γ2 subunit in a yeast two-hybrid assay, we identified a 23-amino acid motif that mediates self-association, residues 389–411. We confirmed this interaction motif by inhibiting the interaction in a glutathione-S-transferase pull-down assay by adding a corresponding γ2-derived peptide. Using similar approaches, we identified the interaction motif in the γ2 subunit mediating interaction with the β2 subunit as a 47-amino acid motif that includes the γ2 self-interacting motif. The identified γ2 self-association motif is identical to the interaction motif reported between GABAA receptor and GABAA receptor-associated protein (GABARAP). We propose a model for GABAA receptor clustering based on GABARAP and GABAA receptor subunit–subunit interaction.

Chronic administration of ethanol to rats on an intermittent regimen, for 60 repeated intoxicating doses and repeated withdrawal episodes, results in a long-lasting kindling phenomenon. This involves an increasing severity of withdrawal, including a reduced threshold to seizures produced by the GABA(A) antagonist, pentylenetetrazol. We have shown previously that muscimol-evoked 36Cl- efflux and paired-pulse inhibition (involving GABA(A)-mediated recurrent inhibition) were decreased persistently in the CA1 region of hippocampal slices from chronic intermittent ethanol (CIE)-treated rats. We now report elevated levels of mRNA in forebrain for the alpha4 subunit of the GABA(A) receptor (GABAR), considered to be a constituent of pharmacologically and physiologically novel subtypes of GABARs. Using in situ hybridization with digoxigenin-labeled RNA probes, we show that at 2 days withdrawal, 60-dose CIE leads to a significant 30% increase in alpha4 subunit mRNA levels in the dentate gyrus, 46% increase in the CA3, and 26% increase in the CA1 regions. In contrast, there was no significant change in the mRNAs for the alpha5 subunit or glutamic acid decarboxylase 67 in the same regions. This study suggests that GABAR subunit-selective alterations occur after CIE treatment, possibly resulting in the alteration of the subunit composition of GABARs, with presumably altered physiological functions. This plasticity of GABARs may contribute to the increased withdrawal severity, reduced hippocampal inhibition, and increased seizure susceptibility of this animal model of human alcohol dependence.

The gamma-aminobutyric acid receptor type A (GABA(A)) receptor-associated protein (GABARAP) is a member of a growing family of intracellular membrane trafficking and/or fusion proteins and has been implicated in plasma membrane targeting and clustering of GABA(A) receptors. GABARAP interacts with microtubules and the gamma2 subunit of GABA(A) receptor and modulates channel kinetics. From crystal structures of GABARAP in high salt concentration it has been proposed that oligomerization of GABARAP might take place in a head-to-tail fashion. In this study, we report that GABARAP self-associates and dimerizes in physiological salt concentrations. We find no evidence for higher order complex larger than a dimer. By using deletion constructs of GABARAP we show that interaction takes place between amino acid 36 and 68. We further narrow the interacting domain by inhibiting the self-association, by adding GABARAP-derived synthetic peptides in GST pull-down assays and shows that the interaction specifically takes place in the previously identified GABARAP-GABA(A) receptor interaction domain from amino acid 41-51. The identification of binding domains in GABARAP allows for the study of GABARAP functions, including GABA(A) receptor dynamics.

Summary: Purpose: Mice with a targeted disruption of the γ-aminobutyric acid (A) receptor (GABAAR) δ subunit exhibited spontaneous seizures and a strikingly selective attenuation of responses to neuroactive steroids, but not to other neuromodulators. This study further characterized the behavior and physiology of the δ mutants. Methods: Seizure susceptibility to pentylenetetrazol (PTZ) injection, intracellular recordings, and whole-cell patch recordings in dentate granule neurons was determined. Results: Susceptibility to PTZ-induced convulsive seizures was significantly higher in −/− versus +/+ mice. In intracellular recordings, the evoked inhibitory postsynaptic potentials (IPSPs) had much faster decay τ in −/− mutants (25 ± 3.5 ms) compared with +/+ controls (51 ± 5.2 ms). In whole-cell patch recordings, the decay τ of pharmacologically isolated miniature spontaneous GABAAR-mediated synaptic currents (mIPSCs) from −/− mice was only slightly, but significantly faster (5.7 ± 0.13 ms; n = 18) than that of +/+ controls (6.8 ± 0.32 ms; n = 20). Furthermore, mIPSC prolongation by bath application of alphaxalone (10 μM), was much smaller in −/− mice (52 ± 12%; n = 3) compared with +/+ littermates (192 ± 14%; n = 3). Conclusions: Faster decay of evoked IPSPs and mIPSCs is consistent with altered GABAAR subunit composition in the δ mutants, but suggests predominant extrasynaptic δ subunit location in the dentate gyrus of normal mice. Faster-decaying IPSPs likely contribute to the proepileptic phenotype of the δ mutants. Reduced neurosteroid sensitivity might also contribute to seizure susceptibility. It remains to be determined whether δ subunit–containing GABAARs represent the actual target of neurosteroids or whether the behavioral and physiologic sensitivity to neurosteroids is indirectly altered in the δ−/− mice.

The delta subunit is a novel subunit of the pentameric gamma-aminobutyric acid (GABA)(A) receptor that conveys special pharmacological and functional properties to recombinant receptors and may be particularly important in mediating tonic inhibition. Mice that lack the delta subunit have been produced by gene-targeting technology, and these mice were studied with immunohistochemical and immunoblot methods to determine whether changes in GABA(A) receptors were limited to deletion of the delta subunit or whether alterations in other GABA(A) receptor subunits were also present in the delta subunit knockout (delta-/-) mice. Immunohistochemical studies of wild-type mice confirmed the restricted distribution of the delta subunit in the forebrain. Regions with moderate to high levels of delta subunit expression included thalamic relay nuclei, caudate-putamen, molecular layer of the dentate gyrus, and outer layers of the cerebral cortex. Virtually no delta subunit labeling was evident in adjacent regions, such as the thalamic reticular nucleus, hypothalamus, and globus pallidus. Comparisons of the expression of other subunits in delta-/- and wild-type mice demonstrated substantial changes in the alpha4 and gamma2 subunits of the GABA(A) receptor in the delta-/- mice. gamma2 Subunit expression was increased, whereas alpha4 subunit expression was decreased in delta-/- mice. Importantly, alterations of both the alpha4 and the gamma2 subunits were confined primarily to brain regions that normally expressed the delta subunit. This suggests that the additional subunit changes are directly linked to loss of the delta subunit and could reflect local changes in subunit composition and function of GABA(A) receptors in delta-/- mice.

GABARAP (GABA(A) receptor-associated protein) interacts with both microtubules and GABA(A) receptors in vitro and in vivo and is capable of modulating receptor channel kinetics. In this study, we use the intracellular loop of 15 GABA(A) receptor subunits to show that the interaction between GABARAP and GABA(A) receptor is specific for the gamma subunits. Pharmacological characterization of proteins purified by GABARAP affinity column indicates that native GABA(A) receptors interact with GABARAP. Quantitative yeast two-hybrid assays were used to identify the interaction domain in the gamma2 subunit for GABARAP binding, and to identify the interaction domain in GABARAP for GABA(A) receptor binding. A peptide corresponding to the GABARAP interaction domain in the gamma2 subunit was used to inhibit the interaction between GABARAP and the gamma2 subunit. In addition, the ability of GABARAP to promote cluster formation of recombinant receptors expressed in QT-6 fibroblasts was inhibited by a membrane-permeable form of this peptide in a time-dependent manner. The establishment of a model for GABARAP-induced clustering of GABA(A) receptors in living cells and the identification of subunit specificity and interaction domains in the interaction between GABARAP and GABA(A) receptors is a step in dissecting the function of GABARAP in GABA(A) receptor clustering and/or targeting.

GABA(A) receptor-associated protein (GABARAP) was isolated on the basis of its interaction with the gamma2 subunit of GABA(A) receptors. It has sequence similarity to light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. This suggests that GABARAP may link GABA(A) receptors to the cytoskeleton. GABARAP associates with tubulin in vitro. However, little is known about the mechanism for the interaction, and it is not clear whether the interaction occurs in vivo. Here, we report that GABARAP interacts directly with both tubulin and microtubules in a salt-sensitive manner, indicating the association is mediated by ionic interactions. GABARAP coimmunoprecipitates with tubulin and associates with both microtubules and microfilaments in intact cells. The cellular distribution is altered by treatment with taxol, nocodazole, and cytochalasin D. The tubulin binding domain was located at the N terminus of GABARAP by using synthetic peptides and deletion constructs and is marked by a specific arrangement of basic amino acids. The interaction between GABARAP and actin might be mediated by other proteins. These results demonstrate the GABARAP interacts with the cytoskeleton both in vitro and in cells and suggest a role of GABARAP in the interaction between GABA(A) receptors and the cytoskeleton. Such interactions are presumably needed for receptor trafficking, anchoring, and/or synaptic clustering. The structural arrangement of the basic amino acids present in the tubulin binding domain of GABARAP may aid in recognition of the potential of tubulin binding activity in other known proteins.

The nucleus accumbens (NAcc) is a key structure of the mesolimbic dopaminergic reward system and plays an important role in mediating alcohol-seeking behaviors. Alterations in glutamatergic and GABAergic signaling were recently demonstrated in the NAcc of rats after chronic intermittent ethanol (CIE) treatment, a model of alcohol dependence. Here we studied dopamine (DA) modulation of GABAergic signaling and how this modulation might be altered by CIE treatment. We show that the tonic current ( Itonic) mediated by extrasynaptic γ-aminobutyric acid type A receptors (GABAARs) of medium spiny neurons (MSNs) in the NAcc core is differentially modulated by DA at concentrations in the range of those measured in vivo (0.01–1 μM), without affecting the postsynaptic kinetics of miniature inhibitory postsynaptic currents (mIPSCs). Use of selective D1 receptor (D1R) and D2 receptor (D2R) ligands revealed that Itonic potentiation by DA (10 nM) is mediated by D1Rs while Itonic depression by DA (0.03–1 μM) is mediated by D2Rs in the same MSNs. Addition of guanosine 5′- O-(2-thiodiphosphate) (GDPβS) to the recording pipettes eliminated Itonic decrease by the selective D2R agonist quinpirole (5 nM), leaving intact the quinpirole effect on mIPSC frequency. Recordings from CIE and vehicle control (CIV) MSNs during application of D1R agonist (SKF 38393, 100 nM) or D2R agonist (quinpirole, 2 nM) revealed that SKF 38393 potentiated Itonic to the same extent, while quinpirole reduced Itonic to a similar extent, in both groups of rats. Our data suggest that the selective modulatory effects of DA on Itonic are unaltered by CIE treatment and withdrawal.

Acute and chronic ethanol (EtOH) administration is known to affect function, surface expression, and subunit composition of γ-aminobutyric acid (A) receptors (GABAARs) in different parts of the brain, which is believed to play a major role in alcohol dependence and withdrawal symptoms. The basolateral amygdala (BLA) participates in anxiety-like behaviors including those induced by alcohol withdrawal. In the present study we assessed the changes in cell surface levels of select GABAAR subunits in the BLA of a rat model of alcohol dependence induced by chronic intermittent EtOH (CIE) treatment and long-term (>40 days) withdrawal and investigated the time-course of such changes after a single dose of EtOH (5 g/kg, gavage). We found an early decrease in surface expression of α4 and d subunits at 1 h following single dose EtOH treatment. At 48 h post-EtOH and after CIE treatment there was an increase in α4 and γ2, while α1, α2, and δ surface expression were decreased. To relate functional changes in GABAARs to changes in their subunit composition we analyzed miniature inhibitory postsynaptic currents (mIPSCs) and the picrotoxin-sensitive tonic current (Itonic) 48 h after EtOH intoxication. The Itonic magnitude and most of the mIPSC kinetic parameters (except faster mIPSC decay) were unchanged at 48 h post-EtOH. At the same time, Itonic potentiation by acute EtOH was greatly reduced, whereas mIPSCs became significantly more sensitive to potentiation by acute EtOH. These results suggest that EtOH intoxication-induced GABAAR plasticity in the BLA might contribute to the diminished sedative/hypnotic and maintained anxiolytic effectiveness of EtOH. © Springer Science+Business Media 2014.

Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create large problems both for society and for the drinkers themselves. To date, no therapeutic can effectively solve these problems. Understanding the underlying mechanisms leading to AUD is critically important for developing effective and safe pharmacological therapies. Benzodiazepines (BZs) are used to reduce the symptoms of alcohol withdrawal syndrome. However, frequent use of BZs causes cross-tolerance, dependence, and cross-addiction to alcohol. The FDA-approved naltrexone and acamprosate have shown mixed results in clinical trials. Naltrexone is effective to treat alcohol dependence (decreased length and frequency of drinking bouts), but its severe side effects, including withdrawal symptoms, are difficult to overcome. Acamprosate showed efficacy for treating alcohol dependence in European trials, but two large US trials have failed to confirm the efficacy. Another FDA-approved medication, disulfiram, does not diminish craving, and it causes a peripheral neuropathy. Kudzu is the only natural medication mentioned by the National Institute on Alcohol Abuse and Alcoholism, but its mechanisms of action are not yet established. It has been recently shown that dihydromyricetin, a flavonoid purified from Hovenia, has unique effects on GABAA receptors and blocks ethanol intoxication and withdrawal in alcoholic animal models. In this article, we review the role of GABAA receptors in the treatment of AUD and currently available and potentially novel pharmacological agents.

GABA(A) receptors the major sites of fast synaptic inhibition in the brain are composed predominately of alpha, beta, and gamma2 subunits. The receptor gamma2 subunit interacts with a 17-kDa microtubule associated protein GABARAP, but the significance of this interaction remains unknown. Here we demonstrate that GABARAP, which immunoprecipitates with GABA(A) receptors, is not found at significant levels within inhibitory synapses, but is enriched within the Golgi apparatus and postsynaptic cisternae. We also demonstrate that GABARAP binds directly to N-ethylmaleimide-sensitive factor (NSF), a protein critical for intracellular membrane trafficking events. NSF and GABARAP complexes could be detected in neurons and these two proteins also colocalize within intracellular membrane compartments. Together our observations suggest that GABARAP may play a role in intracellular GABA(A) receptor transport but not synaptic anchoring, via its ability to interact with NSF. GABARAP may therefore have an important role in the production of GABAergic synapses.

Two γ-aminobutyric acidA (GABAA) receptor chimeras were designed in order to elucidate the structural requirements for GABAA receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.

BACKGROUND Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompanied by reduced inhibitory synaptic transmission in the hippocampus and changes in gamma-aminobutyric acid type A (GABAA) receptors. Further information is needed on the detailed changes in GABAA receptors and their time course and persistence, as is comparison to changes after chronic, continuous ethanol. METHODS GABAA receptors were analyzed in the rat brain after chronic intermittent ethanol (CIE) by using radioligand binding, photoaffinity labeling of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS CIE rats were confirmed to have increased GABAA receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the alpha6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in alpha6 or in several other receptor subunits in several brain regions, but a decrease in the ratio of the long and short splice variants (L/S) of the gamma2 subunit was detected in the hippocampus, especially the CA1 region. CONCLUSIONS Changes in GABAA receptors were found in rats given CIE. Increased alpha6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [3H]Ro15-4513 and increased levels of the 57-kDa alpha6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to normal 1 week after ethanol was discontinued. The transient change in cerebellar alpha6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hyperexcitable, kindled, seizure-susceptible state seen in CIE. However, the significant decrease in gamma2 subunit L/S splice variant ratio in the hippocampus implies changes in GABAA receptor function, possibly involving protein phosphorylation by protein kinase C. Altered receptor trafficking and turnover associated with synaptic plasticity may contribute to the observed reduced inhibition in the hippocampus and other signs of alcohol dependence produced by CIE.

Fetal alcohol exposure (FAE) can lead to a variety of behavioral and physiological disturbances later in life. Understanding how alcohol (ethanol, EtOH) affects fetal brain development is essential to guide the development of better therapeutics for FAE. One of EtOH's many pharmacological targets is the γ-aminobutyric acid type A receptor (GABAAR), which plays a prominent role in early brain development. Acute EtOH potentiates inhibitory currents carried by certain GABAAR subtypes, whereas chronic EtOH leads to persistent alterations in GABAAR subunit composition, localization and function. We recently introduced a flavonoid compound, dihydromyricetin (DHM), which selectively antagonizes EtOH's intoxicating effects in vivo and in vitro at enhancing GABAAR function as a candidate for alcohol abuse pharmacotherapy. Here, we studied the effect of FAE on physiology, behavior and GABAAR function of early adolescent rats and tested the utility of DHM as a preventative treatment for FAE-induced disturbances. Gavage administration of EtOH (1.5, 2.5, or 5.0 g/kg) to rat dams on day 5, 8, 10, 12, and 15 of pregnancy dose-dependently reduced female/male offspring ratios (largely through decreased numbers of female offspring) and offspring body weights. FAE (2.5 g/kg) rats tested on postnatal days (P) 25-32 also exhibited increased anxiety and reduced pentylenetetrazol (PTZ)-induced seizure threshold. Patch-clamp recordings from dentate gyrus granule cells (DGCs) in hippocampal slices from FAE (2.5 g/kg) rats at P25-35 revealed reduced sensitivity of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and tonic current (Itonic) to potentiation by zolpidem (0.3 μM). Interestingly, potentiation of mIPSCs by gaboxadol increased, while potentiation of Itonic decreased in DGCs from FAE rats. Co-administration of EtOH (1.5 or 2.5 g/kg) with DHM (1.0 mg/kg) in pregnant dams prevented all of the behavioral, physiological, and pharmacological alterations observed in FAE offspring. DHM administration alone in pregnant rats had no adverse effect on litter size, progeny weight, anxiety level, PTZ seizure threshold, or DGC GABAAR function. Our results indicate that FAE induces long-lasting alterations in physiology, behavior, and hippocampal GABAAR function and that these deficits are prevented by DHM co-treatment of EtOH-exposed dams. The absence of adverse side effects and the ability of DHM to prevent FAE consequences suggest that DHM is an attractive candidate for development as a treatment for prevention of fetal alcohol spectrum disorders.

The α4 subunit of the GABAA receptor (GABAAR) is highly expressed in the thalamus where receptors containing the α4 and δ subunits are major mediators of tonic inhibition. The α4 subunit also exhibits considerable plasticity in a number of physiological and pathological conditions, raising questions about the expression of remaining GABAAR subunits when the α4 subunit is absent. Immunohistochemical studies of an α4 subunit knockout (KO) mouse revealed a substantial decrease in δ subunit expression in the ventrobasal nucleus of the thalamus as well as other forebrain regions where the α4 subunit is normally expressed. In contrast, several subunits associated primarily with phasic inhibition, including the α1 and γ2 subunits, were moderately increased. Intracellular localization of the δ subunit was also altered. While δ subunit labeling was decreased within the neuropil, some labeling remained in the cell bodies of many neurons in the ventrobasal nucleus. Confocal microscopy demonstrated co-localization of this labeling with an endoplasmic reticulum marker, and electron microscopy demonstrated increased immunogold labeling near the endoplasmic reticulum in the α4 KO mouse. These results emphasize the strong partnership of the δ and α4 subunit in the thalamus and suggest that the α4 subunit of the GABAAR plays a critical role in trafficking of the δ subunit to the neuronal surface. The findings also suggest that previously observed reductions in tonic inhibition in the α4 subunit KO mouse are likely to be related to alterations in δ subunit expression, in addition to loss of the α4 subunit.

GABAA receptors (GABARs) have long been the focus for acute alcohol actions with evidence for behaviorally relevant low millimolar alcohol actions on tonic GABA currents and extrasynaptic α4/6, δ, and β3 subunit-containing GABARs. Using recombinant expression in oocytes combined with two electrode voltage clamp, we show with chimeric β2/β3 subunits that differences in alcohol sensitivity among β subunits are determined by the extracellular N-terminal part of the protein. Furthermore, by using point mutations, we show that the β3 alcohol selectivity is determined by a single amino acid residue in the N-terminus that differs between GABAR β subunits (β3Y66, β2A66, β1S66). The β3Y66 residue is located in a region called “loop D” which in γ subunits contributes to the imidazobenzodiazepine (iBZ) binding site at the classical α+γ2− subunit interface. In structural homology models β3Y66 is the equivalent of γ2T81 which is one of three critical residues lining the benzodiazepine binding site in the γ2 subunit loop D, opposite to the “100H/R-site” benzodiazepine binding residue in GABAR α subunits. We have shown that the α6R100Q mutation at this site leads to increased alcohol-induced motor in-coordination in alcohol non-tolerant rats carrying the α6R100Q mutated allele. Based on the identification of these two amino acid residues α6R100 and β66 we propose a model in which β3 and δ containing GABA receptors contain a unique ethanol site at the α4/6+β3− subunit interface. This site is homologous to the classical benzodiazepine binding site and we propose that it not only binds ethanol at relevant concentrations (EC50– 17 mM), but also has high affinity for a few selected benzodiazepine site ligands including alcohol antagonistic iBZs (Ro15-4513, RY023, RY024, RY80) which have in common a large moiety at the C7 position of the benzodiazepine ring. We suggest that large moieties at the C7-BZ ring compete with alcohol for its binding pocket at a α4/6+β3− EtOH/Ro15-4513 site. This model reconciles many years of alcohol research on GABARs and provides a plausible explanation for the competitive relationship between ethanol and iBZ alcohol antagonists in which bulky moieties at the C7 position compete with ethanol for its binding site. We conclude with a critical discussion to suggest that much of the controversy surrounding this issue might be due to fundamental species differences in alcohol and alcohol antagonist responses in rats and mice.

A microtubule-associated protein, γ-aminobutyric acid type A (GABA A ) receptor-associated protein (GABARAP), was previously identified as binding to the intracellular domain of GABA A receptors by using the yeast two-hybrid screen. In the present work, immunofluorescent staining and green fluorescent protein-tagged receptor subunits showed that GABARAP is associated with and promotes the clustering of GABA A receptors in QT-6 quail fibroblasts. The tubulin-binding motif of GABARAP and the γ2 subunit of the receptor are required. Disruption of microtubules prevents the clustering in a time-dependent manner. When green fluorescent protein-tagged α1 or γ2 subunit coexpressed with β2, γ2L, and GABARAP was used, recordings from visually identified cells revealed that clustered GABA A receptor had an EC 50 of about 20 μM, vs. 5.7 μM for the diffuse receptor. Clustered receptors deactivated faster and desensitized slower than the diffuse receptors, because of decrease in the apparent affinity of GABA binding. Different properties for clustered receptors relative to unclustered receptors in heterologous cells suggest that homologous differences between extrasynaptic and synaptic clustered receptors in neurons may be due to the organization of the postsynaptic machinery.

Site-directed mutagenesis of the gamma-aminobutyric acid type A (GABA(A)) receptor beta(2) subunit has demonstrated that conversion of a conserved glycine residue located at the entrance to the first transmembrane domain into the homologous rho(1) residue phenylalanine alters the modulating effects of four different i.v. anesthetics: pentobarbital, alphaxalone, etomidate, and propofol. Using the baculovirus expression system in Spodoptera frugiperda 9 cells, anesthetic-induced enhancement of [(3)H]muscimol and [(3)H]flunitrazepam binding in receptors containing the beta(2)(G219F) point mutation displayed a significantly reduced efficacy in modulation by all four i.v. anesthetics tested. Furthermore, GABA(A) receptors containing the alpha(1)(G223F) point mutation also significantly decreased the maximal effect of etomidate- and propofol-induced enhancement of ligand binding. Conversely, the homologous point mutation in rho(1) receptors (F261G) changed the i.v. anesthetic-insensitive receptor to confer anesthetic modulation of [(3)H]muscimol binding. Consistent with the binding, functional analysis of pentobarbital-enhanced GABA currents recorded with whole-cell patch clamp demonstrated the beta(2)(G219F) subunit mutation eliminated the potentiating effect of the anesthetic. Similarly, propofol-enhanced GABA currents were potentiated less in alpha(1)beta(2)(G219F)gamma(2) receptors than in alpha(1)beta(2)gamma(2) receptors. Although ligand binding displayed comparable K(D) values for muscimol among wild-type, alpha(1)beta(2)gamma(2), and mutant receptors, patch-clamp recordings showed that alpha(1)beta(2)(G219F)gamma(2) receptors had a significantly more potent response to GABA than did alpha(1)beta(2)gamma(2) or alpha(1)(G223F)beta(2)gamma(2). The alpha(1)beta(2)(G219F)gamma(2) receptors also were more sensitive to direct channel activation by pentobarbital and propofol in the absence of GABA. These results suggest that the first transmembrane glycine residue on the beta(2) subunit may be important for conformational or allosteric interactions of channel gating by both GABA and anesthetics.

Spodoptera frugiperda insect cells (Sf-9 cells) were used to study GABA(A) receptor assembly. Time courses of the expression level of alpha1beta2 and alpha1beta2gamma2 receptor protein showed [(3)H]muscimol binding to appear 2 hr before [(3)H]flunitrazepam and [(35)S]TBPS binding. This indicates that muscimol may bind to pentamers with an immature conformation or to molecules smaller than the pentamer. Binding studies performed on fractions from sucrose gradients loaded with solubilized alpha1beta2 or alpha1beta2gamma2 containing membranes revealed no binding other than to the pentameric fractions. Western blotting on fractionated sucrose gradients, however, clearly revealed the existence of GABA(A) receptor intermediates. The alpha1 subunit was seen in fractions corresponding to molecules smaller than the pentamer only when co-expressed with gamma2, indicating that the gamma2 subunit is needed for the alpha1 to form relatively long lasting intermediates. Moreover, Western blots revealed multiple isoforms for each subunit. In general, it was primarily the lower molecular weight forms that were detected in the pentameric fractions. The exception being for the alpha1 and gamma2 forms in subunit combinations that did not contain both of these subunits (i.e., alpha1, gamma2, alpha1beta2, beta2gamma2), where higher molecular weight forms were strongly represented. These findings show that alpha1 and gamma2 prefer specific protein forms when expressed together.

PURPOSE This study evaluated the modulatory effects of topiramate (TPM) on various subtypes of recombinant rat gamma-aminobutyric acid A (GABA(A)) receptors expressed in Xenopus oocytes. METHODS Specific subunits of GABA(A) receptors were expressed in Xenopus oocytes. Voltage-clamp recordings of currents were performed after application of TPM (1-100 microM) to these oocytes in the presence or absence of GABA. RESULTS In a concentration-dependent fashion, TPM (1-100 microM) reversibly inhibited GABA-evoked Cl- currents in oocytes expressing either alpha1beta2gamma2S and alpha2beta2gamma2S recombinant GABA(A) receptors and reduced the current-fading rate in alpha1beta2gamma2S-expressing oocytes. Topiramate was effective at GABA concentrations of 1-10 microM but not at 100 microM. Topiramate (1-100 microM) potentiated GABA-evoked Cl- currents and increased the fading rate in oocytes expressing the alpha6beta2gamma2S GABA(A) receptor. It had no effect on Cl- currents mediated through the alpha4beta2gamma2S receptor or through the mixed population of GABA(A) receptors expressed from rat brain mRNA. In general, the observed effects of TPM were more pronounced on fading rates than on peak Cl- currents. CONCLUSIONS These results indicate that TPM may affect desensitization of GABA(A) receptors as assessed by changes in the fading rates of GABA-evoked Cl- currents, possibly by effects on second-messenger systems.

Peptide mapping and microsequencing were used to infer the site of photoaffinity labeling by the gamma-aminobutyric acidA receptor modulator [3H]flunitrazepam. Peptide mapping with and without N-deglycosylation was used to restrict the domain for photoaffinity labeling to residues 74-123 of the bovine alpha1 subunit, in agreement with a previously predicted labeling domain between residues 59-148 based on cyanogen bromide fragmentation. Edman degradation of partially purified photolabeled peptides gave release of 3H counts in the ninth cycle of a tryptic peptide sequence. A second V8/chymotryptic peptide produced an impure sequence with release of 3H counts in the seventh through ninth cycle of sequence. The combined data support those previously reported, i.e., that the primary site for photoaffinity labeling by [3H]flunitrazepam is His102 of the bovine alpha1 subunit. In addition we also detected possible secondary labeling of Pro97.

To overcome the difficulties of locating the molecular sites of general anesthetic action, we synthesized a novel photoactivable general anesthetic, 3-(2-hydroxyethyl)-3-n-pentyldiazirine (3-diazirinyloctanol), which anesthetized tadpoles with an ED(50) of 160 microM. Subanesthetic concentrations of 3-diazirinyloctanol enhanced GABA-induced currents in GABA(A) receptors, an effect that has been implicated in general anesthetic action. It also enhanced [(3)H]muscimol binding to this receptor. In muscle nicotinic acetylcholine receptors (nAcChoR), it inhibited the response to acetylcholine with an IC(50) of 33 microM. 3-Diazirinyloctanol's pharmacological actions were comparable to those of octanol. 3-(2-Hydroxyethyl)-3-[4,5-(3)H(2)]-n-pentyldiazirine photoincorporated into Torpedo nAcChoR-rich membranes mainly in the alpha subunit with 70% being in a proteolytic fragment containing the M4 transmembrane segment. Agonist enhanced the photolabeling 10-fold in a fragment containing the M1, M2, and M3 transmembrane segments. Thus, 3-diazirinyloctanol is a novel general anesthetic that acts on, and can be photoincorporated into, postsynaptic receptors.

Type-A receptors for the neurotransmitter GABA (gamma-aminobutyric acid) are ligand-gated chloride channels that mediate inhibitory neurotransmission. Each subunit of the pentameric receptor protein has ligand-binding sites in the amino-terminal extracellular domain and four membrane-spanning regions, one of which forms a wall of the ion channel. Each subunit also has a large intracellular loop that may be a target for protein kinases and be required for subcellular targeting and membrane clustering of the receptor, perhaps by anchoring the receptor to the cytoskeleton. Neurotransmitter receptors need to be positioned in high density in the cell membrane at sites postsynaptic to nerve terminals releasing that neurotransmitter. Other members of the superfamily of ligand-gated ion-channel receptors associate in postsynaptic-membrane clusters by binding to the proteins rapsyn or gephyrin. Here we identify a new cellular protein, GABA(A)-receptor-associated protein (GABARAP), which can interact with the gamma2 subunit of GABA(A) receptors. GABARAP binds to GABA(A) receptors both in vitro and in vivo, and co-localizes with the punctate staining of GABA(A) receptors on cultured cortical neurons. Sequence analysis shows similarity between GABARAP and light chain-3 of microtubule-associated proteins 1A and 1B. Moreover, the N terminus of GABARAP is highly positively charged and features a putative tubulin-binding motif. The interactions among GABA(A) receptors, GABARAP and tubulin suggest a mechanism for the targeting and clustering of GABA(A) receptors.

γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain. A deficiency of GABAergic inhibition mediated via the GABAA receptor complex has for a long time been suspected to be a central factor in epileptogenesis. Status epilepticus is a condition of sustained and prolonged excitation of neuronal circuits, as detected by epileptiform discharges in the electroencephalogram (EEG). Reduction of GABAA receptor-mediated hippocampal inhibition has been implicated in the development of status epilepticus. The present study provides direct evidence of a link between the GABAA receptor and epilepsy. We show that selective inhibition of the expression of the GABAA receptor γ2 subunit in the rat hippocampus by means of antisense oligonucleotides leads to spontaneous electrographic seizures that evolve into profound limbic status epilepticus, ultimately resulting in severe neurodegenerative changes. Concurrent treatment with diazepam prevents the development of status epilepticus and markedly reduces neuronal cell loss. These findings strongly support the hypothesis that the GABAA receptor is critically involved in the pathogenesis of seizures and status epilepticus. J. Neurosci. Res. 54:863–869, 1998. © 1998 Wiley-Liss, Inc.

Chronic intermittent ethanol (CIE)-treated rats exhibited a kindling-like persistent increase in withdrawal hyperexcitability. The alteration of GABA(A) receptor (GABA(A)R) function in the hippocampus was suggested as a possible mechanism underlying the hyperexcitability observed in CIE rats, because (1) GABA(A)R agonist (muscimol)-evoked 36Cl- efflux was decreased; (2) paired-pulse inhibition in the CA1 area, predominantly due to GABA(A)R-mediated recurrent inhibition, was persistently decreased; and (3) GABA(A)R subunit expression was altered in the hippocampus from CIE rats. To further characterize the functional alteration of GABA(A)R after CIE treatment, their sensitivity to acute ethanol, a steroid anesthetic (alphaxalone), and a benzodiazepine inverse agonist (DMCM; methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) were studied using either synaptically evoked GABA(A)R responses or exogenously applied muscimol-evoked responses in hippocampal slices. Bath application of ethanol (60 mM) enhanced the area of GABA(A)R-mediated inhibitory postsynaptic potentials in the hippocampal CA1 region from control and CIE rats, and this potentiation was significantly (p = 0.027) greater in CIE rats (98%) than in control rats (53%). The positive modulatory effect of alphaxalone (1 microM) on GABA(A)R-inhibitory postsynaptic potentials was not significantly different between control and CIE rats (p = 0.375), whereas alphaxalone allosterically increased [3H]flunitrazepam binding in the CA1 area only in CIE rats (by 20 to 25%, p < 0.01), but not in controls. On the other hand, the negative modulatory effect of DMCM (1 microM) on muscimol-evoked responses was significantly larger in CIE rats (p = 0.002). These results suggest that the sensitization of GABA(A)R to acute ethanol and benzodiazepine inverse agonists, and possibly neurosteroids, may underlie ethanol dependence after multiple ethanol withdrawal episodes. These altered pharmacological properties are most consistent with changes in the subunit composition in the CA1 area of this rat model of alcohol dependence.

Modification of GABAA receptor mRNA levels by seizure activity can regulate general neuronal excitability. The possibility of absence seizure-induced alteration in GABAA receptor alpha 1, alpha 4, beta 2, and gamma 2 subunit gene expression in thalamic relay nuclei was studied in a rat model of absence seizures induced by gamma-hydroxybutyric acid (GHB). We observed a marked increase in alpha 1 mRNA and a corresponding decrease in alpha 4 mRNA in thalamic relay nuclei 2-4 h after the onset of GHB-induced absence seizures (when the seizures were terminating). These changes were selective to these alpha isoforms as neither beta 2 nor gamma 2 mRNA changed following seizures and occurred only in thalamic relay nuclei but not in hippocampus, a structure from which absence seizures do not evolve. The alterations in alpha 1 and alpha 4 mRNA persisted until about 12 h, and by 24 h after the seizure-onset the mRNA levels normalized. Blocking GHB-seizures produced no change in the levels of alpha 1 and alpha 4 mRNA in thalamic relay nuclei, suggesting that seizures themselves were responsible for mRNA alterations. In order to determine if absence seizure-induced changes in alpha 1 and alpha 4 mRNA had any physiological significance, GHB was readministered in rats 6 and 24 h after the onset of seizures. The total duration of GHB-seizures was found to be significantly decreased when GHB was readministered at 6 h but not 24 h after the seizure-onset. These results suggest that absence seizures regulate GABAA receptor alpha 1 and alpha 4 gene expression in thalamic relay nuclei as a compensatory mechanism by which absence seizures are terminated.

(1) Considerable evidence has accumulated that the molecular target of general anesthetics in the central nervous system is the GABA(A) receptor, the major mediator of inhibitory synaptic transmission. This receptor is actually a family of ligand-gated chloride channel proteins, each a heteropentameric membrane-spanning structure. (2) Regional variation in anesthetic actions on the central nervous system may parallel a corresponding regional variation in pharmacological subtypes of GABA(A) receptors. These result from differential regional expression of approximately 18 subunit genes. (3) Receptors of varying subunit composition show differential sensitivity to GABA, modulatory drugs, and biological regulatory mechanisms. Regional variation in allosteric modulation of GABA(A) receptor binding and function can be reconstituted in certain recombinant receptor subunit combinations expressed in heterologous cells. (5) Differential sensitivity to anesthetics for various GABA(A) receptor subunits also allows the use of the chimeric and site-directed mutagenesis approach in attempting to define domains of the protein which participate in the binding and actions of anesthetics.

Primary cultures of cerebellar granule cells, prepared from cerebella of 7-day-old rats and cultured for 4 or 8 days, were used to study the neurodifferentiative effect of a GABAA receptor agonist, 4,5,6,7-tetrahydroisoxazol[5,4-c]pyridin-3-ol (THIP), on the expression of the α6 GABAA receptor subunit. Membranes prepared from these cultures were photolabeled with the imidazobenzodiazepine [3H]Ro15-4513. In THIP-treated cultures at 4 days in vitro (DIV), photolabeled [3H]Ro15-4513 binding in membranes was significantly increased for both the 51 kilodalton, kDa, (α1 subunit) and 56-kDa (α6 subunit) radioactive peaks in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). In contrast, THIP-treated granule cells at 8 DIV demonstrated a small but significant decrease from control cultures in the photoincorporation of [3H]Ro15-4513 in the 51-kDa peak; however, no significant change in [3H]Ro15-4513 binding was observed for the 56-kDa polypeptide. Immunolabeling of the α6 subunit using silver-enhanced, immuno-gold staining of granule cells showed a significant effect with THIP treatment only at 4 DIV and not at 8 DIV. Examination by light microscopy demonstrated that the major effect of THIP was to increase α6 subunit clustering on granule cell bodies as well as neurites, 15-fold and sixfold, respectively. Using in situ hybridization, a small THIP-induced increase in α6 mRNA was detected at 4 DIV; however, no effect was apparent at 8 DIV. These data suggest that THIP has a trophic effect on α6 subunit expression, and this effect occurs only at an early developmental stage. Moreover, this study presents further evidence for the role of GABAA agonists, and thus the neurotransmitter, GABA, in regulating the expression of GABAA receptor subunits in the developing cerebellum. J. Neurosci. Res. 50:1053–1062, 1997. © 1997 Wiley-Liss, Inc.

Etomidate is a potent general anesthetic that acts as an allosteric co-agonist at GABAA receptors. Photoreactive etomidate derivatives labeled αMet-236 in transmembrane domain M1, which structural models locate in the β+/α- subunit interface. Other nearby residues may also contribute to etomidate binding and/or transduction through rearrangement of the site. In human α1β2γ2L GABAA receptors, we applied the substituted cysteine accessibility method to α1-M1 domain residues extending from α1Gln-229 to α1Gln-242. We used electrophysiology to characterize each mutant's sensitivity to GABA and etomidate. We also measured rates of sulfhydryl modification by p-chloromercuribenzenesulfonate (pCMBS) with and without GABA and tested if etomidate blocks modification of pCMBS-accessible cysteines. Cys substitutions in the outer α1-M1 domain impaired GABA activation and variably affected etomidate sensitivity. In seven of eight residues where pCMBS modification was evident, rates of modification were accelerated by GABA co-application, indicating that channel activation increases water and/or pCMBS access. Etomidate reduced the rate of modification for cysteine substitutions at α1Met-236, α1Leu-232 and α1Thr-237. We infer that these residues, predicted to face β2-M3 or M2 domains, contribute to etomidate binding. Thus, etomidate interacts with a short segment of the outer α1-M1 helix within a subdomain that undergoes significant structural rearrangement during channel gating. Our results are consistent with in silico docking calculations in a homology model that orient the long axis of etomidate approximately orthogonal to the transmembrane axis.

Alcohol abuse and dependence have a staggering socio-economic impact, yet current therapeutic strategies are largely inadequate to treat these disorders. Thus, the development of new strategies that can effectively prevent alcohol use disorders is of paramount importance. Currently approved medications attempt to deter alcohol intake by blocking EtOH metabolism or by targeting the neurochemical systems downstream of the cascades leading to craving and dependence. Unfortunately, these medications have provided only limited success as indicated by the continued high rates of alcohol abuse and alcoholism. The lack of currently available effective treatment strategies is highlighted by the urgent call by the NIAAA to find new and paradigm-changing therapeutics to either prevent or treat alcohol-related problems. The present mini-review highlights recent findings from four laboratories with a focus on compounds that have the potential to be novel therapeutic agents that can be developed for the prevention and/or treatment of alcohol use disorders.

GABA(A) receptor-mediated function was studied in rats treated with chronic intermittent ethanol (CIE). Rats were given 60 doses of 6g/kg ethanol every 24 h by gastric intubation, with repeated intoxicating and withdrawal episodes leading to a kindling-like increase in seizure susceptibility (Kokka et al., Alcohol: Clin. Exp. Res., 17 (1993) 525-531). Efflux of 36Cl-, evoked by application of muscimol, a measure of GABA(A) receptor function, was examined in 300 mu m slices obtained from frontal, parietal, and temporal cortex, hippocampus, and inferior colliculus, one day after the last administration of ethanol. Compared to controls, the 36Cl- efflux in hippocampal slices of CIE rats was significantly reduced by 29%, while there were no changes in the other brain regions studied. In hippocampal slices, paired-pulse inhibition in CA1 pyramidal neurons, measured extracellularly using homosynaptic orthodromic stimulation at an interval of 10 ms, was significantly reduced in CIE rats. A significant decrease by 40% both at 2 and 40 days after 60 doses of ethanol was found, implying a persistent decrease in GABA(A) receptor-mediated inhibition in CIE rats. These reductions in paired-pulse inhibition are consistent with the decrease in the pentylenetetrazol (PTZ) seizure threshold which was previously observed in CIE rats. Therefore, we suggest that this reduction of GABA(A) receptor-mediated inhibition contributes to the persistent increase in seizure susceptibility of CIE rats.

The effects of varied durations of cocaine (1, 3 or 5 days) on muscarinic (MSC) and benzodiazepine (BZD) binding sites in striatum and hippocampus were investigated using homogenate receptor binding. The progressive alterations in these receptor sites from a 5 day cocaine administration were also examined 12 h, 2 days or 21 days after drug exposure. Neither a one nor a three day exposure to cocaine produced any long-term alteration in BZD binding in either structure whereas a 5 day administration produced significant increases in binding. Decreases in MSC receptor binding were apparent in striatum from either a 3 or 5 day cocaine exposure and in hippocampus from a 5 day exposure. The 5 day cocaine exposure produced immediate increases in striatal and hippocampal BZD binding which persisted for 21 days. Conversely, 5 days of cocaine produced a short-term increase in MSC receptor binding in both structures which then became significantly decreased 21 days later. Based on the divergent pattern of changes in BZD and MSC receptor types over time in these structures, it appears that cocaine may induce such changes via separate mechanisms. In addition, it is apparent that changes in the numbers of these receptor sites after cocaine exposure can be quite dynamic, changing rapidly over time.