Involvement of membrane GABA transporter in alpha-latrotoxin-stimulated [3H]GABA release

Alpha-latrotoxin evokes massive [3H]GABA release from rat brain synaptosomes by stimulating exocytosis and outflow from non-vesicular pool. In the present study, GABA transporter-mediated [3H]GABA release was shown to be involved in alpha-latrotoxin-triggered release of [3H]GABA from non-vesicular pool. The following agents have been exploited as tools: (1) a protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and bafilomycin A1 for evoking depletion of synaptic vesicle [3H]GABA and enlargement of non-vesicular pool; (2) a non-substrate high-affinity GABA transport blocker NO-711 for determining participation of GABA carrier in the toxin-stimulated GABA release; (3) a competitive inhibitor of GABA reuptake nipecotic acid for heteroexchange [3H]GABA release. As shown by the experiments with nipecotic acid, FCCP and bafilomycin A1 considerably increase the content of non-vesicular [3H]GABA. The treatment of the synaptosomes with these agents modified the response to alpha-latrotoxin, particularly to its subnanomolar concentrations: the lack or substantial lowering of the toxin-evoked release during the first 2 min after the toxin addition and substantial enhancement of release up to the 5th minute were observed. Only the step of enhanced release was sensitive to GABA transporter blocker NO-711. Distinct sensitivity to NO-711 was shown to be characteristic for different steps of alpha-latrotoxin-stimulated [3H]GABA release from the control, untreated synaptosomes: lack of any effect of NO-711 during the first 2 min and powerful inhibition in 10 min after the toxin application. Taken together these data appear to indicate that the toxin non-simultaneously from vesicular and non-vesicular origins releases the neurotransmitter, the first rapid step reflects exocytosis stimulation, and the second tardy step is at least in part due to the release mediated by GABA transporters. The incomplete inhibition with NO-711 of the tardy step of the release evoked by nanomolar toxin concentrations suggests the participation not only of the GABA transporters.     

[3H]GABA binding in developing rabbit retina

We have studied the developmental sequence of the GABA system in the rabbit retina using an in vitro binding assay to monitor developmental changes in the post-synaptic receptor. A variety of tissue treatments including perchlorate and Triton X-100 were employed to optimize binding and remove endogenous factors which inhibit binding. Pre-treatment of the tissue with 0.05% Triton X-100 revealed high affinity binding for [³H]GABA which increased in a sigmoidal fashion with the post-natal age of the animal. A constant level of binding, at about 16% of adult levels, was noted until day 8, at which time a rapid increase occurred. At 16 days post-natal, the amount of specific binding reached a plateau near adult levels. Kinetic analysis of the GABA receptor showed an increase in the number of receptors (B_max) with little or no change in the apparent affinity (K_D). Our results suggest that the onset of post-synaptic receptor activity is delayed approximately 1 to 2 days, relative to the pre-synaptic components, and the period of rapid increase in GABA receptor binding coincides with the period of maximum increase in retinal synaptic density.     

Inactivation of GABA aminotransferase by 3-nitro-1-propanamine

3-Nitro-1-propanamine is a close structural analog of the neuro-transmitter GABA. The nitro compound is a good substrate for the GABA aminotransferase from porcine brain. However, it inactivates the GABA aminotransferase from GABA-grown Pseudomonas fluorescens in a slowly reversible reaction. Both enzymes are inactivated by the homolog 4-nitro-1-butanamine.     

GABA binding in mammalian brain: Inhibition by endogenous GABA

Sodium-independent binding of gamma aminobutyric acid (GABA) to receptor-like sites in mammalian brain homogenates was much greater in membrane fractions which had been thoroughly washed with buffer, or detergent, and frozen and thawed several times, than in fresh unwashed membranes. As previously shown (Greenlee, Van Ness, & Olsen, Life Sciences $\text{22}$, 1653 (1978), the washing procedure removed endogenous inhibitors of GABA binding which led to an apparent improvement in GABA binding affinity to a low affinity class of sites (K_D ? 170 nM), and, additionally, the appearance of a high affinity (K_D ? 10 nM) class of sites. This endogenous inhibitory material was found to inhibit both classes of GABA binding sites, but with greater potency towards the high affinity sites for GABA. Biochemical characterization of the inhibitor fraction revealed that the activity was heat-stable, insensitive to trypsin and disulfide reducing compounds, dialyzeable through membrane sieves which would retain molecules with a molecular weight of 5000, and eluted 100% from a molecular sieve column in the position of small molecules (salt volume), clearly separated from a 16,000 molecular weight marker. The inhibitor was over 80% inactivated by the enzyme GABAse, indicating that most, and perhaps all of the endogenous inhibitor of GABA binding was indeed GABA itself. The difficulty in removing endogenous GABA from brain membranes must be considered in studies on benzodiazepine receptors (since they are affected in vitro by GABA) and in any comparison of GABA or benzodiazepine receptors in human neuropsychiatric disorders, drug treatment or lesion studies.     

Chemical integrity of [3H]GABA used in binding studies

A method which is claimed to be able to determine the proportion of true GABA within radiolabeled GABA used in binding studies was tested using [³H]GABA. The method was found to be unsuitable for³H-labeled GABA and, furthermore, both theoretical considerations and the present experimental data indicated that it could also produce misleading results with [¹⁴C]GABA.     

Effects of GABA antagonist-induced seizures on 3H-muscimol and 3H-diazepam binding in the rat striatum]

The binding of 3H-muscimol and 3H-diazepam to rat striatum membranes after picrotoxin- and bicuculline-induced seizures was characterized. No alteration in the maximal binding capacity (Bmax) of 3H-muscimol was observed. However, bicuculline produced a 27% decrease in Kd. Both picrotoxin and bicuculline increased the binding capacity of 3H-diazepam. Bicuculline produced a 86% increase in Kd. These results suggest that the GABA antagonists-induced seizures may modulate 3H-muscimol and 3H-diazepam binding in rat striatum.     

Effects of Valeriana Officinalis Extracts on [3H]Flunitrazepam Binding,Synaptosomal [3H]GABA Uptake,and Hippocampal [3H]GABA Release

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with -aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [³H]flunitrazepam binding (EC₅₀ 4.13 × 10^–10 mg/ml). However, this increased [³H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC₅₀ of 4.82 × 10^–1 mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [³H]flunitrazepam binding sites. Valerian extracts also potentiate K⁺ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [³H]GABA. Finally, inhibition of synaptosomal [³H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA_A receptors, but can also interact at other presynaptic components of GABAergic neurons.     

GABA agonists

Summary This review describes the development of GABA receptor agonists with no detectable affinity for other recognition sites in GABA-mediated synapses. The key compounds are THIP, isoguvacine, and piperidine-4-sulphonic acid (P4S), developed via extensive structural modifications of the potent but not strictly specific GABA agonist muscimol. The structural parameters, which have to be considered in the design of GABA agonists are discussed on the basis of the structures and biological activities of these GABA agonists and a number of related compounds.A model, which summarizes our present knowledge of the structure of the postsynaptic GABA receptor complex, is presented, and the interaction of GABA agonists with various sites in this complex is discussed. Of particular interest are the effects of GABA agonists on the binding of diazepam to the benzodiazepine binding site, assumed to be a structural unit of the GABA receptor complex. While rigid molecules like THIP are capable of activating the GABA receptors, a certain degree of conformational mobility of GABA agonists apparently is a prerequisite for stimulation of diazepam binding in vitro at 0 °C. These findings suggest that GABA receptor functions involve conformational changes of certain elements of the receptor complex.Some aspects of the pharmacology of GABA agonists are discussed, including the attempts to develop GABA agonists with desirable pharmacokinetic and toxicological characteristics. While muscimol is a toxic compound, THIP is well tolerated by animals, and in contrast to isoguvacine, THIP penetrates into the brain after systemic administration to animals, a difference which can be explained on the basis of their protolytic properties. The attempts to develop pro-drugs of isoguvacine capable of penetrating the blood-brain barrier with subsequent decomposition in the brain tissue to isoguvacine are described.     

Preferential release of newly synthesized [3H]GABA from striatal slices to preloaded [3H]GABA

The release of [³H]GABA which is newly synthesized from [³H]l-glutamic acid (GLU) has been examined using striatal slices obtained from the rat brain. It was found that 8–10% of [³H]GLU transported was converted to [³H]GABA during the incubation of striatal slices in the presence of nipecotic acid (5 × 10^?5 M). Nipecotic acid was added to the medium in order to prevent possible reuptake of [³H]GABA released during its synthesis, and it was found to have no significant effect on the formation of [³H]GABA from [³H]GLU as well as on the uptake of [³H]GLU. The application of high potassium (60 mM) stimulation exhibited a significant enhancement of the release of this newly synthesized [³H]GABA in a Ca²⁺ dependent manner. Kinetic analysis revealed that the evoked release of newly synthesized [³H]GABA was approximately two times greater than that of previously-loaded [³H]GABA, whereas no significant difference was observed in the spontaneous release. An immobilization stress in water failed to affect the release of newly synthesized [³H]GABA from striatal slices despite the occurrence of a significant enhancement of GABA formation in this structure.These results suggest that newly synthesized GABA may be preferentially released from its nerve terminals in response to the excitation of neurons at least in the striatum as compared with previously accumulated GABA.     

Measurement of release of endogenous GABA and catabolites of [3H]GABA from synaptosomal preparations using ion-exchange chromatography

Picomole quantities of endogenous GABA in acidified superfusates of synaptosomal preparations have been measured using micro-bore ion-exchange chromatography and post-column formation of the fluorescent iso-indole derivative. Using this technique superfusates have been analyzed directly, without further manipulations, to investigate the release of endogenous GABA. Spontaneous release of GABA was 2–5 pmol/200 l superfusate increasing to 20 pmol/200 l with potassium stimulation. When -vinyl GABA (RMI 71754), an inhibitor of GABA-T was injected into rats (750 mg/kg) and synaptosomes prepared the potassium-evoked release of GABA was increased 3-fold compared to controls. Chromatographic separations and measurement of release of endogenous and radiolabeled GABA allowed the real specific activity of released GABA to be calculated. Only when 500 M amino-oxyacetic acid was added during isolation of synaptosomes was the specific activity of released GABA the same as the initial specific activity.     

Effects of GABA receptor stimulation on the release of [3H]GABA from slices of rat neostriatum.

Slices of rat neostriatum were incubated in Krebs-Henseleit medium. Modulation of [3H]GABA release by GABA agonists and antagonists was investigated. The GABAA receptor agonists muscimol (0.1 microM) and isoguvacine (5 microM) enhanced the stimulated release of [3H]GABA. The antagonists picrotoxin (1 microM) and bicuculline (50 microM) prevented the effects of the agonists. In the presence of naloxone (1 microM), which blocked the effects of enkephalinergic neurons within the slice preparation, muscimol (1 microM) no longer affected the release of [3H]GABA.     

Editing modifies the GABA(A) receptor subunit alpha3

Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain development.     

Gestational changes of GABA levels and GABA binding in the human uterus

The concentrations of gamma-aminobutyric acid (GABA), the activities of L-glutamate decarboxylase and GABA-transaminase, and the nature of the sodium-independent binding of GABA were examined in uterine tissue pieces obtained surgically from pregnant and non-pregnant women. GABA concentrations were reduced, while the activity of GABA-transaminase and the specific binding of [3H]GABA significantly increased in specimens from pregnant subjects. These findings suggest some gestation-related functional role for the GABA system in the human uterus.     

Celsr3 is required for normal development of GABA circuits in the inner retina

The identity of the specific molecules required for the process of retinal circuitry formation is largely unknown. Here we report a newly identified zebrafish mutant in which the absence of the atypical cadherin, Celsr3, leads to a specific defect in the development of GABAergic signaling in the inner retina. This mutant lacks an optokinetic response (OKR), the ability to visually track rotating illuminated stripes, and develops a super-normal b-wave in the electroretinogram (ERG). We find that celsr3 mRNA is abundant in the amacrine and ganglion cells of the retina, however its loss does not affect synaptic lamination within the inner plexiform layer (IPL) or amacrine cell number. We localize the ERG defect pharmacologically to a late-stage disruption in GABAergic modulation of ON-bipolar cell pathway and find that the DNQX-sensitive fast b1 component of the ERG is specifically affected in this mutant. Consistently, we find an increase in GABA receptors on mutant ON-bipolar terminals, providing a direct link between the observed physiological changes and alterations in GABA signaling components. Finally, using blastula transplantation, we show that the lack of an OKR is due, at least partially, to Celsr3-mediated defects within the brain. These findings support the previously postulated inner retina origin for the b1 component and reveal a new role for Celsr3 in the normal development of ON visual pathway circuitry in the inner retina.     

Sites in TM2 and 3 are critical for alcohol-induced conformational changes in GABA receptors

Abstract gamma-Aminobutyric acid type A (GABA(A)) receptors are molecular targets for alcohols. Previous work suggests that S270 and A291 residues in the transmembrane (TM) 2 and 3 domains of the GABA(A) receptor alpha subunit are components of an alcohol-binding pocket, and S270I and A291W mutants abolished ethanol potentiation. Our results showed that A295C and F296C residues in the TM3 of the GABA(A) receptor alpha1 subunit are accessible to hexylmethanethiosulfonate (HMTS) in the alcohol-bound state, but not in the resting state. Thus, the A295C and F296C sites become water-accessible as a result of alcohol-induced conformational changes. If S270 or A291 residues are sites of alcohol binding, then S270I or A291W mutations should prevent alcohol-induced conformational movements within the TM3 domain. To investigate this question, the accessibility of HMTS reagent to double mutants (A291W/A295C, A291W/F296C, S270I/A295C or S270I/F296C) in the presence of ethanol or hexanol was tested. The A291W or S270I mutations markedly reduced the accessibility of HMTS to all the double mutants in the ethanol-bound state, and to S270I/F296C, A291W/A295C and A291W/F296C double mutants in the hexanol-bound state, suggesting that the A291 or S270 residues are critical sites for alcohol binding and alcohol-induced conformational changes.     

Graded response to GABA by native extrasynaptic GABA receptors

GABA is the main inhibitory neurotransmitter in the mammalian CNS. GABA in the brain is commonly associated with a fast, point-to-point form of signalling called synaptic transmission (phasic inhibition), but there is growing evidence that GABA participates in another, slower and more diffuse form of signalling often referred to as tonic inhibition. Unresolved questions regarding tonic neuronal inhibition concern activation and functional properties of extrasynaptic GABAA receptors (GABARex) present on neurones. Extrasynaptic receptors are exposed to submicromolar GABA concentrations and may modulate the overall excitability of neurones and neuronal networks. Here, we examined GABA-activated single-channel currents in dentate gyrus granule neurones in rat hippocampal slices. We activated three types (I, II, III) of GABARex channels by nanomolar GABA concentrations (EC50 I: 27 +/- 12; II: 4 +/- 3; III: 43 +/- 19 nm). The channels opened after a delay and the single-channel conductance was graded (gammamax I: 61 +/- 3; II: 85 +/- 8, III: 40 +/- 3 pS). The channels were differentially modulated by 1 microm diazepam, 200 nm zolpidem, 1 microm flumazenil and 50 nm THDOC (3alpha, 21-dihydroxy-5alpha-pregnan-20-one), consistent with the following minimal subunit composition of GABARex I alpha1betagamma2, GABARex II alpha4betagamma2 and GABARex III alphabetadelta channels.     

Number of GABA immunonegative and GABA immunopositive neurons in human epileptic temporal cortex

The number of neurons, both GABA immunopositive and immunonegative, was determined in temporal epileptic foci of 7 patients after temporal lobectomy, and compared to neuronal numbers in temporal cortex of two controls taken from tumor operated patients. The thickness of the cortex of the epileptic cortex diminished by about 10%, while the number of nerve cells decreased to 67% of that of the control value: it was 19.000/mm(3) vs. 28.000/mm(3) found in the control. This decline was due to cell degeneration, which, however, was more severe for non-GABAergic nerve cells. Accordingly, the proportion of the GABA-positive neurons in the otherwise diminished neuronal population increased to 36.4% from the 32% control value. The number of GABAergic terminals, however, decreased even further, explaining the resulting disinhibition during epileptic seizures.     

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 γ-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.     

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.