HPLC conditions are critical for the detection of GABA by microdialysis

In microdialysis studies, neither exocytotic release of gamma-aminobutyric acid (GABA), nor the presence of GABA type B (GABA(B)) autoreceptors, have been clearly established. It was investigated whether the chromatographic separation of GABA may have contributed to discrepancies in the literature. After extending the profile of the HPLC chromatogram to a retention time of 60 min, it was observed that various unknown compounds of biological origin co-eluted near the GABA peak. The retention time of GABA appeared to be extremely sensitive to pH; even at a retention time of around 60 min there was only a small pH window (5.26 +/- 0.01) where GABA was consistently well separated from co-eluting compounds. GABA determined by the improved assay was sensitive to tetrodotoxin (TTX), calcium depletion and the GABA(B) autoreceptor agonist baclofen. The present results illustrate that if the proper analytical conditions are applied, extracellular GABA can be sampled and quantified by microdialysis in free-moving animals. However, when the time-curves are considered, there is a striking delay of about 15-30 min before the effects of TTX, calcium depletion or baclofen are observed, as compared to the reported response of neurotransmitters such as dopamine (less than 5 min). It is assumed that the glial cells serve as a buffer between the GABA synapse and the microdialysis probes. It is proposed that microdialysis samples measure synaptic GABA indirectly, through glial cells surrounding the synapses.     

Identification of a domain in the delta subunit (S238-V264) of the alpha4beta3delta GABAA receptor that confers high agonist sensitivity

We have expressed the alpha4beta3delta and alpha4beta3gamma2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the alpha4beta3delta receptor (EC50 approximately 1.4 micromol/L) than of the alpha4beta3gamma2L subtype (EC50 approximately 27.6 micromol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, imidazole-4-amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co-expressing chimeric delta/gamma2L subunits with alpha4 and beta3 subunits. A stretch of amino acids in the delta subunit, S238-V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the delta and gamma2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.     

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.     

Replacement of histidine in position 105 in the α₅ subunit by cysteine stimulates zolpidem sensitivity of α₅β₂γ₂ GABA(A) receptors

Zolpidem is a positive allosteric modulator of GABA_A receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA_A receptors containing the α₁ subunit, it has a lower affinity to GABA_A receptors containing α₂, α₃, or α₅ subunits and has a very weak efficacy at receptors containing the α₅ subunit. Here, we show that replacing histidine in position 105 in the α₅ subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the α₅ subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to α₅H105 in α₁, α₂, and α₃ are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines α₁H101, α₂H101, and α₃H126 by arginine, as naturally present in α₄ and α₆, leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in α₅ containing receptors also for the action of zolpidem.     

Regulation of Transmitter γ-Aminobutyric Acid (GABA) Synthesis and Metabolism Illustrated by the Effect of γ-Vinyl GABA and Hypoglycemia

The effect of different treatments on amino acid levels in neostriatum was studied to throw some light on the synthesis and metabolism of gamma-aminobutyric acid (GABA). Irreversible inhibition of GABA transaminase by microinjection of gamma-vinyl GABA (GVG) led to a decrease in aspartate, glutamate, and glutamine levels and an increase in the GABA level, such that the nitrogen pool remained constant. The results indicate that a large part of brain glutamine is derived from GABA. Hypoglycemia led to an increase in the aspartate level and a decrease in glutamate, glutamine, and GABA levels. The total amino acid pool was decreased compared with amino acid levels in normoglycemic rats. GVG treatment of hypoglycemic rats led to a decrease in the aspartate level and a further reduction in glutamate and glutamine levels. In this case, GABA accumulation continued, although the glutamine pool was almost depleted. The GABA level increased postmortem, but there were no detectable changes in levels of the other amino acids. Pretreatment of the rats with hypoglycemia reduced both glutamate and glutamine levels with a subsequent decreased postmortem GABA accumulation. The half-maximal GABA synthesis rate was obtained when the glutamate level was reduced by 50% and the glutamine level was reduced by 80%.     

Metabotropic glutamate receptor subtype 4 selectively modulates both glutamate and GABA transmission in the striatum: implications for Parkinson's disease treatment

Alterations of striatal synaptic transmission have been associated with several motor disorders involving the basal ganglia, such as Parkinson's disease. For this reason, we investigated the role of group-III metabotropic glutamate (mGlu) receptors in regulating synaptic transmission in the striatum by electrophysiological recordings and by using our novel orthosteric agonist (3 S )-3-[(3-amino-3-carboxypropyl(hydroxy)phosphinyl)-hydroxymethyl]-5-nitrothiophene (LSP1-3081) and l -2-amino-4-phosphonobutanoate (L-AP4). Here, we show that both drugs dose-dependently reduced glutamate- and GABA-mediated post-synaptic potentials, and increased the paired-pulse ratio. Moreover, they decreased the frequency, but not the amplitude, of glutamate and GABA spontaneous and miniature post-synaptic currents. Their inhibitory effect was abolished by ( RS )-α-cyclopropyl-4-phosphonophenylglycine and was lost in slices from mGlu4 knock-out mice. Furthermore, ( S )-3,4-dicarboxyphenylglycine did not affect glutamate and GABA transmission. Finally, intrastriatal LSP1-3081 or L-AP4 injection improved akinesia measured by the cylinder test. These results demonstrate that mGlu4 receptor selectively modulates striatal glutamate and GABA synaptic transmission, suggesting that it could represent an interesting target for selective pharmacological intervention in movement disorders involving basal ganglia circuitry.     

gamma-Aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development

Profound alterations in the function of GABA occur over the course of postnatal development. Changes in GABA(A) receptor expression are thought to contribute to these differences in GABAergic function, but how subunit changes correlate with receptor function in individual developing neurons has not been defined precisely. In the current study, we correlate expression of 14 different GABA(A) receptor subunit mRNAs with changes in the pharmacological properties of the receptor in individual hippocampal dentate granule cells over the course of postnatal development in rat. We demonstrate significant developmental differences in GABA(A) receptor subunit mRNA expression, including greater than two-fold lower expression of alpha1-, alpha4- and gamma2-subunit mRNAs and 10-fold higher expression of alpha5-mRNA in immature compared with adult neurons. These differences correlate both with regional changes in subunit protein level and with alterations in GABA(A) receptor function in immature dentate granule cells, including two-fold higher blockade by zinc and three-fold lower augmentation by type-I benzodiazepine site modulators. Further, we find an inverse correlation between changes in GABA(A) receptor zinc sensitivity and abundance of vesicular zinc in dentate gyrus during postnatal development. These findings suggest that developmental differences in subunit expression contribute to alterations in GABA(A) receptor function during postnatal development.     

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.     

Flumazenil selectively prevents the increase in alpha(4)-subunit gene expression and an associated change in GABA(A) receptor function induced by ethanol withdrawal

The actions of ethanol on gamma-aminobutyric acid type A (GABA(A)) receptors are still highly controversial issues but it appears that some of its pharmacological effects may depend on receptor subunit composition. Prolonged ethanol exposure produces tolerance and dependence and its withdrawal alters GABA(A) receptor subunit gene expression and function. Whereas benzodiazepines are clinically effective in ameliorating ethanol withdrawal symptoms, work in our laboratory showed that benzodiazepines also prevent, in vitro, some of the ethanol withdrawal-induced molecular and functional changes of the GABA(A) receptors. In the present work, we investigated the effects, on such changes, of the benzodiazepine receptor antagonist flumazenil that can positively modulate alpha(4)-containing receptors. We here report that flumazenil prevented both the ethanol withdrawal-induced up-regulation of the alpha(4)-subunit and the increase in its own modulatory action. In contrast, flumazenil did not inhibit ethanol withdrawal-induced decrease in alpha(1)- and delta-subunit expression as well as the corresponding decrease in the modulatory action on GABA(A) receptor function of both the alpha(1)-selective ligand zaleplon and the delta-containing receptor preferentially acting steroid allopregnanolone. These observations are the first molecular and functional evidence that show a selective inhibition by flumazenil of the up-regulation of alpha(4)-subunit expression elicited by ethanol withdrawal.     

Understanding your inhibitions: effects of GABA and GABAA receptor modulation on brain cortical metabolism

A targeted neuropharmacological, ¹H/¹³C NMR spectroscopy and multivariate statistical approach was used to examine the effects of exogenous GABA and ligands at the GABA_A receptor family on brain metabolism in the Guinea pig cortical tissue slice. All ligands at GABA_A receptors generated metabolic patterns which were distinct from one another with the major variance in the data arising because of metabolic work (shown by net flux into Krebs cycle byproducts and increased metabolic pool sizes). Three major clusters of metabolic signatures were identified which corresponded to: (i) activity at phasic (synaptic) GABA_A receptors, dominated by α1-containing receptors and responsive to GABA at 10 μmol/L; (ii) activity at perisynaptic receptors, dominated by response to high (40 μmol/L) GABA and the superagonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride, and C, activity at extrasynaptic receptors, dominated by response to low (0.1–1.0 μmol/L) GABA, zolpidem (400 nmol/L) and the non-specific allosteric modulator RO19-4603 (1 nmol/L). These results highlight the utility of a different but robust approach to study of the GABAergic system using metabolic systems analysis.     

Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Kinase C Phosphorylate a Synthetic Peptide Corresponding to a Sequence That Is Specific for the γ2L Subunit of the GABAA Receptor

Abstract: The γ₂ subunit of the GABA_A receptor (GABA_A-R) is alternatively spliced. The long variant (γ_2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca²⁺/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ_2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K _m values for PKC-and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 μM , respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the γ_2L subunit has been shown to be necessary for ethanol potentiation of the GABA_A-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.     

Social isolation-induced increase in alpha and delta subunit gene expression is associated with a greater efficacy of ethanol on steroidogenesis and GABA receptor function

Previously we have demonstrated that social isolation of rats reduces both the cerebrocortical and plasma concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and potentiates the positive effects of acute ethanol administration on the concentrations of this neurosteroid. We now show that the ethanol-induced increase in 3alpha,5alpha-TH PROG is more pronounced in the brain than in the plasma of isolated rats. The ability of ethanol to inhibit isoniazid-induced convulsions is greater in isolated rats than in group-housed animals and this effect is prevented by treatment with finasteride. Social isolation modified the effects of ethanol on the amounts of steroidogenic regulatory protein mRNA and protein in the brain. Moreover, ethanol increased the amplitude of GABA(A) receptor-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurones with greater potency in hippocampal slices prepared from socially isolated rats than in those from group-housed rats, an effect inhibited by finasteride. The amounts of the alpha(4) and delta subunits of the GABA(A) receptor in the hippocampus were increased in isolated rats as were GABA(A) receptor-mediated tonic inhibitory currents in granule cells of the dentate gyrus. These results suggest that social isolation results in changes in GABA(A) receptor expression in the brain, and in an enhancement of the stimulatory effect of ethanol on brain steroidogenesis, GABA(A) receptor function and associated behaviour.     

Subunit composition and quantitative importance of GABA(A) receptor subtypes in the cerebellum of mouse and rat

In cerebellum, 13 different GABA(A) receptor subunits are expressed. The number of different receptor subtypes formed in this tissue, their subunit composition and their quantitative importance so far has not been determined. In the present study, immunodepletion by immunoaffinity chromatography, as well as immunoprecipitation and western blot analysis was performed using 13 different subunit-specific antibodies to provide an overview on the subunit composition and abundance of GABA(A) receptor subtypes in mouse and rat cerebellum. Results obtained indicate that alpha1betaxgamma2, alpha1alpha6betaxgamma2, alpha6betaxgamma2, alpha6betaxdelta and alpha1alpha6betaxdelta are the major GABA(A) receptor subtypes present in the cerebellum. In addition, small amounts of alpha1betaxdelta receptors and a series of minor receptor subtypes containing alpha2, alpha3, alpha4, alpha5, gamma1 or gamma3 subunits are also present in the cerebellum. Whereas the abundance of alpha1alpha6betaxgamma2, alpha6betaxdelta and alpha1alpha6betaxdelta receptors is different in mouse and rat cerebellum, that of other receptors is quite similar in these tissues. Data obtained for the first time provide an overview on the GABA(A) receptor subtypes present in the cerebellum and represent the basis for further studies investigating changes in receptor expression and composition under pathological conditions.     

Identification of amino acid residues important for assembly of GABA receptor alpha1 and gamma2 subunits

Comparative models of GABA(A) receptors composed of alpha1 beta3 gamma2 subunits were generated using the acetylcholine-binding protein (AChBP) as a template and were used for predicting putative engineered cross-link sites between the alpha1 and the gamma2 subunit. The respective amino acid residues were substituted by cysteines and disulfide bond formation between subunits was investigated on co-transfection into human embryonic kidney (HEK) cells. Although disulfide bond formation between subunits could not be observed, results indicated that mutations studied influenced assembly of GABA(A) receptors. Whereas residue alpha1A108 was important for the formation of assembly intermediates with beta3 and gamma2 subunits consistent with its proposed location at the alpha1(+) side of GABA(A) receptors, residues gamma2T125 and gamma2P127 were important for assembly with beta3 subunits. Mutation of each of these residues also caused an impaired expression of receptors at the cell surface. In contrast, mutated residues alpha1F99C, alpha1S106C or gamma2T126C only impaired the formation of receptors at the cell surface when co-expressed with subunits in which their predicted interaction partner was also mutated. These data are consistent with the prediction that the mutated residue pairs are located close to each other.     

黑腹果蝇GABA受体GRD亚基和LCCH3 亚基的克隆、表达与鉴定

昆虫GABA受体（γ-aminobutyric acid receptor, GABAR）是杀虫剂的重要靶标之一。本研究以黑腹果蝇Drosophila melanogaster整体组织的cDNA作为模板, 采用RT-PCR技术扩增了黑腹果蝇GABA受体LCCH3亚基和GRD亚基的cDNA序列, 并克隆至pET-32a表达载体上, 测序结果表明获得的序列与基因库中已发表的序列一致性在99%以上, 无移码突变。在IPTG的诱导下, LCCH3基因成功在大肠杆菌Escherichia coli中表达, 而GRD基因未表达。通过包涵体洗涤、变性、Ni²⁺亲合层析纯化、稀释复性获得纯化的重组表达的LCCH3蛋白, 并用圆二色谱测定了目标蛋白的二级结构, 主要富含β结构。该研究结果为研究昆虫GABAR的结构和功能关系提供了重要的参考数据。     

Developing oligodendrocytes express functional GABA(B) receptors that stimulate cell proliferation and migration

GABA(B) receptors (GABA(B)Rs) are involved in early events during neuronal development. The presence of GABA(B)Rs in developing oligodendrocytes has not been established. Using immunofluorescent co-localization, we have identified GABA(B)R proteins in O4 marker-positive oligodendrocyte precursor cells (OPCs) in 4-day-old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABA(B1abcdf) and GABA(B2) subunits of the GABA(B)R. Using semiquantitative PCR analysis with GABA(B)R isoform-selective primers we found that the expression level of GABA(B1abd) was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABA(B2) isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABA(B1) and GABA(B2) subunits are under independent control during oligodendroglial development. Activation of GABA(B)Rs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABA(B)R activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber-based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABA(B)Rs in oligodendrocyte development. The presence of GABA(B)Rs in developing oligodendrocytes provides a new mechanism for neuronal-glial interactions during development and may offer a novel target for promoting remyelination following white matter injury.     

Interaction between GABAA receptor subunit intracellular loops: implications for higher order complex formation

The majority of fast inhibitory neurotransmission in the CNS is mediated by the GABA type-A (GABAA) receptor, a ligand-gated chloride channel. Of the approximately 20 different subunits composing the hetero-pentameric GABAA receptor, the gamma2 subunit in particular seems to be important in several aspects of GABAA receptor function, including clustering of the receptor at synapses. In this study, we report that the intracellular loop of the gamma2 subunit interacts with itself as well as with gamma1, gamma3 and beta1-3 subunits, but not with the alpha subunits. We further show that gamma2 subunits interact with photolabeled pentameric GABAA receptors composed of alpha1, beta2/3 and gamma2 subunits, and calculate the dissociation constant to be in the micromolar range. By using deletion constructs of the gamma2 subunit in a yeast two-hybrid assay, we identified a 23-amino acid motif that mediates self-association, residues 389-411. We confirmed this interaction motif by inhibiting the interaction in a glutathione-S-transferase pull-down assay by adding a corresponding gamma2-derived peptide. Using similar approaches, we identified the interaction motif in the gamma2 subunit mediating interaction with the beta2 subunit as a 47-amino acid motif that includes the gamma2 self-interacting motif. The identified gamma2 self-association motif is identical to the interaction motif reported between GABAA receptor and GABAA receptor-associated protein (GABARAP). We propose a model for GABAA receptor clustering based on GABARAP and GABAA receptor subunit-subunit interaction.