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.     

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.     

The Ataxia (ax J) Mutation Causes Abnormal GABAA Receptor Turnover in Mice

Ataxia represents a pathological coordination failure that often involves functional disturbances in cerebellar circuits. Purkinje cells (PCs) characterize the only output neurons of the cerebellar cortex and critically participate in regulating motor coordination. Although different genetic mutations are known that cause ataxia, little is known about the underlying cellular mechanisms. Here we show that a mutated ax ^J gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover. Ax ^J mouse mutants, characterized by cerebellar ataxia, display both increased GABA_A receptor (GABA_AR) levels at PC surface membranes accompanied by enlarged IPSCs. Accordingly, we identify physical interaction of Usp14 and the GABA_AR α1 subunit. Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABA_AR turnover at cerebellar synapses contributes to ax ^J-mediated behavioural impairment.     

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.     

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.     

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.     

Altered Cortical GABAA Receptor Composition,Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome

Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABA_A receptors (GABA_ARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Het_α1KO) of the human epilepsy gene, the GABA_AR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Het_α1KO on the expression and physiology of GABA_ARs in the mouse cortex. We found that Het_α1KO caused modest reductions in the total and surface expression of the β2 subunit but did not alter β1 or β3 subunit expression, results consistent with a small reduction of GABA_ARs. Cortices partially compensated for Het_α1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Het_α1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3βγ receptors. Patch clamp electrophysiology studies showed that Het_α1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Het_α1KO reduced base-line GABA_AR endocytosis, an effect that probably contributes to the observed changes in GABA_AR expression. These findings demonstrate that Het_α1KO exerts two principle disinhibitory effects on cortical GABA_AR-mediated inhibitory neurotransmission: 1) a modest reduction of GABA_AR number and 2) a partial compensation with GABA_AR isoforms that possess physiological properties different from those of the otherwise predominant α1βγ GABA_ARs.     

Differential Modulation of GABAA Receptors Underlies Postsynaptic Depolarization- and Purinoceptor-Mediated Enhancement of Cerebellar Inhibitory Transmission: A Non-Stationary Fluctuation Analysis Study

Cerebellar GABAergic inhibitory transmission between interneurons and Purkinje cells (PCs) undergoes a long-lasting enhancement following different stimulations, such as brief depolarization or activation of purinergic receptors of postsynaptic PCs. The underlying mechanisms, however, are not completely understood. Using a peak-scaled non-stationary fluctuation analysis, we therefore aimed at characterizing changes in the electrophysiological properties of GABA_A receptors in PCs of rat cerebellar cortex during depolarization-induced “rebound potentiation (RP)” and purinoceptor-mediated long-term potentiation (PM-LTP), because both RP and PM-LTP likely depend on postsynaptic mechanisms. Stimulation-evoked inhibitory postsynaptic currents (eIPSCs) were recorded from PCs in neonatal rat cerebellar slices. Our analysis showed that postsynaptic membrane depolarization induced RP of eIPSCs in association with significant increase in the number of synaptic GABA_A receptors without changing the channel conductance. By contrast, bath application of ATP induced PM-LTP of eIPSCs with a significant increase of the channel conductance of GABA_A receptors without affecting the receptor number. Pretreatment with protein kinase A (PKA) inhibitors, H-89 and cAMPS-Rp, completely abolished the PM-LTP. The CaMKII inhibitor KN-62 reported to abolish RP did not alter PM-LTP. These results suggest that the signaling mechanism underlying PM-LTP could involve ATP-induced phosphorylation of synaptic GABA_A receptors, thereby resulting in upregulation of the channel conductance by stimulating adenylyl cyclase-PKA signaling cascade, possibly via activation of P2Y₁₁ purinoceptor. Thus, our findings reveal that postsynaptic GABA_A receptors at the interneuron-PC inhibitory synapses are under the control of two distinct forms of long-term potentiation linked with different second messenger cascades.     

Time- and Behavioral State-Dependent Changes in Posterior Hypothalamic GABAA Receptors Contribute to the Regulation of Sleep

Sleep-wake behavior is regulated by a circadian rhythm, homeostatically and by additional mechanisms that determine the timing of slow-wave sleep and rapid eye movement sleep (REMS) episodes. The posterior hypothalamus coordinates the neural and humoral signals with the rest-activity cycle. It contains wake-active neurons, and is a site where stimulation of inhibitory GABA_A receptors promotes sleep, whereas their antagonism enhances wakefulness. We explored whether GABAergic mechanisms present in the posterior hypothalamus contribute to the homeostatic and other aspects of sleep-wake regulation. Using micropunches of tissue extracted from either the perifornical (PF) or dorsomedial (DM) regions of the posterior hypothalamus of rats, we determined that mRNA levels for selected subunits of GABA_A receptors (β1, β3 and ε) were higher at the end of the active period or following sleep deprivation, when the need for sleep is high, than after several hours of sleep, when sleep need is partially fulfilled. Such a pattern was present in the PF region only, and was consistent with changes in β1 subunit and GABA synthesizing enzyme (GAD) protein levels. In contrast, in the DM region, the levels of GABA_A receptor subunit mRNAs and proteins (α1, α2, β1) and GAD varied with circadian time, but were not responsive to sleep deprivation. Separate experiments with sleep-wake monitoring and local perfusion of the PF region with the GABA_A receptor antagonist bicuculline revealed that the antagonist had a weaker sleep-reducing effect when sleep need was enhanced by sleep deprivation and that the increased amount of REMS characteristic of the late sleep period was dependent on endogenous GABAergic inhibition. These results support the concept that a varying magnitude of GABAergic inhibition exerted within the PF region contributes to the homeostatic regulation of sleep and shapes its temporal pattern, whereas GABAergic mechanisms in the DM region contribute to circadian regulation.     

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.     

The cell adhesion molecule neuroplastin-65 is a novel interaction partner of γ-aminobutyric acid type A receptors

γ-Aminobutyric acid type A (GABA_A) receptors are pentameric ligand-gated ion channels that mediate fast inhibition in the central nervous system. Depending on their subunit composition, these receptors exhibit distinct pharmacological properties and differ in their ability to interact with proteins involved in receptor anchoring at synaptic or extra-synaptic sites. Whereas GABA_A receptors containing α1, α2, or α3 subunits are mainly located synaptically where they interact with the submembranous scaffolding protein gephyrin, receptors containing α5 subunits are predominantly found extra-synaptically and seem to interact with radixin for anchorage. Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that is involved in hippocampal synaptic plasticity. Our results reveal that neuroplastin and GABA_A receptors can be co-purified from rat brain and exhibit a direct physical interaction as demonstrated by co-precipitation and Förster resonance energy transfer (FRET) analysis in a heterologous expression system. The brain-specific isoform neuroplastin-65 co-localizes with GABA_A receptors as shown in brain sections as well as in neuronal cultures, and such complexes can either contain gephyrin or be devoid of gephyrin. Neuroplastin-65 specifically co-localizes with α1 or α2 but not with α3 subunits at GABAergic synapses. In addition, neuroplastin-65 also co-localizes with GABA_A receptor α5 subunits at extra-synaptic sites. Down-regulation of neuroplastin-65 by shRNA causes a loss of GABA_A receptor α2 subunits at GABAergic synapses. These results suggest that neuroplastin-65 can co-localize with a subset of GABA_A receptor subtypes and might contribute to anchoring and/or confining GABA_A receptors to particular synaptic or extra-synaptic sites, thus affecting receptor mobility and synaptic strength.     

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.     

Structure of Excitatory Synapses and GABAA Receptor Localization at Inhibitory Synapses Are Regulated by Neuroplastin-65

Formation, maintenance, and activity of excitatory and inhibitory synapses are essential for neuronal network function. Cell adhesion molecules (CAMs) are crucially involved in these processes. The CAM neuroplastin-65 (Np65) highly expressed during periods of synapse formation and stabilization is present at the pre- and postsynaptic membranes. Np65 can translocate into synapses in response to electrical stimulation and it interacts with subtypes of GABA_A receptors in inhibitory synapses. Here, we report that in the murine hippocampus and in hippocampal primary culture, neurons of the CA1 region and the dentate gyrus (DG) express high Np65 levels, whereas expression in CA3 neurons is lower. In neuroplastin-deficient (Np^−/−) mice the number of excitatory synapses in CA1 and DG, but not CA3 regions is reduced. Notably this picture is mirrored in mature Np^−/− hippocampal cultures or in mature CA1 and DG wild-type (Np^+/+) neurons treated with a function-blocking recombinant Np65-Fc extracellular fragment. Although the number of GABAergic synapses was unchanged in Np^−/− neurons or in mature Np65-Fc-treated Np^+/+ neurons, the ratio of excitatory to inhibitory synapses was significantly lower in Np^−/− cultures. Furthermore, GABA_A receptor composition was altered at inhibitory synapses in Np^−/− neurons as the α1 to α2 GABA_A receptor subunit ratio was increased. Changes of excitatory and inhibitory synaptic function in Np^−/− neurons were confirmed evaluating the presynaptic release function and using patch clamp recording. These data demonstrate that Np65 is an important regulator of the number and function of synapses in the hippocampus.     

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid,Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes

Propofol, an intravenous anesthetic, is a positive modulator of the GABA_A receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABA_A receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABA_A receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABA_A receptor subunit protection. This investigation demonstrates striking interfacial GABA_A receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity.     

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.     

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.     

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.