GABA Binding Research Articles

PCDH19 interplay with GABA(A) receptors: a window to DEE9 pathogenetic mechanisms.

PCDH19 interplay with GABA(A) receptors: a window to DEE9 pathogenetic mechanisms.

Punicalagin and ellagic acid containing Punica granatum L. fruit rind extract prevents vincristine-induced neuropathic pain in rats: an in silico and in vivo evidence of GABAergic action and cytokine inhibition

ABSTRACT Objectives: We aimed to investigate the protective potential of Punica granatum L. fruit rind extract (PFE) containing punicalagin (10.3% W/W), ellagic acid (EA) (2.7%W/W) in vincristine (75 µg/kg i.p.)- induced neuropathic pain in Wistar rats. Methods: Docking simulation studies were done on the three-dimensional (3D) structure of the GABAA and PPAR γ receptor for the binding of EA as well as punicalagin docking studies on TNF-α, and IL-6. The Present Study conceptualized a test battery to evaluate the behavioral, biochemical and histological changes. Results: Vincristine -induced significant cold allodynia, mechanical hyperalgesia, and functional deficit on 12th and 21st days. It also increased in the levels of TNF-α (Tumor necrosis factor-α), IL-6 (Interleukin-6), and MPO (Myeloperoxidase). Administration of PFE (100 and 300 mg/kg, p.o.), EA (50 mg/kg), and gabapentin (100 mg/kg) attenuated Vincristine-induced behavioral and biochemical changes significantly (P < .05). PFE showed better antinociceptive activity to EA. The histopathological evaluation also revealed the protective effects of PFE. Pretreatment of bicuculline (selective antagonist of GABAA receptors) reversed antinociceptive action of PFE, but administration of γ aminobutyric acid potentiated the action of PFE. PPAR-γ antagonist BADGE did not modify the effect of PFE. Docking results revealed that EA properly positioned into GABA and PPARγ binding site and acts as a partial agonist. Docking score of Punicalagin found to be − 9.02 kcal/mol and - 8.32 kcal/mol on IL-6 and TNFα respectively. Discussion: Conclusively, the attenuating effect of PFE may be attributed to the GABAergic system, cytokine inhibition, and anti-inflammatory activities.

Neuroepithelial cells (NECs) and mucous cells express a variety of neurotransmitters and neurotransmitter receptors in the gill and respiratory air-sac of the catfish Heteropneustes fossilis (Siluriformes, Heteropneustidae): a possible role in local immune defence

Heteropneustes fossilis is an air-breathing teleost inhabiting environments with very poor O2 conditions, and so it has evolved to cope with hypoxia. In the gills and respiratory air-sac, the sites for O2 sensing and the response to hypoxia rely on the expression of acetylcholine (Ach) acting via its nicotinic receptor (nAChR). This study examined the expression patterns of neuronal markers and some compounds in the NECs of the gills and respiratory air sac having an immunomodulatory function in mammalian lungs. Mucous cells, epithelial cells and neuroepithelial cells (NECs) were immunopositive to a variety of both neuronal markers (VAChT, nAChR, GABA-B-R1 receptor, GAD679) and the antimicrobial peptide piscidin, an evolutionary conserved humoral component of the mucosal immune system in fish. We speculate that Ach release via nAChR from mucous cells may be modulated by GABA production in the NECs and it is required for the induction of mucus production in both normoxic and hypoxic conditions. The presence of piscidin in mucous cells may act in synergy with the autocrine/paracrine signals of Ach and GABA binding to GABA B R1B receptor that may play a local immunomodulatory function in the mucous epithelia of the gills and the respiratory air sac. The potential role of the NECs in the immunobiological behaviour of the gill/air-sac is at moment a matter of speculation. The extent to which the NECs as such may participate is elusive at this stage and waits investigation.

Fluoroquinolones-Associated Disability: It Is Not All in Your Head

Fluoroquinolones (FQs) are a broad class of antibiotics typically prescribed for bacterial infections, including infections for which their use is discouraged. The FDA has proposed the existence of a permanent disability (Fluoroquinolone Associated Disability; FQAD), which is yet to be formally recognized. Previous studies suggest that FQs act as selective GABAA receptor inhibitors, preventing the binding of GABA in the central nervous system. GABA is a key regulator of the vagus nerve, involved in the control of gastrointestinal (GI) function. Indeed, GABA is released from the Nucleus of the Tractus Solitarius (NTS) to the Dorsal Motor Nucleus of the vagus (DMV) to tonically regulate vagal activity. The purpose of this review is to summarize the current knowledge on FQs in the context of the vagus nerve and examine how these drugs could lead to dysregulated signaling to the GI tract. Since there is sufficient evidence to suggest that GABA transmission is hindered by FQs, it is reasonable to postulate that the vagal circuit could be compromised at the NTS-DMV synapse after FQ use, possibly leading to the development of permanent GI disorders in FQAD.

The β2 subunit E155 residue as a proton sensor at the binding site on GABA type A receptors

GABA type A receptor plays a key role in inhibitory signaling in the adult central nervous system. This receptor can be modulated by protons but the underlying molecular mechanisms have not been fully explored. To find possible pH-sensor residues, a comparative study for proton-activated GLIC channel and α1β2γ2 GABA receptor was performed and pK ′s of respective residues were estimated by numerical algorithms which consider local interactions. β E155, located at the GABA binding site, showed pKa values close to physiological values and dependence on the receptor state and ligation, suggesting a role in modulation by pH. To validate this prediction, pH sensitivity of current responses to GABA was investigated using patch-clamp technique for WT and mutated (β2E155[C, S, Q, L]) GABA receptors. Cysteine mutation preserved pH sensitivity. However, for remaining mutants, the sensitivity to acidification (pH = 6.0) was reduced becoming not statistically significant. The effect of alkaline pH (8.0) was maintained for all mutants with exception for β2E155L for which it was nearly abolished. To further explore the impact of considered mutations, molecular docking was performed which indicated that pH modulation is probably affected by interplay between binding site residues, zwitterion GABA and protons. These data, altogether, indicate that mutation of β2E155 to hydrophobic residue (L) maximally impaired pH modulation while for polar substitutions the effect was smaller. In conclusion, our data provide evidence that a key binding site residue β2E155 plays an important role in proton sensitivity of GABA receptor.

Nephrotoxicity and neurotoxicity following star fruit (Averrhoa carambola) ingestion: a narrative review.

In recent times, star fruit (Averrhoa carambola) nephrotoxicity and neurotoxicity have been increasingly reported, both in individuals with pre-existing renal disease and those with previously normal renal function. We summarise the clinical findings of star fruit toxicity in humans and outline the important pathogenetic insights provided by animal studies. Google Scholar, EMBASE, Scopus and PubMed were searched from 1995 through July 2020 for case reports/series on renal or neurological manifestations of star fruit toxicity in humans and mechanisms of star fruit toxicity in animal studies. Ten case series and 28 case reports in humans (total number of individuals=136) were included and 8 animal studies were analysed. Ninety-four (69.1%) patients had prior renal impairment. Renal histology showed acute oxalate nephropathy with tubulointerstitial nephritis or tubular necrosis. Neurotoxicity manifestations ranged from hiccups to status epilepticus. Oxalate and caramboxin are considered the main substances causing nephrotoxicity and neurotoxicity. Caramboxin inhibits GABA binding and activates the glutamatergic receptors. Haemodialysis improved outcomes in neurotoxicity. Nephrotoxicity and neurotoxicity need to be looked for with star fruit toxicity, both in individuals with abnormal or normal renal function. Once star fruit intoxication is identified, early renal replacement therapy should be considered. Further studies on the mechanisms of star fruit toxicity are needed.

Efficient expression of concatenated α1β2δ and α1β3δ GABAA receptors, their pharmacology and stoichiometry.

GABAA receptors containing δ-subunits are notorious for being difficult to study in vitro due to heterogeneity of expressed receptor populations and low GABA-evoked current amplitudes. Thus, there are some published misconceptions and contradictory conclusions made regarding the pharmacology and stoichiometry of δ-containing receptors. The aim of this study was to obtain robust homogenous expression of α1βδ receptors for in-depth investigation. Novel δ-containing pentameric concatenated constructs were designed. The resulting α1β2δ and α1β3δ GABAA receptor concatemers were investigated by two-electrode voltage-clamp electrophysiology using Xenopus laevis oocytes. First, while homogenous α1βδ GABAA receptor pools could not be obtained by manipulating the ratio of injected cRNAs of free α1, β2/3, and δ subunits, concatenated pentameric α1β2δ and α1β3δ constructs resulted in robust expression levels of concatemers. Second, by using optimised constructs that give unidirectional assembly of concatemers, we found that the δ subunit cannot directly participate in GABA binding and receptor activation. Hence, functional δ-containing receptors are likely to all have a conventional 2α:2β:1δ stoichiometry arranged as βαβαδ when viewed counterclockwise from the extracellular side. Third, α1β2/3δ receptors were found to express efficiently in X. laevis oocytes but have a low estimated open probability of ~0.5% upon GABA activation. Because of this, these receptors are uniquely susceptible to positive allosteric modulation by, for example, neurosteroids. Our data answer important outstanding questions regarding the pharmacology and stoichiometry of α1δ-containing GABAA receptors and pave the way for future analysis and drug discovery efforts.

The desensitization pathway of GABAA receptors, one subunit at a time

GABAA receptors mediate most inhibitory synaptic transmission in the brain of vertebrates. Following GABA binding and fast activation, these receptors undergo a slower desensitization, the conformational pathway of which remains largely elusive. To explore the mechanism of desensitization, we used concatemeric α1β2γ2 GABAA receptors to selectively introduce gain-of-desensitization mutations one subunit at a time. A library of twenty-six mutant combinations was generated and their bi-exponential macroscopic desensitization rates measured. Introducing mutations at the different subunits shows a strongly asymmetric pattern with a key contribution of the γ2 subunit, and combining mutations results in marked synergistic effects indicating a non-concerted mechanism. Kinetic modelling indeed suggests a pathway where subunits move independently, the desensitization of two subunits being required to occlude the pore. Our work thus hints towards a very diverse and labile conformational landscape during desensitization, with potential implications in physiology and pharmacology.

Structural Basis for Activation of the Heterodimeric GABAB Receptor

The neurotransmitter γ-aminobutyric acid (GABA) activates the metabotropic GABAB receptor to generate slow, prolonged inhibitory signals that regulate the neural circuitry. The GABAB receptor is an obligate heterodimeric G protein-coupled receptor (GPCR) comprised of GBR1 and GBR2 subunits, each with extracellular, seven-helix transmembrane (7TM), and coiled-coil domains. To understand how GABA-driven conformational changes in the extracellular domain are transmitted to the 7TM domain during signal transduction, we determined cryo-electron microscopy (EM) structures of GABAB in two different states: an antagonist-bound inactive state, and an active state in which both the GABA agonist and a positive allosteric modulator (PAM) are bound. In the inactive state, the TM3 and TM5 helices in the two 7TM domains engage in cholesterol-mediated as well as direct interactions, resulting in an open conformation. GABA binding forces the extracellular domains of GBR1 and GBR2 into a compact form, relocating the linkers that connect the extracellular and 7TM domains closer to each other. The movement of the linker along with the associated extracellular loop 2 of the 7TM domain reorients the two 7TM domains and creates a new interface with the TM5, TM6 and TM7 helices in a closed conformation. PAM binding to the interface between the TM6 and TM6 helices stabilizes the active 7TM domain conformation. The relayed structural rearrangement results in significant conformational changes in the TM helices, as well as intracellular loop 3 in GBR2, which may promote the binding and activation of the Gi/o proteins.

New insights in the mode of action of (+)-erythravine and (+)-11α-hydroxy-erythravine alkaloids

Erythrinian alkaloids ((+)-erythravine and (+)-11-α-hydroxy-erythravine) have been pointed as the main responsible agents for the anticonvulsant and anxiolytic properties of Erythrina mulungu Mart ex Benth. The present work provides a new set of information about the mode of action of these alkaloids by the use of a complementary approach of neurochemical and electrophysiological assays. We propose here that the antiepileptic and anxiolytic properties exhibited by both alkaloids appear not to be related to the inhibition of glutamate binding or GABA uptake, or even to the increase of glutamate uptake or GABA binding, as investigated here by the use of rat cortical synaptosomes. Similarly, and even in a high concentration, (+)-erythravine and (+)-11-α-hydroxy-erythravine did not modulate the main sodium and potassium channel isoforms checked by the use of voltage-clamp studies on Xenopus laevis oocytes. However, unlike (+)-11-α-hydroxy-erythravine, which presented a little effect, it was possible to observe that the (+)-erythravine alkaloid produced a significant inhibitory modulation on α4β2, α4β4 and α7 isoforms of nicotinic acetylcholine receptors also checked by the use of voltage-clamp studies, which could explain at least partially its anxiolytic and anticonvulsant properties. Since (+)-11-α-hydroxy-erythravine and (+)-erythravine modulated nicotinic acetylcholine receptors to different extents, it is possible to reinforce that small differences between the chemical structure of these alkaloids can affect the selectivity and affinity of target-ligand interactions, conferring distinct potency and/or pharmacological properties to them, as previously suggested by differential experimental comparison between different erythrinian alkaloids.

Chronic aerobic exercise: Autoradiographic assessment of GABA(a) and mu-opioid receptor binding in adult rats

Exercise programs have shown great potential for both the prevention and treatment of substance use disorder (SUD). As exercise has been shown to have potent effects on physical and psychological health, it is reasonable to examine the mechanism of how exercise can be used as an adjunct treatment for addiction. The present study examined the effects of chronic aerobic (treadmill) exercise on both GABA(a) and mu-opioid receptor levels in the brains of male and female rats. GABA(a) receptor binding, measured by [3H] Flunitrazepam, was increased in the cingulate cortex following exercise, but only in females. Mu-opioid receptor expression, measured by [3H] ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) (DAMGO), showed no effect of exercise while showing an effect of sex, with increased [3H] DAMGO binding in the brains of sedentary males compared to that of sedentary females. Our findings support the potential role for GABA(a) signaling in the cingulate cortex as part of the mechanism of action of aerobic exercise. These data, along with prior reports, aid our understanding of the neurochemical impact and mechanism of chronic aerobic exercise on neuropsychiatric disease, particularly regarding addiction.

Acute Toxicity and Sedative-hypnotic Effects of Ethanol Stem Bark Extract and Fractions of Milicia excelsa (Moraceae) in Mice

Aim: Milicia excelsa stem bark is used as sedative and for treating mental illnesses among the Hausa tribe of Northern Nigeria, but there is no scientific rationale for its use. Hence, this study investigated the oral acute toxicity and sedative potential of ethanol stem bark extract, n-hexane, ethylacetate, n-butanol and aqueous fractions of the stem bark extract in mice. The phytoconstituents in the extract and fractions were quantified. Methodology: The acute toxicity of the extract and fractions were investigated using OECD guidelines 425 of 2008. The sedative effects of the extract and fractions were investigated using pentobarbital-, and ketamine-induced sleep tests. Results and discussion: The results obtained showed that the acute toxicity of the extract and fractions were > 5000 mg/kg, suggesting that the extract and fractions may be safe. The extract, n-hexane, ethylacetate and aqueous fractions significantly (p<0.05) reduced sleep latencies, indicative of sedative effects, effective for sleep induction, while the extract and all the fractions significantly (p<0.05) prolonged the sleep durations, suggesting sedative effects, effective for sleep maintenance. Conclusion: This study therefore, concluded that the extract and fractions may be safe. The study further concluded that the sedative effect of the extract and fractions may be due to the abundance of flavonoids in the extract and fractions. Thus, providing scientific rationale for its ethnomedicinal use. However, further study may be warranted to isolate and characterize the sleep promoting bioactive principles as well as carry out GABA binding assay of the isolated compound(s) in ESB and its various fractions.

Mutations of α1F45 residue of GABAA receptor loop G reveal its involvement in agonist binding and channel opening/closing transitions

GABAA receptors (GABAARs) mediate inhibitory neurotransmission in the mammalian brain. Recently, numerous GABAAR static structures have been published, but the molecular mechanisms of receptor activation remain elusive. Loop G is a rigid β-strand belonging to an extensive β-sheet that spans the regions involved in GABA binding and the interdomain interface which is important in receptor gating. It has been reported that loop G participates in ligand binding and gating of GABAARs, however, it remains unclear which specific gating transitions are controlled by this loop. Analysis of macroscopic responses revealed that mutation at the α1F45 residue (loop G midpoint) resulted in slower macroscopic desensitization and accelerated deactivation. Single-channel analysis revealed that these mutations also affected open and closed times distributions and reduced open probability. Kinetic modeling demonstrated that mutations affected primarily channel opening/closing and ligand binding with a minor effect on preactivation. Thus, α1F45 residue, in spite of its localization close to binding site, affects late gating transitions. In silico structural analysis suggested an important role of α1F45 residue in loop G stability and rigidity as well as in general structure of the binding site. We propose that the rigid β-sheet comprising loop G is well suited for long range communication within GABAAR but this mechanism becomes impaired when α1F45 is mutated. In conclusion, we demonstrate that loop G is crucial in controlling both binding and gating of GABAARs. These data shed new light on GABAAR activation mechanism and may also be helpful in designing clinically relevant modulators.

GABA-mediated tonic inhibition differentially modulates gain in functional subtypes of cortical interneurons

The binding of GABA (γ-aminobutyric acid) to extrasynaptic GABAA receptors generates tonic inhibition that acts as a powerful modulator of cortical network activity. Despite GABA being present throughout the extracellular space of the brain, previous work has shown that GABA may differentially modulate the excitability of neuron subtypes according to variation in chloride gradient. Here, using biophysically detailed neuron models, we predict that tonic inhibition can differentially modulate the excitability of neuron subtypes according to variation in electrophysiological properties. Surprisingly, tonic inhibition increased the responsiveness (or gain) in models with features typical for somatostatin interneurons but decreased gain in models with features typical for parvalbumin interneurons. Patch-clamp recordings from cortical interneurons supported these predictions, and further in silico analysis was then performed to seek a putative mechanism underlying gain modulation. We found that gain modulation in models was dependent upon the magnitude of tonic current generated at depolarized membrane potential-a property associated with outward rectifying GABAA receptors. Furthermore, tonic inhibition produced two biophysical changes in models of relevance to neuronal excitability: 1) enhanced action potential repolarization via increased current flow into the dendritic compartment, and 2) reduced activation of voltage-dependent potassium channels. Finally, we show theoretically that reduced potassium channel activation selectively increases gain in models possessing action potential dynamics typical for somatostatin interneurons. Potassium channels in parvalbumin-type models deactivate rapidly and are unavailable for further modulation. These findings show that GABA can differentially modulate interneuron excitability and suggest a mechanism through which this occurs in silico via differences of intrinsic electrophysiological properties.

Functional movements of the GABA type A receptor.

We have performed a parallel tempering crankshaft motion Monte Carlo simulation on a model of the GABA type A receptor with the aim of exploring a wide variety of local conformational space. We develop a novel method to analyse the protein movements in terms of a correlation tensor and use this to explore the gating process, that is, how agonist binding could cause ion channel opening. We find that simulated binding impulses to varying clusters of GABA binding site residues produce channel opening, and that equivalent impulses to single GABA sites produce partial opening.

GABA&lt;sub&gt;A&lt;/sub&gt; receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The GABAA receptor is a ligand-gated ion channel of the Cys-loop family that includes the nicotinic acetylcholine, 5-HT3 and strychnine-sensitive glycine receptors. GABAA receptor-mediated inhibition within the CNS occurs by fast synaptic transmission, sustained tonic inhibition and temporally intermediate events that have been termed ‘GABAA, slow’ [41]. GABAA receptors exist as pentamers of 4TM subunits that form an intrinsic anion selective channel. Sequences of six α, three β, three γ, one δ, three ρ, one ε, one π and one θ GABAA receptor subunits have been reported in mammals [273, 232, 231, 278]. The π-subunit is restricted to reproductive tissue. Alternatively spliced versions of many subunits exist (e.g. α4- and α6- (both not functional) α5-, β2-, β3- and γ2), along with RNA editing of the α3 subunit [67]. The three ρ-subunits, (ρ1-3) function as either homo- or hetero-oligomeric assemblies [354, 46]. Receptors formed from ρ-subunits, because of their distinctive pharmacology that includes insensitivity to bicuculline, benzodiazepines and barbiturates, have sometimes been termed GABAC receptors [354], but they are classified as GABAA receptors by NC-IUPHAR on the basis of structural and functional criteria [14, 232, 231].Many GABAA receptor subtypes contain α-, β- and γ-subunits with the likely stoichiometry 2α.2β.1γ [164, 232]. It is thought that the majority of GABAA receptors harbour a single type of α- and β -subunit variant. The α1β2γ2 hetero-oligomer constitutes the largest population of GABAA receptors in the CNS, followed by the α2β3γ2 and α3β3γ2 isoforms. Receptors that incorporate the α4- α5-or α6-subunit, or the β1-, γ1-, γ3-, δ-, ε- and θ-subunits, are less numerous, but they may nonetheless serve important functions. For example, extrasynaptically located receptors that contain α6- and δ-subunits in cerebellar granule cells, or an α4- and δ-subunit in dentate gyrus granule cells and thalamic neurones, mediate a tonic current that is important for neuronal excitability in response to ambient concentrations of GABA [205, 268, 79, 17, 283]. GABA binding occurs at the β+/α- subunit interface and the homologous γ+/α- subunits interface creates the benzodiazepine site. A second site for benzodiazepine binding has recently been postulated to occur at the α+/β- interface ([250]; reviewed by [277]). The particular α-and γ-subunit isoforms exhibit marked effects on recognition and/or efficacy at the benzodiazepine site. Thus, receptors incorporating either α4- or α6-subunits are not recognised by ‘classical’ benzodiazepines, such as flunitrazepam (but see [351]). The trafficking, cell surface expression, internalisation and function of GABAA receptors and their subunits are discussed in detail in several recent reviews [48, 136, 184, 311] but one point worthy of note is that receptors incorporating the γ2 subunit (except when associated with α5) cluster at the postsynaptic membrane (but may distribute dynamically between synaptic and extrasynaptic locations), whereas as those incorporating the d subunit appear to be exclusively extrasynaptic. NC-IUPHAR [14, 232] class the GABAA receptors according to their subunit structure, pharmacology and receptor function. Currently, eleven native GABAA receptors are classed as conclusively identified (i.e., α1β2γ2, α1βγ2, α3βγ2, α4βγ2, α4β2δ, α4β3δ, α5βγ2, α6βγ2, α6β2δ, α6β3δ and ρ) with further receptor isoforms occurring with high probability, or only tentatively [232, 231]. It is beyond the scope of this Guide to discuss the pharmacology of individual GABAA receptor isoforms in detail; such information can be gleaned in the reviews [14, 91, 164, 169, 140, 273, 212, 232, 231] and [8, 7]. Agents that discriminate between α-subunit isoforms are noted in the table and additional agents that demonstrate selectivity between receptor isoforms, for example via β-subunit selectivity, are indicated in the text below. The distinctive agonist and antagonist pharmacology of ρ receptors is summarised in the table and additional aspects are reviewed in [354, 46, 141, 219].Several high-resolution cryo-electron microscopy structures have been described in which the full-length human α1β3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam [194].

Discovery of the Oral Leukotriene C4 Synthase Inhibitor (1S,2S)-2-({5-[(5-Chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic Acid (AZD9898) as a New Treatment for Asthma

While bronchodilators and inhaled corticosteroids are the mainstay of asthma treatment, up to 50% of asthmatics remain uncontrolled. Many studies show that the cysteinyl leukotriene cascade remains highly activated in some asthmatics, even those on high-dose inhaled or oral corticosteroids. Hence, inhibition of the leukotriene C4 synthase (LTC4S) enzyme could provide a new and differentiated core treatment for patients with a highly activated cysteinyl leukotriene cascade. Starting from a screening hit (3), a program to discover oral inhibitors of LTC4S led to (1S,2S)-2-({5-[(5-chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic acid (AZD9898) (36), a picomolar LTC4S inhibitor (IC50 = 0.28 nM) with high lipophilic ligand efficiency (LLE = 8.5), which displays nanomolar potency in cells (peripheral blood mononuclear cell, IC50,free = 6.2 nM) and good in vivo pharmacodynamics in a calcium ionophore-stimulated rat model after oral dosing (in vivo, IC50,free = 34 nM). Compound 36 mitigates the GABA binding, hepatic toxicity signal, and in vivo toxicology findings of an early lead compound 7 with a human dose predicted to be 30 mg once daily.

GABAAReceptors Are Well Preserved in the Hippocampus of Aged Mice

GABA is the primary inhibitory neurotransmitter in the nervous system. GABAA receptors (GABAARs) are pentameric ionotropic channels. Subunit composition of the receptors is associated with the affinity of GABA binding and its downstream inhibitory actions. Fluctuations in subunit expression levels with increasing age have been demonstrated in animal and human studies. However, our knowledge regarding the age-related hippocampal GABAAR expression changes is limited and based on rat studies. This study is the first analysis of the aging-related changes of the GABAAR subunit expression in the CA1, CA2/3, and dentate gyrus regions of the mouse hippocampus. Using Western blotting and immunohistochemistry we found that the GABAergic system is robust, with no significant age-related differences in GABAAR α1, α2, α3, α5, β3, and γ2 subunit expression level differences found between the young (6 months) and old (21 months) age groups in any of the hippocampal regions examined. However, we detected a localized decrease of α2 subunit expression around the soma, proximal dendrites, and in the axon initial segment of pyramidal cells in the CA1 and CA3 regions that is accompanied by a pronounced upregulation of the α2 subunit immunoreactivity in the neuropil of aged mice. In summary, GABAARs are well preserved in the mouse hippocampus during normal aging although GABAARs in the hippocampus are severely affected in age-related neurological disorders, including Alzheimer’s disease.

Altered inhibitory synapses in de novo GABRA5 and GABRA1 mutations associated with early onset epileptic encephalopathies.

We performed next generation sequencing on 1696 patients with epilepsy and intellectual disability using a gene panel with 480 epilepsy-related genes including all GABAA receptor subunit genes (GABRs), and we identified six de novo GABR mutations, two novel GABRA5 mutations (c.880G>T, p.V294F and c.1238C>T, p.S413F), two novel GABRA1 mutations (c.778C>T, p.P260S and c.887T>C, p.L296S/c.944G>T, p.W315L) and two known GABRA1 mutations (c.335G>A, p.R112Q and c.343A>G, p.N115D) in six patients with intractable early onset epileptic encephalopathy. The α5(V294F and S413F) and α1(P260S and L296S/W315L) subunit residue substitutions were all in transmembrane domains, while the α1(R112Q and N115R) subunit residue substitutions were in the N-terminal GABA binding domain. Using multidisciplinary approaches, we compared effects of mutant GABAA receptor α5 and α1 subunits on the properties of recombinant α5β3γ2 and α1β3γ2 GABAA receptors in both neuronal and non-neuronal cells and characterized their effects on receptor clustering, biogenesis and channel function. GABAA receptors containing mutant α5 and α1 subunits all had reduced cell surface and total cell expression with altered endoplasmic reticulum processing, impaired synaptic clustering, reduced GABAA receptor function and decreased GABA binding potency. Our study identified GABRA5 as a causative gene for early onset epileptic encephalopathy and expands the mutant GABRA1 phenotypic spectrum, supporting growing evidence that defects in GABAergic neurotransmission contribute to early onset epileptic encephalopathy phenotypes.

The Functional Role of Spontaneously Opening GABAA Receptors in Neural Transmission.

Ionotropic type of γ-aminobutyric acid receptors (GABAARs) produce two forms of inhibitory signaling: phasic inhibition generated by rapid efflux of neurotransmitter GABA into the synaptic cleft with subsequent binding to GABAARs, and tonic inhibition generated by persistent activation of extrasynaptic and/or perisynaptic GABAARs by GABA continuously present in the extracellular space. It is widely accepted that phasic and tonic GABAergic inhibition is mediated by receptor groups of distinct subunit composition and modulated by different cytoplasmic mechanisms. Recently, however, it has been demonstrated that spontaneously opening GABAARs (s-GABAARs), which do not need GABA binding to enter an active state, make a significant input into tonic inhibitory signaling. Due to GABA-independent action mode, s-GABAARs promise new safer options for therapy of neural disorders (such as epilepsy) devoid of side effects connected to abnormal fluctuations of GABA concentration in the brain. However, despite the potentially important role of s-GABAARs in neural signaling, they still remain out of focus of neuroscience studies, to a large extent due to technical difficulties in their experimental research. Here, we summarize present data on s-GABAARs functional properties and experimental approaches that allow isolation of s-GABAARs effects from those of conventional (GABA-dependent) GABAARs.