Gamma-aminobutyric Acid Binding Research Articles

Elucidating the binding mechanisms of GABA and Muscimol as an avenue to discover novel GABA-mimetic small molecules

Gamma-aminobutyric acid (GABA) signaling is the principal inhibitory pathway in the central nervous system. It is critical in neuronal cell proliferation and fate determination. Any aberration in GABA inhibition results in psychiatric and neurological diseases. Thus, modulating GABAergic neurotransmission has become the basis of drug therapy for psychiatric and several neurological diseases. Though GABA and muscimol are classical inhibitors of GABA receptors, the search for novel inhibitors continues unabated. In this study, the binding mechanism of GABA and muscimol was elucidated and applied in the search for small molecule GABAergic inhibitors using comprehensive computational techniques. It was revealed that a high-affinity binding of GABA and muscimol was mediated by a water molecule involving α1Thr129 and then stabilized by strong interactions including salt bridges with β2Glu155 and α1Arg66 amidst hydrogen bonds, π-π stacking, and π -cation interactions with other residues. The binding of GABA and muscimol was also characterized by stability and deeper penetration into the hydrophobic core of the protein which resulted in conformational changes of the binding pocket and domain, by inducing correlated motions of the residues. Thermodynamics analysis showed GABA and muscimol exhibited total binding free energies of −19.85 ± 8.83 Kcal/mol and −26.55 ± 3.42 Kcal/mol, respectively. A pharmacophore model search, based on the energy contributions of implicating binding residues, resulted in the identification of ZINC68604167, ZINC19735138, ZINC04202466, ZINC00901626, and ZINC01532854 as potential GABA-mimetic compounds from metabolites and natural products libraries. This study has elucidated the binding mechanisms of GABA and muscimol and successfully applied in the identification of GABA-mimetic compounds. Communicated by Ramaswamy H. Sarma

Metabolomics-Based Effects of a Natural Product on Remyelination After Cerebral Ischemia Injury Via GABABR–pCREB–BDNF Pathway

Background Yi-Qi-Tong-Luo Granules (YQTLs) is a natural compound of Traditional Chinese Medicine authorized by China Food and Drug Administration (CFDA). These granules are employed in the convalescent stage of cerebral infarction and render notable clinical efficacy. This study aims to uncover the underlying mechanisms of YQTLs on remyelination after cerebral ischemia injury. Materials and Methods We established cerebral ischemia model in rats using microsphere-induced multiple cerebral infarction (MCI). We evaluated the pharmacological effects of YQTLs on MCI rats, through Morri’s water maze test, open field test, hematoxylin and eosin staining, and glycine silver immersion. We employed liquid chromatography mass spectrometry metabolomics to identify differential metabolites. Enzyme-linked immunosorbent assay was utilized to measure the release of neurotrophins, while immunofluorescence staining was used to assess oligodendrocyte precursor cells differences and myelin regeneration. We used Western blotting to validate the protein expression of remyelination-associated signaling pathways. Results YQTLs significantly improves cognitive function following cerebral ischemia injury. Pathological tissue staining revealed that YQTLs administration inhibits neuronal denaturation and neurofibrillary tangles. We identified 141 differential metabolites among the sham, MCI, and YQTLs-treated MCI groups. Among these metabolites, neurotransmitters were identified, and notably, gamma-aminobutyric acid (GABA) showed marked improvement in the YQTLs group. The induction of neurotrophins, such as brain-derived neurotrophic factor (BDNF) and PDGFAA, upregulation of olig2 and MBP expression, and promotion of remyelination were evident in YQTLs-treated MCI groups. Gamma-aminobutyric acid B receptors (GABABR), pERK/extracellular regulated MAP kinase, pAKT/protein kinase B, and pCREB/cAMP response element-binding were upregulated following YQTLs treatment. Conclusion YQTLs enhance the binding of GABA to GABABR, thereby activating the pCREB/BDNF signaling pathway, which in turn increases the expression of downstream myelin-associated proteins and promotes remyelination and cognitive function.

GABA-enriched rice bran inhibits inflammation in LPS-stimulated macrophages via suppression of TLR4-MAPK/NF-κB signaling cascades.

Gamma-aminobutyric acid (GABA)-enriched products (GEP) exhibited a wide range of pharmaceutical properties. In this study, anti-inflammatory activity of GEP from Lactobacillus fermentum-fermented water solution of rice bran was evaluated on lipopolysaccharide-activated macrophage model. GABA content in L. fermentum-fermented rice bran solution was determined up to 1.27 g/L. GEP was shown to inhibit the expression levels of inducible nitric oxide synthase and cyclooxygenase-2 enzymes. Moreover, pretreatment of GEP attenuated the generation level of interleukin (IL)-6, IL-1β, tumor necrosis factor α, and monocyte chemoattractant protein-1. Especially, the activation of signaling pathways due to nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) was interrupted in GEP-exposed cells. Notably, molecular docking result showed a potential binding of GABA to Toll-like receptor 4 with a binding energy of -3.88 kcal/mol, suggesting the role of GABA in suppression of Toll-like receptor 4-MAPK/NF-κB signaling cascades. As the result, GEP from L. fermentum-fermented rice bran solution could be suggested as a promising food for suppression of inflammatory responses. PRACTICAL APPLICATIONS: GABA-enriched products have been evidenced to possess various pharmaceutical properties and health beneficial effects. In this study, GABA-enriched product from L. fermentum-fermented rice bran solution exhibited the inhibition on inflammatory response in macrophages. Hence, it could be used as a potential ingredient for the mitigation of inflammatory responses.

Neurodevelopmental phenotypes associated with pathogenic variants in SLC6A1

BackgroundSLC6A1 encodes GAT-1, a major gamma-aminobutyric acid (GABA) transporter in the brain. GAT-1 maintains neurotransmitter homeostasis by removing excess GABA from the synaptic cleft. Pathogenic variants in SLC6A1 disrupt the...

Subunit compositions of GABAA receptors determining the diversity of physiological processes and neurotropic properties of medicines

Gamma-aminobutyric acid (GABA) became known as a potentially important chemical in the brain 50 years ago, but its significance as a neurotransmitter was fully found 16 years later. It is now known that at least 40 % of the inhibitory synaptic activity in the mammalian brain is accounted for by GABA. Аim. To analyze achievements in the study of the physiological and pharmacological role of GABA receptor subtypes, their potential applications in drug development and updated information on the clinical development of subtype-selective GABA receptor compounds. Results. The GABAA-receptor complex (GABA-RC) is ligand-gated ion channels with chloride conductance. These receptors contain α, β, and γ subunits, but δ, ε, θ, and ρ can be also present. The GABA binding site is located at the interface between α and β subunits where a number of important amino acids are also found. GABA-RC is sensitive to a wide range of drugs, e.g. benzodiazepines (BDZ), which are often used for their sedative/hypnotic and anxiolytic effects. Classical BDZ interact non-selectively with α1,3,5 βγ2 GABA-RС in the binding site located at the α+γ− interface. Conclusions. In addition to the potent and rapid pharmacotherapeutic action BDZ also possess some addictive potential (drug dependence), which appears after the interaction of molecules with α1-receptors. Using the selective targeting to separate subgroups not only the main effect of BDZ without side effects can be provided, but also one can use this approach in creating new analgesic medicines; we have demonstrated it on the example of propoxazepam (full agonist GABA-R).

Diverse role of γ-aminobutyric acid in dynamic plant cell responses.

Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is found in most prokaryotic and eukaryotic organisms. Although, ample research into GABA has occurred in mammals as it is a major inhibitory neurotransmitter; in plants, a role for GABA has often been suggested as a metabolite that changes under stress rather than as a signal, as no receptor or motif for GABA binding was identified until recently and many aspects of its biological function (ranging from perception to function) remain to be answered. In this review, flexible properties of GABA in regulation of plant responses to various environmental biotic and abiotic stresses and its integration in plant growth and development either as a metabolite or a signaling molecule are discussed. We have elaborated on the role of GABA in stress adaptation (i.e., salinity, hypoxia/anoxia, drought, temperature, heavy metals, plant-insect interplay and ROS-related responses) and its contribution in non-stress-related biological pathways (i.e., involvement in plant-microbe interaction, contribution to the carbon and nitrogen metabolism and governing of signal transduction pathways). This review aims to represent the multifunctional contribution of GABA in various biological and physiological mechanisms under stress conditions; the objective is to review the current state of knowledge about GABA role beyond stress-related responses. Our effort is to place findings about GABA in an organized and broader context to highlight its shared metabolic and biologic functions in plants under variable conditions. This will provide potential modes of GABA crosstalk in dynamic plant cell responses.

GABAergic signaling in Alzheimer's disease

GABAergic signaling in Alzheimer's disease

Aluminum-Activated Malate Transporters Can Facilitate GABA Transport.

Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABAA receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA]i) in both wheat (Triticum aestivum) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA]i because TaALMT1 facilitates GABA efflux but GABA does not complex Al3+ TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14C-GABA uptake into TaALMT1-expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1F213C) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA]i We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other's transport, suggestive of a role for ALMTs in communicating metabolic status.

Use of subcutaneous flumazenil preparations for the treatment of idiopathic hypersomnia: A case report

Idiopathic hypersomnia (IH) is a rare sleep disorder, recently hypothesized to be related to the production of a molecule that facilitates the binding of gamma-aminobutyric acid (GABA) to the GABA receptor. This paper reports on the treatment of a patient with IH who was treated with a 96-hour continuous low dose (4 mg/day) infusion of a benzodiazepine antagonist, flumazenil, followed by a slow-release flumazenil implant. The use of flumazenil mitigated the patient's IH symptoms including excessive daytime sleepiness, sleep drunkenness and self-reported cognitive problems. The case both provides a possible treatment and system for short (subcutaneous (SC) administration) and longer term (implant) flumazenil delivery. Current data supports the need for further research into the use of flumazenil for the treatment of IH and to develop long term flumazenil delivery systems.

Small-Angle X-Ray Scattering Study for Conformational Changes of Unusual Transcription Factor at the Operator

The chimeric transcription factors of the GntR family in bacteria contain a N-terminal winged helix-turn-helix (WHTH) DNA-binding domain and a C-terminal effector-binding/oligomerisation (EB/O) domain. The GntR family regulator GabR controls the expression of genes that are essential for glutamic acid metabolism. It represents an unusual transcription factor in that its EB/O domain is homologous to type I aminotransferases and is thought to form a head-to-tail dimer. Simultaneous binding of effector molecules such gamma-aminobutyric acid (GABA) and the co-enzyme pyridoxal-5'-phosphate (PLP) lead to activation of gene expression. This regulation is expected to involve conformational changes and/or changes in the number of transcription factors bound to DNA. Here we present the effect of such conformational change on small-angle X-ray scattering (SAXS) data, furthering the understanding of the linkage between the structure, dynamics, and function of GabR at the operator. The scattering observations are quantitatively investigated with respect to the average spatial extent of structural variations occurring in this transcription factor under diverse experimental conditions including scattering of the protein alone. The analyses of scattering data support that the spatial structural extent of this transcription factor at the operator is significant.

Altered Gamma-Aminobutyric Acid Type B Receptor Subunit 1 Splicing In Alcoholics

Chronic alcohol exposure can change splice variant expression. The gamma-aminobutyric acid type B (GABAB) receptor undergoes splicing and is an alcoholism treatment target, but there is little information about splicing changes in this receptor in alcoholics. We studied GABAB receptor subunit 1 (GABAB1) splicing in alcoholic postmortem brains. To maximize GABAB1 splice junction identification, we combined gene specific libraries with RNA-seq. Splice junctions and mapped reads were also found from intronic and intergenic regions. We compared GABAB1 splice junctions in prefrontal cortices from 14 alcoholic and 15 control subjects and introduced new strategies, reads per kilobase of splice junction model per million mapped reads and reads per kilobase of gene model per million mapped reads, for quantitating splice junction and gene expression. Novel splice junction detection indicated that the GABAB1 gene is at least two times longer than the previously reported gene length. GABAB1 exon and intron expression data showed low expression at the 5' end exons and exon grouping. This indicated that there are short splicing variants in addition to GABAB receptor subunit GABAB1a, the longest known major transcript. We found that chronic alcohol altered exon/intron expression and splice junction levels. Decreased expression of the gamma-aminobutyric acid binding site, a transmembrane domain and a microRNA binding site may decrease normal GABAB1 transcript population and thereby decrease normal signal transduction in alcoholics. We discovered novel, complex splicing of GABAB1 in human brain and showed that chronic alcohol produces additional splicing complexity.

Discovery of novel insomnia leads from screening traditional Chinese medicine database

Insomnia is a prominent modern disease that affects an increasing population. Undesirable side effects of commercial drugs highlight the need to develop novel insomnia drugs. Virtual screening of traditional chinese medicine Database@Taiwan (TCM Database@Taiwan) identified 2-O-Caffeoyl tartaric acid (1), 2-O-Feruloyl tartaric acid (2), and Mumefural (3) as potential agonists for both gamma-amino butyric acid (GABA) or benzodiazepine (BZ) binding sites. The TCM candidates exhibited higher affinity than GABA and Zolpidem, a phenomenon that could be attributed to higher quantity of stabilizing H-bonds. Efficacy profiles using support vector machines and pharmacophore contour also suggest drug potential of the TCM candidates. Fragments added to the de novo derivatives 3a, 3b, 3c for GABA binding site, and 1a, 2a, and 3d for BZ binding site contributed to new binding sites and structural stability, further optimizing binding to GABA or BZ binding sites. Increased opening of the ion channel by candidate ligands provide strong support for their potential biological functions. The dual binding properties of the TCM candidates present a unique opportunity to develop twin-targeting drugs with less side effects. Derivative structures can be used as starting points for developing high affinity GABAA receptor agonists with specificity towards GABA binding site and BZ binding site.

GABAA Receptor Imaging With Positron Emission Tomography in the Human Newborn: A Unique Binding Pattern

Flumazenil is a specific, reversibly bound antagonist at benzodiazepine binding sites of gamma-aminobutyric acid A receptors; these sites can be imaged using positron emission tomography with 11C-flumazenil. We reported an exponential decline of flumazenil volume of distribution (proportional to receptor binding) of gamma-aminobutyric acid A receptors in children 2 to 17 years. Six newborns (33.3-46.7 weeks' postconception) were studied. All had experienced epileptic seizures and undergone 60-minute dynamic 11C-flumazenil-positron emission tomography imaging after injection of 0.4 mCi/kg of 11C-flumazenil. All newborns were scanned during their natural sleep. Binding potential (indicating flumazenil receptor binding) was calculated using Logan-plot analysis. Visual and quantitative analyses showed highest receptor binding in the amygdala-hippocampus region, sensory-motor cortex, thalamus, brainstem and basal ganglia, in that order. Cerebellum and most of the cerebral cortex showed relatively low binding. This is the first demonstration of gamma-aminobutyric acid A receptor binding in human neonates and is strikingly different from that in older children/adults, showing a programmed pattern of expression. The ontogeny data of flumazenil receptor binding from children may contribute to understanding regional differences in synaptic plasticity and improve rational therapeutic use of drugs acting at the gamma-aminobutyric acid A receptor in the pediatric population.

Epilepsy in succinic semialdehyde dehydrogenase deficiency, a disorder of GABA metabolism

Objectives Succinic semialdehyde dehydrogenase (SSADH) deficiency is a gamma-aminobutyric acid (GABA) degradative defect. Epilepsy affects half of patients. The murine model is associated with a transition from absence to convulsive seizures in the third week, with fatal status epilepticus. Methods The clinical phenotype is reported from a patient database. Flumazenil-Positron Emission Topography (FMZ-PET) and Transcranial Magnetic Stimulation (TMS) were used to study GABA neurotransmission. Electrocorticography, single cell electrophysiology, and radioligand binding studies are reported from animal studies. Results Generalized seizures predominate, including tonic–clonic, atypical absence, and myoclonic. EEG discharges are typically generalized spike-wave. MRI shows a dentatopallidoluysian pattern. Sudden Unexpected Death in Epilepsy Patients (SUDEP) has occurred and the associated neuropathology reveals chronic excitotoxic injury in gloubus pallidus. Investigations using FMZ-PET and TMS support downregulation of GABA A and GABA B activity, respectively, in patients. Gamma-hydroxybutyrate (GHB) induces spike-wave discharges in homozygous null mice via GHB and GABA B-mediated mechanisms. These resemble absence seizures and are abolished by a GABA B receptor antagonist. Decreased binding of GABA A and GABA B receptor antagonists has been demonstrated in P19 and P14 null mice, respectively. Downregulation of GABA A and GABA B receptor subunits is observed by P14. GABA A and GABA B mediated potentials are reduced from P8-P14. Conclusion Generalized epilepsy and epileptiform discharges are characteristic of SSADH deficiency. Spontaneous absence seizures appear in null mice by the third week, which may be induced by GHB or GABA B activity. Subsequent overuse dependent downregulation of GABA A and GABA B receptor activity may be associated with hyperexcitability concomitant with the transition to generalized seizures.

The effects of zolpidem treatment and withdrawal on the in vitro expression of recombinant α1β2γ2s GABAA receptors expressed in HEK 293 cells

Zolpidem, a widely used hypnotic drug which acts through benzodiazepine binding sites, is a positive allosteric modulator of gamma-aminobutyric acid (GABA) action with preferential affinity for GABA(A) receptors containing alpha1 subunit. The pharmacological profile of zolpidem is different from that of classical benzodiazepines. The aim of this study was to find out whether zolpidem treatment triggers adaptive changes in the recombinant alpha1 subunit-containing GABA(A) receptors other than those observed following treatment with classical benzodiazepine-diazepam. Radioligand binding studies showed that 2-day exposure of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors to zolpidem (10 muM) up-regulated the maximum number (B (max)) of [(3)H]flunitrazepam, [(3)H]muscimol, and [(3)H]t-butylbicycloorthobenzoate ([(3)H]TBOB) binding sites without changing their affinity (K (d)), suggesting an increase in total GABA(A) receptor number. Semi-quantitative RT-PCR analysis demonstrated increased levels of alpha1 subunit mRNA, while Western blot demonstrated up-regulated gamma2 subunit proteins, suggesting that zolpidem induced de novo synthesis of receptors proteins, at both the transcriptional and translational levels. GABA-induced potentiation of [(3)H]flunitrazepam binding to membranes obtained from zolpidem-treated cells was markedly reduced, indicating allosteric uncoupling between GABA and benzodiazepine binding sites. The number of benzodiazepine and convulsant binding sites as well as the functional coupling between GABA and benzodiazepine binding sites normalized in 24 h following discontinuation of zolpidem treatment. The results of our in vitro studies suggest that a 2-day exposure of recombinant alpha1 subunit-containing GABA(A) receptors stably transfected in HEK 293 cells to zolpidem induces adaptive changes in this selective GABA(A) receptor subtype, which are not substantially different from those obtained after prolonged exposure of cells to high concentrations of diazepam.

Generation of a 3D model for human GABA transporter hGAT-1 using molecular modeling and investigation of the binding of GABA

A three-dimensional model of the human Na(+)/Cl(-)-dependent gamma-aminobutyric acid (GABA) transporter hGAT-1 was developed by homology modeling and refined by subsequent molecular modeling using the crystal structure of a bacterial homologue leucine transporter from Aquifex aeolicus (LeuT(Aa)) as the template. Protein structure quality checks show that the resulting structure is particularly suited for the analysis of the substrate binding pocket and virtual screening experiments. Interactions of GABA and the substrate binding pocket were investigated using docking studies. The difference of 6 out of 13 substrate interacting side chains between hGAT-1 and LeuT(Aa) lead to the different substrate preference which can be explained using our three-dimensional model of hGAT-1. In particular the replacement of serine 256 and isoleucine 359 in LeuT(Aa) with glycine and threonine in hGAT-1 seems to facilitate the selection of GABA as the main substrate by changing the hydrogen bonding pattern in the active site to the amino group of the substrate. For a set of 12 compounds flexible docking experiments were performed using LigandFit in combination with the Jain scoring function. With few exceptions the obtained rank order of potency was in line with experimental data. Thus, the method can be assumed to give at least a rough estimate of the potency of the potential of GABA uptake inhibitors.

Agonist‐ and antagonist‐induced conformational changes of loop F and their contributions to the ρ1 GABA receptor function

Binding of gamma-aminobutyric acid (GABA) to its receptor initiates a conformational change to open the channel, but the mechanism of the channel activation is not well understood. To this end, we scanned loop F (K210-F227) in the N-terminal domain of the rho1 GABA receptor expressed in Xenopus oocytes using a site-specific fluorescence technique. We detected GABA-induced fluorescence changes at six positions (K210, K211, L216, K217, T218 and I222). At these positions the fluorescence changes were dose dependent and highly correlated to the current dose-response, but with lower Hill coefficients. The competitive antagonist 3-aminopropyl(methyl)phosphinic acid (3-APMPA) induced fluorescence changes in the same direction at the four middle or lower positions. The non-competitive antagonist picrotoxin blocked nearly 50% of GABA-induced fluorescence changes at T218 and I222, but only <20% at K210 and K217 and 0% at K211 and L216 positions. Interestingly, the picrotoxin-blocked fraction of the GABA-induced fluorescence changes was highly correlated to the Hill coefficient of the GABA-induced dose-dependent fluorescence change. The PTX-insensitive mutant L216C exhibited the lowest Hill coefficient, similar to that in binding. Thus, the PTX-sensitive fraction reflects the conformational change related to channel gating, whereas the PTX-insensitive fraction represents a binding effect. The binding effect is further supported by the picrotoxin resistance of a competitive antagonist-induced fluorescence change. A cysteine accessibility test further confirmed that L216C and K217C partially line the binding pocket, and I222C became more exposed by GABA. Our results are consistent with a mechanism that an outward movement of the lower part of loop F is coupled to the channel activation.

Enantiomers of 4-Amino-3-fluorobutanoic Acid as Substrates for γ-Aminobutyric Acid Aminotransferase. Conformational Probes for GABA Binding

Gamma-aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5'-phosphate (PLP) to pyridoxamine 5'-phosphate (PMP). The enzyme then catalyzes the conversion of alpha-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches.

GABA A receptor heterogeneity, function, and implications for epilepsy

Gamma-aminobutyric acid (GABA) is the neurotransmitter of the local inhibitory neurons that control neuronal excitability and network interactions in the cerebral cortex. Most of the cortical inhibitory effects of GABA are mediated by GABAA receptors. Cortical GABAergic mechanisms are important in epileptogenesis and provide a target of antiepileptic drug therapy. Over the past several years, there has been increasing understanding of the heterogeneous molecular structure, function, and modulation of the GABAA receptors and the functional consequences of mutations affecting specific receptor subunits linked with various epilepsy syndromes. This new information provides a further insight into the mechanisms of epileptogenesis and potential for development of subunit-selective antiepileptic drugs. The GABAA receptors are pentameric chloride (Cl−) channels formed by various combinations of different types of α (α1 to α6), β (β1 to β3), γ (γ1 to γ3), δ, e, π, θ, and ρ (ρ1 to ρ3) subunits1,2 (figure 1). The different subunit combinations result in a great functional heterogeneity of GABAA receptors.2,3 GABA binding triggers opening of an intrinsic Cl− channel, allowing the rapid flux of Cl−. Most neurons in the adult brain maintain a low intracellular Cl− concentration due to the activity of a K+/Cl− cotransporter, and therefore in most cases, GABAA receptor activation produces a net entry of Cl−. This elicits inhibitory postsynaptic potentials due to both the hyperpolarizing effect of Cl− influx and shunting of excitatory synaptic inputs, resulting in a decreased probability of generation of an action potential. The GABAA receptors have several modulatory allosteric binding sites for benzodiazepines, neurosteroids, barbiturates, and general anesthetics, and their function is also affected by phosphorylation and the presence of divalent ions, such as zinc. …

Long-term alteration of gamma-aminobutyric acid B receptor subunits in immature rats after recurrent febrile seizures

Febrile seizure (FS) is closely related to an altered transmission of gamma-aminobutyric acid (GABA). GABA exerts its effects through ionotropic receptors (GABA(AR) and GABA(CR)) and metabotropic receptors (GABA(BR)). GABA(BRs) are located at pre- and postsynaptic sites. Stimulation of postsynaptic receptors generates long-lasting inhibitory postsynaptic potentials (IPSPs) that are important for the fine-tuning of inhibitory neurotransmission and caused by an increase in K(+) conductance. At presynaptic sites, GABA(BRs) mediate a suppression on the release of neurotransmitters such as of GABA or glutamate by inhibiting voltage-sensitive Ca(2+) channels. The present study aimed to explore the long-term changes of GABA(B) receptor subunits in immature rats after recurrent febrile seizures. Rats were randomly divided into control group and hyperthermia treatment group. The control rats (n = 64) were put into 37 degrees C water for 5 minutes. Rats with hyperthermia treatment were put into 44.8 degrees C water for 5 minutes. If a rat in hyperthermia treatment group showed seizure within 5 min, the rat was taken out of the water as soon as the seizure occurred. Water-immersion was carried out 10 times, once every 2 days. Rats showing 10 seizures (FS(10), n = 64) were studied. Rats exposed to hyperthermia for 10 times without seizure were also studied as hyperthermia-only (H, n = 64) group. Rats showing one seizure at the last time of 10 times of hyperthermia treatment were studied as one-seizure group (FS(1), n = 64). The other rats were studied for other research. The changes of GABA(B)R(1) and GABA(B)R(2) co-localization were detected by double fluorescence;the quantitative alteration of GABA(B)R(1) and GABA(B)R(2) were detected by quantitative RT-PCR; the binding of GABA(B)R(2) to GABA(B)R(1) was detected by immunoprecipitation/Western blot. GABA(B)R(1), GABA(B)R(2), and the binding of GABA(B)R(2) to GABA(B)R(1) decreased after the last febrile seizure in FS(10) group, the expression of GABA(B)R(1) returned to normal in later phase while GABA(B)R(2) and the binding of them did not. Recurrent FS down-regulated the expression of GABA(B)R subunits in a long term.