GABAA Receptor α1 Subunit Mutation A322D Associated with Autosomal Dominant Juvenile Myoclonic Epilepsy Reduces the Expression and Alters the Composition of Wild Type GABAA Receptors

A GABA_A receptor (GABA_AR) α1 subunit mutation, A322D (AD), causes an autosomal dominant form of juvenile myoclonic epilepsy (ADJME). Previous studies demonstrated that the mutation caused α1(AD) subunit misfolding and rapid degradation, reducing its total and surface expression substantially. Here, we determined the effects of the residual α1(AD) subunit expression on wild type GABA_AR expression to determine whether the AD mutation conferred a dominant negative effect. We found that although the α1(AD) subunit did not substitute for wild type α1 subunits on the cell surface, it reduced the surface expression of α1β2γ2 and α3β2γ2 receptors by associating with the wild type subunits within the endoplasmic reticulum and preventing them from trafficking to the cell surface. The α1(AD) subunit reduced surface expression of α3β2γ2 receptors by a greater amount than α1β2γ2 receptors, thus altering cell surface GABA_AR composition. When transfected into cultured cortical neurons, the α1(AD) subunit altered the time course of miniature inhibitory postsynaptic current kinetics and reduced miniature inhibitory postsynaptic current amplitudes. These findings demonstrated that, in addition to causing a heterozygous loss of function of α1(AD) subunits, this epilepsy mutation also elicited a modest dominant negative effect that likely shapes the epilepsy phenotype.     

Agonist-dependent Endocytosis of γ-Aminobutyric Acid Type A (GABAA) Receptors Revealed by a γ2(R43Q) Epilepsy Mutation

GABA-gated chloride channels (GABA_ARs) trafficking is involved in the regulation of fast inhibitory transmission. Here, we took advantage of a γ2(R43Q) subunit mutation linked to epilepsy in humans that considerably reduces the number of GABA_ARs on the cell surface to better understand the trafficking of GABA_ARs. Using recombinant expression in cultured rat hippocampal neurons and COS-7 cells, we showed that receptors containing γ2(R43Q) were addressed to the cell membrane but underwent clathrin-mediated dynamin-dependent endocytosis. The γ2(R43Q)-dependent endocytosis was reduced by GABA_AR antagonists. These data, in addition to a new homology model, suggested that a conformational change in the extracellular domain of γ2(R43Q)-containing GABA_ARs increased their internalization. This led us to show that endogenous and recombinant wild-type GABA_AR endocytosis in both cultured neurons and COS-7 cells can be amplified by their agonists. These findings revealed not only a direct relationship between endocytosis of GABA_ARs and a genetic neurological disorder but also that trafficking of these receptors can be modulated by their agonist.     

Complex Role of Collybistin and Gephyrin in GABAA Receptor Clustering

Gephyrin and collybistin are key components of GABA_A receptor (GABA_AR) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA_AR subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA_AR α2 and α3 subunit intracellular M3–M4 domain (but not α1, α4, α5, α6, β1–3, or γ1–3) with gephyrin. Curiously, GABA_AR α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA_AR α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA_AR α3 interacts with gephyrin, GABA_AR α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA_AR α2 and collybistin or GABA_AR α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA_AR α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA_AR α2 is capable of “activating ” collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA_AR α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA_AR and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABA_ARs, but not GlyRs or other GABA_AR subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA_ARs containing the α2 subunit.     

Synaptic Competition Sculpts the Development of GABAergic Axo-Dendritic but Not Perisomatic Synapses

The neurotransmitter GABA regulates many aspects of inhibitory synapse development. We tested the hypothesis that GABA_A receptors (GABA_ARs) work together with the synaptic adhesion molecule neuroligin 2 (NL2) to regulate synapse formation in different subcellular compartments. We investigated mice (“γ2 knockdown mice”) with an engineered allele of the GABA_AR γ2 subunit gene which produced a mosaic expression of synaptic GABA_ARs in neighboring neurons, causing a strong imbalance in synaptic inhibition. Deletion of the γ2 subunit did not abolish synapse formation or the targeting of NL2 to distinct types of perisomatic and axo-dendritic contacts. Thus synaptic localization of NL2 does not require synaptic GABA_ARs. However, loss of the γ2 subunit caused a selective decrease in the number of axo-dendritic synapses on cerebellar Purkinje cells and cortical pyramidal neurons, whereas perisomatic synapses were not significantly affected. Notably, γ2-positive cells had increased axo-dendritic innervation compared with both γ2-negative and wild-type counterparts. Moreover heterologous synapses on spines, that are found after total deletion of GABA_ARs from all Purkinje cells, were rare in cerebella of γ2 knockdown mice. These findings reveal a selective role of γ2 subunit-containing GABA_ARs in regulating synapse development in distinct subcellular compartments, and support the hypothesis that the refinement of axo-dendritic synapses is regulated by activity-dependent competition between neighboring neurons.     

Ring Finger Protein 34 (RNF34) Interacts with and Promotes γ-Aminobutyric Acid Type-A Receptor Degradation via Ubiquitination of the γ2 Subunit

We have found that the large intracellular loop of the γ2 GABA_A receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABA_ARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABA_AR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2. Mutating several lysines of the γ2IL into arginines makes the γ2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the γ2 subunit when α1 and β3 subunits are co-assembled with γ2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of γ2 GABA_AR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased γ2 GABA_AR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic γ2-GABA_AR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the γ2-GABA_AR subunit promoting GABA_AR degradation.     

Fragrant Dioxane Derivatives Identify β1-Subunit-containing GABAA Receptors

Nineteen GABA_A receptor (GABA_AR) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of β1-subunit-containing GABA_ARs is unknown. Here we report the discovery of a new structural class of GABA_AR positive modulators with unique β1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed α1βxγ2L (x-for 1,2,3) GABA_AR FDD were 6 times more potent at β1- versus β2- and β3-containing receptors. Serine at position 265 was essential for the high sensitivity of the β1-subunit to FDD and the β1N286W mutation nearly abolished modulation; vice versa the mutation β3N265S shifted FDD sensitivity toward the β1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to β1-negative cerebellar Purkinje neurons. Immunostaining for the β1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by β1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of β1-containing GABA_ARs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA_ARs.     

γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains

The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABA_A receptors (GABA_ARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABA_ARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type of α/β subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, β2, and γ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs and α1/β2/γ2-expressing HEK293 cells, the α1, β2, or γ2 ECD each caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABA_ARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific.     

GABAA Receptor α and γ Subunits Shape Synaptic Currents via Different Mechanisms

Synaptic GABA_A receptors (GABA_ARs) mediate most of the inhibitory neurotransmission in the brain. The majority of these receptors are comprised of α1, β2, and γ2 subunits. The amygdala, a structure involved in processing emotional stimuli, expresses α2 and γ1 subunits at high levels. The effect of these subunits on GABA_AR-mediated synaptic transmission is not known. Understanding the influence of these subunits on GABA_AR-mediated synaptic currents may help in identifying the roles and locations of amygdala synapses that contain these subunits. Here, we describe the biophysical and synaptic properties of pure populations of α1β2γ2, α2β2γ2, α1β2γ1 and α2β2γ1 GABA_ARs. Their synaptic properties were examined in engineered synapses, whereas their kinetic properties were studied using rapid agonist application, and single channel recordings. All macropatch currents activated rapidly (<1 ms) and deactivated as a function of the α-subunit, with α2-containing GABA_ARs consistently deactivating ∼10-fold more slowly. Single channel analysis revealed that the slower current decay of α2-containing GABA_ARs was due to longer burst durations at low GABA concentrations, corresponding to a ∼4-fold higher affinity for GABA. Synaptic currents revealed a different pattern of activation and deactivation to that of macropatch data. The inclusion of α2 and γ1 subunits slowed both the activation and deactivation rates, suggesting that receptors containing these subunits cluster more diffusely at synapses. Switching the intracellular domains of the γ2 and γ1 subunits substantiated this inference. Because this region determines post-synaptic localization, we hypothesize that GABA_ARs containing γ1 and γ2 use different mechanisms for synaptic clustering.     

Increases in [3H]Muscimol and [3H]Flumazenil Binding in the Dorsolateral Prefrontal Cortex in Schizophrenia Are Linked to α4 and γ2S mRNA Levels Respectively

Background

GABA_A receptors (GABA_AR) are composed of several subunits that determine sensitivity to drugs, synaptic localisation and function. Recent studies suggest that agonists targeting selective GABA_AR subunits may have therapeutic value against the cognitive impairments observed in schizophrenia. In this study, we determined whether GABA_AR binding deficits exist in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia and tested if changes in GABA_AR binding are related to the changes in subunit mRNAs. The GABA orthosteric and the benzodiazepine allosteric binding sites were assessed autoradiographically using [³H]Muscimol and [³H]Flumazenil, respectively, in a large cohort of individuals with schizophrenia (n = 37) and their matched controls (n = 37). We measured, using qPCR, mRNA of β (β1, β2, β3), γ (γ1, γ2, γ2S for short and γ2L for long isoform, γ3) and δ subunits and used our previous measurements of GABA_AR α subunit mRNAs in order to relate mRNAs and binding through correlation and regression analysis.

Results

Significant increases in both [³H]Muscimol (p = 0.016) and [³H]Flumazenil (p = 0.012) binding were found in the DLPFC of schizophrenia patients. Expression levels of mRNA subunits measured did not show any significant difference in schizophrenia compared to controls. Regression analysis revealed that in schizophrenia, the [³H]Muscimol binding variance was most related to α4 mRNA levels and the [³H]Flumazenil binding variance was most related to γ2S subunit mRNA levels. [³H]Muscimol and [³H]Flumazenil binding were not affected by the lifetime anti-psychotics dose (chlorpromazine equivalent).

Conclusions

We report parallel increases in orthosteric and allosteric GABA_AR binding sites in the DLPFC in schizophrenia that may be related to a “shift” in subunit composition towards α4 and γ2S respectively, which may compromise normal GABAergic modulation and function. Our results may have implications for the development of treatment strategies that target specific GABA_AR receptor subunits.     

Methyl CpG Binding Protein 2 Gene Disruption Augments Tonic Currents of γ-Aminobutyric Acid Receptors in Locus Coeruleus Neurons: IMPACT ON NEURONAL EXCITABILITY AND BREATHING*

People with Rett syndrome and mouse models show autonomic dysfunction involving the brain stem locus coeruleus (LC). Neurons in the LC of Mecp2-null mice are overly excited, likely resulting from a defect in neuronal intrinsic membrane properties and a deficiency in GABA synaptic inhibition. In addition to the synaptic GABA receptors, there is a group of GABA_A receptors (GABA_ARs) that is located extrasynaptically and mediates tonic inhibition. Here we show evidence for augmentation of the extrasynaptic GABA_ARs in Mecp2-null mice. In brain slices, exposure of LC neurons to GABA_AR agonists increased tonic currents that were blocked by GABA_AR antagonists. With 10 μm GABA, the bicuculline-sensitive tonic currents were ∼4-fold larger in Mecp2-null LC neurons than in the WT. Single-cell PCR analysis showed that the δ subunit, the principal subunit of extrasynaptic GABA_ARs, was present in LC neurons. Expression levels of the δ subunit were ∼50% higher in Mecp2-null neurons than in the WT. Also increased in expression in Mecp2-null mice was another extrasynaptic GABA_AR subunit, α6, by ∼4-fold. The δ subunit-selective agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride and 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]]benzamide activated the tonic GABA_A currents in LC neurons and reduced neuronal excitability to a greater degree in Mecp2-null mice than in the WT. Consistent with these findings, in vivo application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride alleviated breathing abnormalities of conscious Mecp2-null mice. These results suggest that extrasynaptic GABA_ARs seem to be augmented with Mecp2 disruption, which may be a compensatory response to the deficiency in GABAergic synaptic inhibition and allows control of neuronal excitability and breathing abnormalities.     

Characteristic Expressions of GABA Receptors and GABA Producing/Transporting Molecules in Rat Kidney

Gamma-aminobutyric acid (GABA) is an important neurotransmitter, but recent reports have revealed the expression of GABAergic components in peripheral, non-neural tissues. GABA administration induces natriuresis and lowers blood pressure, suggesting renal GABA targets. However, systematic evaluation of renal GABAergic components has not been reported. In this study, kidney cortices of Wistar-Kyoto rats (WKY) were used to assay for messenger RNAs of GABA-related molecules using RT-PCR. In WKY kidney cortex, GABA_A receptor subunits, α1, β3, δ, ε and π, in addition to both types of GABA_B receptors, R1 and R2, and GABA_C receptor ρ1 and ρ2 subunit mRNAs were detected. Kidney cortex also expressed mRNAs of glutamate decarboxylase (GAD) 65, GAD67, 4-aminobutyrate aminotransferase and GABA transporter, GAT2. Western blot and/or immunohistochemistry were performed for those molecules detected by RT-PCR. By immunofluorescent observation, co-staining of α1, β3, and π subunits was observed mainly on the apical side of cortical tubules, and immunoblot of kidney protein precipitated with π subunit antibody revealed α1 and β3 subunit co-assembly. This is the first report of GABA_A receptor π subunit in the kidney. In summary, unique set of GABA receptor subunits and subtypes were found in rat kidney cortex. As GABA producing enzymes, transporters and degrading enzyme were also detected, a possible existence of local renal GABAergic system with an autocrine/paracrine mechanism is suggested.     

Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABA_ARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABA_AR modulation. [³H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[³H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABA_AR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([³H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABA_ARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [³H]AziPm photoincorporation into GABA_AR subunits maximally by ∼50%. When the amino acids photolabeled by [³H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [³H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [³H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[³H]mTFD-MPAB. The propofol-inhibitable [³H]AziPm photolabeling in the GABA_AR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABA_AR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.     

Homeostatic Competition between Phasic and Tonic Inhibition

The GABA_A receptors are the major inhibitory receptors in the brain and are localized at both synaptic and extrasynaptic membranes. Synaptic GABA_A receptors mediate phasic inhibition, whereas extrasynaptic GABA_A receptors mediate tonic inhibition. Both phasic and tonic inhibitions regulate neuronal activity, but whether they regulate each other is not very clear. Here, we investigated the functional interaction between synaptic and extrasynaptic GABA_A receptors through various molecular manipulations. Overexpression of extrasynaptic α6β3δ-GABA_A receptors in mouse hippocampal pyramidal neurons significantly increased tonic currents. Surprisingly, the increase of tonic inhibition was accompanied by a dramatic reduction of the phasic inhibition, suggesting a possible homeostatic regulation of the total inhibition. Overexpressing the α6 subunit alone induced an up-regulation of δ subunit expression and suppressed phasic inhibition similar to overexpressing the α6β3δ subunits. Interestingly, blocking all GABA_A receptors after overexpressing α6β3δ receptors could not restore the synaptic GABAergic transmission, suggesting that receptor activation is not required for the homeostatic interplay. Furthermore, insertion of a gephyrin-binding-site (GBS) into the α6 and δ subunits recruited α6_GBSβ3δ_GBS receptors to postsynaptic sites but failed to rescue synaptic GABAergic transmission. Thus, it is not the positional effect of extrasynaptic α6β3δ receptors that causes the down-regulation of phasic inhibition. Overexpressing α5β3γ2 subunits similarly reduced synaptic GABAergic transmission. We propose a working model that both synaptic and extrasynaptic GABA_A receptors may compete for limited receptor slots on the plasma membrane to maintain a homeostatic range of the total inhibition.     

The Direct Actions of GABA, 2’-Methoxy-6-Methylflavone and General Anaesthetics at β3γ2L GABAA Receptors: Evidence for Receptors with Different Subunit Stoichiometries

2’-Methoxy-6-methylflavone (2’MeO6MF) is an anxiolytic flavonoid which has been shown to display GABA_A receptor (GABA_AR) β2/3-subunit selectivity, a pharmacological profile similar to that of the general anaesthetic etomidate. Electrophysiological studies suggest that the full agonist action of 2’MeO6MF at α2β3γ2L GABA_ARs may mediate the flavonoid’s in vivo effects. However, we found variations in the relative efficacy of 2’MeO6MF (2’MeO6MF-elicited current responses normalised to the maximal GABA response) at α2β3γ2L GABA_ARs due to the presence of mixed receptor populations. To understand which receptor subpopulation(s) underlie the variations observed, we conducted a systematic investigation of 2’MeO6MF activity at all receptor combinations that could theoretically form (α2, β3, γ2L, α2β3, α2γ2L, β3γ2L and α2β3γ2L) in Xenopus oocytes using the two-electrode voltage clamp technique. We found that 2’MeO6MF activated non-α-containing β3γ2L receptors. In an attempt to establish the optimal conditions to express a uniform population of these receptors, we found that varying the relative amounts of β3:γ2L subunit mRNAs resulted in differences in the level of constitutive activity, the GABA concentration-response relationships, and the relative efficacy of 2’MeO6MF activation. Like 2’MeO6MF, general anaesthetics such as etomidate and propofol also showed distinct levels of relative efficacy across different injection ratios. Based on these results, we infer that β3γ2L receptors may form with different subunit stoichiometries, resulting in the complex pharmacology observed across different injection ratios. Moreover, the discovery that GABA and etomidate have direct actions at the α-lacking β3γ2L receptors raises questions about the structural requirements for their respective binding sites at GABA_ARs.     

Partial Agonism of Taurine at Gamma-Containing Native and Recombinant GABAA Receptors

Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABA_A-receptors (GABA_AR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABA_AR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α_1/2β_1/3 receptors taurine was as efficient as GABA, whereas incorporation of the γ_1/2 subunit reduced taurine efficacy to 60–90% of GABA. The mutation γ_2F77I, which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant α_xβ₁δ-GABA_AR, we generated a chimeric γ₂ subunit carrying the δ subunit motif around F77 (MTVFLH). At α_1/2β₁γ_2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at β₃-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine’s partial agonism at γ-containing GABA_A receptors. Our study sheds new light on the β₁ subunit conferring the widest range of taurine-efficacies modifying GABA_AR function under (patho)physiological conditions.     

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β? Interface

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABA_ARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABA_AR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABA_AR responses. In the α1β3γ2 GABA_AR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ⁺-β⁻ subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β⁺-α⁻ subunit interfaces. GABA inhibits S-[³H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[³H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ⁺-β⁻ site, based upon the distance in GABA_AR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABA_AR function and provide a first demonstration that an intersubunit-binding site in the GABA_AR transmembrane domain binds negative and positive allosteric modulators.     

Neurotoxins from Snake Venoms and α-Conotoxin ImI Inhibit Functionally Active Ionotropic γ-Aminobutyric Acid (GABA) Receptors

Ionotropic receptors of γ-aminobutyric acid (GABA_AR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the α1β3γ2 receptor; and at 10 μm α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC₅₀ = 236 nm) and less potently inhibited α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ GABA_ARs. The α1β3γ2 receptor was also inhibited by some other three-finger toxins, long α-neurotoxin Ls III and nonconventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx-binding sites overlap with the orthosteric sites at the β/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABA_AR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABA_AR is played by the tip of its central loop II accommodating under loop C of the receptors.     

Daily Isoflurane Exposure Increases Barbiturate Insensitivity in Medullary Respiratory and Cortical Neurons via Expression of ε-Subunit Containing GABA ARs

The parameters governing GABA_A receptor subtype expression patterns are not well understood, although significant shifts in subunit expression may support key physiological events. For example, the respiratory control network in pregnant rats becomes relatively insensitive to barbiturates due to increased expression of ε-subunit-containing GABA_ARs in the ventral respiratory column. We hypothesized that this plasticity may be a compensatory response to a chronic increase in inhibitory tone caused by increased central neurosteroid levels. Thus, we tested whether increased inhibitory tone was sufficient to induce ε-subunit upregulation on respiratory and cortical neurons in adult rats. Chronic intermittent increases in inhibitory tone in male and female rats was induced via daily 5-min exposures to 3% isoflurane. After 7d of treatment, phrenic burst frequency was less sensitive to barbiturate in isoflurane-treated male and female rats in vivo. Neurons in the ventral respiratory group and cortex were less sensitive to pentobarbital in vitro following 7d and 30d of intermittent isoflurane-exposure in both male and female rats. The pentobarbital insensitivity in 7d isoflurane-treated rats was reversible after another 7d. We hypothesize that increased inhibitory tone in the respiratory control network and cortex causes a compensatory increase in ε-subunit-containing GABA_ARs.     

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

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