Structure of α6β3δ GABAA receptors and their lack of ethanol sensitivity

Delta (δ) subunit containing GABA_A receptors are expressed extra‐synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with α₆ subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABA_A receptor pentamers by subunit concatenation. These receptors (composed of α₆, β₃ and δ subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one and to 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. α₆‐β₃‐α₆/δ receptors showed a substantial response to GABA alone. Three receptors, β₃‐α₆‐δ/α₆‐β₃, α₆‐β₃‐α₆/β₃‐δ and β₃‐δ‐β₃/α₆‐β₃, were only uncovered in the combined presence of the neurosteroid 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one with GABA. All four receptors were activated by 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the δ subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the δ subunit can assume multiple positions in a receptor pentamer composed of α₆, β₃ and δ subunits.     

Small N-Terminal Deletion by Splicing in Cerebellar α6 Subunit Abolishes GABAA Receptor Function

Abstract: Sequence variation was found in cDNA coding for the extracellular domain of the rat γ-aminobutyric acid type A (GABA_A) receptor α6 subunit. About 20% of polymerase chain reaction (PCR)-amplified α6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226–255 as estimated by counting single-stranded phage plaques hybridized specifically to the short (α6S) and long (wild-type) forms of the α6 mRNA. Genomic PCR revealed an intron located upstream of the 30-nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the α6S subunit with the GABA_A receptor β2 and γ2 subunits in human embryonic kidney 293 cells, were inactive at binding [³H]muscimol and [³H]Ro 15-4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA-induced currents, indistinguishable from those produced by β2γ2 receptors. Therefore, the 10 amino acids encoded by the 30-nucleotide fragment may be essential for the correct assembly or folding of the α6 subunit-containing receptors.     

Analysis by Polymerase Chain Reaction of α1 and α6 GABAA Receptor Subunit mRNAs in Individual Cerebellar Neurons After Whole-Cell Recordings

Abstract: With the use of the single-cell polymerase chain reaction (PCR), the GABA_A receptor subunit mRNA content was analyzed in granule and Purkinje neurons from rat cerebellar slices. We used an experimental protocol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole-cell patch-clamp technique. Based on a computer alignment of the nucleotide sequence corresponding to α1 and α6 GABA_A receptor subunits, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of selective restriction sites within the targeted templates allowed us to identify which receptor subunit mRNAs were coamplified by performing restriction enzyme-mediated cleavage of the amplification products. In all Purkinje neurons assayed, α1 subunit mRNA but not α6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the α1 and α6 GABA_A receptor subunits. A comparison of the results of the PCR amplification and the analysis of GABA-mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic currents that clearly correlate with the presence or the absence of α6 subunit mRNA.     

The α1 and α6 Subunits Can Coexist in the Same Cerebellar GABAA Receptor Maintaining Their Individual Benzodiazepine-Binding Specificities

Abstract: Two GABA_A receptor subunit-specific antibodies anti-α₆ and anti-α₁ have been used for elucidating the relationship between the presence of α₁ and/or α₆ subunits in the cerebellar GABA_A receptors and the benzodiazepine-binding specificity. Receptor immunoprecipitation with the subunit-specific antibodies shows that 39% of the cerebellar GABA_A receptors have α₆, whereas 76% of the receptors have α₁ as determined by [³H]muscimol binding. Results show that 42–45% of the receptors having α₆ also have α₁, whereas 13–15% of the receptors that contain α₁ also have α₆. The immunoprecipitation results as well as immunopurification and immunoblotting experiments reveal the existence of three types of cerebellar GABA_A receptors; i.e., one has both α₁ and α₆ subunits, a second type has α₁ but not α₆, and a third type has α₆ but not α₁ subunits. The results also show that receptors where α₁ and α₆ subunits coexist have two pharmacologically different benzodiazepine-binding properties, each associated with a different α subunit. The α₁ subunit contributes the high-affinity binding of [³H]Ro 15-1788 (flumazenil) and the diazepam-sensitive binding of [³H]Ro 15-4513. The α₆ subunit contributes the diazepam-insensitive binding of [³H]Ro 15-4513, but it does not bind [³H]Ro 15-1788 with high affinity. Thus, in the cerebellar α₁–α₆ GABA_A receptors, there is no dominance of the pharmacology of one α subunit over the other.     

In Situ Analysis of Na,K-ATPase α1- and α3-Isoform mRNAs in Aging Rat Hippocampus

Abstract: Age-related changes in the expression of Na,K-ATPase α1- and α3-isoform mRNAs were analyzed by in situ hybridization in the Fischer-344 rat hippocampus. Quantification of signal density with cRNA probes in rat hippocampus at 3 months of age showed (a) α1 content is 1.5 times higher in granule than in pyramidal cell layers, whereas α3 content shows the opposite ratio and (b) α3 label is found in large clusters related to mossy cells and basket cells and in medium clusters corresponding to interneurons within the dendritic fields of CA1–3. In the 24-month-old rats as compared with the young animals, the α1 signal is increased more than sevenfold in the dendritic fields and is not significantly changed in perikaryal layers. The α3 signal is reduced about threefold ( p < 0.0001, ANOVA, n = 6) in perikaryal layers, is almost completely absent over the interneurons, basket cells, and mossy cells, and is not significantly changed in dendritic fields. These data indicate age-related, cell- and isoform-specific alterations in pretranslational regulation of Na,K-ATPase α isoforms. The striking changes in the dendritic fields, mossy cells, and GABAergic basket cells and interneurons may constitute early and sensitive markers for age-related alterations in hippocampal function, before cell loss.     

Developmental Characterization of Thymosin β4 and β10 Expression in Enriched Neuronal Cultures from Rat Cerebella

Abstract: The β₄ and β₁₀ thymosins are G-actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin β₄ is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin β₁₀ is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the β-thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both β-thymosins were initially expressed in granule cells, although expression, especially of thymosin β₄, was truncated compared with the in vivo time course. As in vivo, thymosin β₄ was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin β₁₀. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the β-thymosins show that modulation of tissue culture conditions could be used to identify factors regulating β-thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the in vivo studies alone.     

Chronic Ethanol Administration Regulates the Expression of GABAA Receptor α1 and α5 Subunits in the Ventral Tegmental Area and Hippocampus

Abstract: Ethanol dependence and tolerance involve perturbation of GABAergic neurotransmission. Previous studies have demonstrated that ethanol treatment regulates the function and expression of GABA_A receptors throughout the CNS. Conceivably, changes in receptor function may be associated with alterations of subunit composition. In the present study, a comprehensive (1–12 weeks) ethanol treatment paradigm was used to evaluate changes in GABA_A receptor subunit expression in several brain regions including the cerebellum, cerebral cortex, ventral tegmental area (VTA) (a region implicated in drug reward/dependence), and the hippocampus (a region involved in memory/cognition). Expression of α₁ and α₅ subunits was regulated by ethanol in a region-specific and time-dependent manner. Following 2–4 weeks of administration, cortical and cerebellar α₁ and α₅ subunit immunoreactivity was reduced. In the VTA, levels of α₁ subunit immunoreactivity were significantly decreased after 12 weeks but not 1–4 weeks of treatment. Hippocampal α₁ subunit immunoreactivity and mRNA content were also significantly reduced after 12 but not after 4 weeks of treatment. In contrast, α₅ mRNA content was increased in this brain region. These data indicate that chronic ethanol administration alters GABA_A receptor subunit expression in the VTA and hippocampus, effects that may play a role in the abuse potential and detrimental cognitive effects of alcohol.     

Molecular determinants for the action of general anesthetics at recombinant α2β3γ2γ-aminobutyric acidA receptors

General anesthetics modulate the activity of ligand-gated ion channels including the GABA(A) receptor. Mutational studies mainly on the benzodiazepine-insensitive alpha(2)beta(1(M286W)) and alpha(6)beta(3(N289M))gamma(2) GABA(A) receptors revealed that a serine in transmembrane domain 2 and a methionine in transmembrane domain 3 are essential for the action of most general anesthetics. We investigated whether these residues would similarly be relevant for their action at the benzodiazepine-sensitive GABA receptor subtype, alpha(2)beta(3)gamma(2). We found that not only the N265M but also the M286W mutation nearly abolished the modulatory effect of etomidate. However, the anti-convulsant loreclezole, a structural homologue of etomidate, was inactive on the N265M mutant, but displayed normal modulatory activity on the M286W mutant. Both mutations did not affect the modulatory action of the neurosteroid alphaxalone. The direct action of alphaxalone, however, was dramatically increased in the M286W mutant to about twice the maximal GABA current but not significantly affected in the N265M mutant. These data demonstrate that the structural requirements for modulatory and direct actions of various general anesthetics are distinct. The molecular switches induced by these mutations can be exploited to identify the molecular determinants for the action of general anesthetics.     

Evidence that GABAA Receptor Subunit mRNA Expression During Development Is Regulated by GABAA Receptor Stimulation

Abstract: The expression of six mRNA species (α2, α3, α5, β2, β3, and γ2) encoding for GABA_A receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. α3, α5, and β3 subunit expression declined, α2 expression increased, whereas β2 and γ2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABA_A receptor stimulation regulates these expression profiles, we tested the effect of a GABA_A receptor positive modulator, allopregnanolone, and a GABA_A receptor noncompetitive antagonist, tert -butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the α3, α5, or β3 subunit mRNA expression declined and prevented the increase in α2 subunit mRNA expression. As well, allopregnanolone down-regulated β2 subunit mRNA expression. TBPS, on the other hand, up-regulated α3, α5, β2, and β3 subunit mRNA expression. It also down-regulated the expression of α2 subunit mRNA. Both allopregnanolone and TBPS had no effect on γ2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABA_A receptor activity.     

Chronic Intermittent Ethanol Treatment in Rats Increases GABAA Receptor α4-Subunit Expression: Possible Relevance to Alcohol Dependence

Abstract: Chronic administration of ethanol to rats on an intermittent regimen, for 60 repeated intoxicating doses and repeated withdrawal episodes, results in a long-lasting kindling phenomenon. This involves an increasing severity of withdrawal, including a reduced threshold to seizures produced by the GABA_A antagonist, pentylenetetrazol. We have shown previously that muscimol-evoked ³⁶Cl⁻ efflux and paired-pulse inhibition (involving GABA_A-mediated recurrent inhibition) were decreased persistently in the CA1 region of hippocampal slices from chronic intermittent ethanol (CIE)-treated rats. We now report elevated levels of mRNA in forebrain for the α4 subunit of the GABA_A receptor (GABAR), considered to be a constituent of pharmacologically and physiologically novel subtypes of GABARs. Using in situ hybridization with digoxigenin-labeled RNA probes, we show that at 2 days withdrawal, 60-dose CIE leads to a significant 30% increase in α4 subunit mRNA levels in the dentate gyrus, 46% increase in the CA3, and 26% increase in the CA1 regions. In contrast, there was no significant change in the mRNAs for the α5 subunit or glutamic acid decarboxylase 67 in the same regions. This study suggests that GABAR subunit-selective alterations occur after CIE treatment, possibly resulting in the alteration of the subunit composition of GABARs, with presumably altered physiological functions. This plasticity of GABARs may contribute to the increased withdrawal severity, reduced hippocampal inhibition, and increased seizure susceptibility of this animal model of human alcohol dependence.     

Developmental Expression of GABAA Receptor Subunit mRNAs in Individual Hippocampal Neurons In Vitro and In Vivo

Abstract: The GABA_A receptor is a heterooligomeric protein complex composed of multiple receptor subunits. Developmental changes in the pattern of expression of 11 GABA_A receptor subunits in individual rat embryonic hippocampal neurons on days 1–21 in culture and acutely dissociated hippocampal neurons from postnatal day (PND) 5 rat pups were investigated using the technique of single-cell mRNA amplification. We demonstrate that multiple GABA_A receptor subunits are expressed within individual hippocampal neurons, with most cells simultaneously expressing α1, α2, α5, β1, and γ2 mRNAs. Further, relative expression of several GABA_A receptor subunit mRNAs changes significantly in embryonic hippocampal neurons during in vitro development, with the relative abundance (compared with β-actin) of α1, α5, and γ2 mRNAs increasing 2.3-, 2.7-, and 3.8-fold, respectively, from days 1 to 14, and β1 increasing 5-fold from days 1 to 21. In situ hybridization with antisense digoxigenin-labeled α1, β1, and γ2 RNA probes demonstrates a similar increase in expression of subunit mRNAs as embryonic hippocampal neurons mature in vitro. Relative abundances of α1, β1, and γ2 subunit mRNAs in acutely dissociated PND 5 hippocampal neurons are also significantly greater than in embryonic day 17 neurons on day 1 in vitro and exceed the peak values seen in cultured neurons on days 14–21, suggesting that GABA_A receptor subunit mRNA expression within individual hippocampal neurons follows a similar, if somewhat delayed, developmental pattern in vitro compared with in vivo. These findings suggest that embryonic hippocampal neuronal culture provides a useful model in which to study the developmental regulation of GABA_A receptor expression and that developmental changes in GABA_A receptor subunit expression may underlie some of the differences in functional properties of GABA_A receptors in neonatal and mature hippocampal neurons.     

Increased Acute Cocaine Sensitivity and Decreased Cocaine Sensitization in GABAA Receptor β3 Subunit knockout Mice

The role of the GABA(A) receptor beta3 subunit in determining acute cocaine sensitivity and behavioral sensitization to repeated cocaine was measured in mice missing both (-/-), one (+/-), or neither (+/+) allele of the beta3 gene. Locomotor stimulation induced by one cocaine injection (20 mg/kg, i.p.) was found to be greater in -/- mice compared with +/+ mice, whereas cocaine-induced behaviors were intermediate in +/- mice. Amphetamine did not cause greater locomotor responses in -/- mice, suggesting that the increased sensitivity of -/- mice to cocaine does not generalize to other psychomotor stimulants. GABA-stimulated chloride uptake was 51% lower in striatum of -/- mice compared with +/+ mice, but only 27% lower in cortex. After 14 daily cocaine injections, the behavioral response to cocaine was increased in +/+ and +/- mice, but was not increased further in -/- mice. Additionally, repeated cocaine exposure decreased striatal GABA(A) receptor function in +/+ and +/- mice. In -/- mice, GABA(A) receptor function was not decreased any further by repeated cocaine injections. Thus, alterations in the beta3 subunit may be responsible for determining the behavioral responses induced by acute and repeated cocaine treatment, as well as mediating the neurochemical adaptation that occurs during sensitization to repeated cocaine.     

γ-Aminobutyric AcidA Receptor α5-Subunit Creates Novel Type II Benzodiazepine Receptor Pharmacology

A cDNA encoding a protein with 70% amino acid identity to the previously characterized gamma-aminobutyric acidA (GABAA) receptor alpha-subunits was isolated from a rat brain cDNA library by homology screening. As observed for alpha 1-, alpha 2-, and alpha 3-subunits, coexpression of this new alpha-subunit (alpha 5) with a beta- and gamma 2-subunit in cultured cells produces receptors displaying high-affinity binding sites for both muscimol, a GABA agonist, and benzodiazepines. Characteristic of GABAA/benzodiazepine type II sites, receptors containing alpha 2-, alpha 3- or alpha 5-subunits have low affinities for several type I-selective compounds. However, alpha 5-subunit-containing receptors have lower affinities for zolpidem (30-fold) and Cl 218 872 (three-fold) than measured previously using recombinantly expressed type II receptors containing either alpha 2- or alpha 3-subunits. Based on these findings, a reclassification of the GABAA/benzodiazepine receptors is warranted.     

Synergistic Interactions Between α1- and α2-Adrenergic Receptors in Activating 3H-Inositol Phosphate Formation in Primary Glial Cell Cultures

Norepinephrine (NE)-stimulated 3H-inositol phosphate (3H-InsP) formation in primary glial cell cultures is thought to be due to alpha 1-adrenergic receptor activation. Surprisingly, the alpha 1-selective agonists phenylephrine and methoxamine showed only 12-21% of the intrinsic activity of NE in activating this response. Although the alpha 2-selective agonist UK 14,304 was itself inactive, inclusion of UK 14,304 increased the response to the alpha 1-selective agonists by about threefold. This increase was concentration-dependent and occurred at all time points examined. 6-Fluoro-NE and alpha-methyl-NE mimicked the effect of NE in glial cultures, although with lower potencies. However, several partial agonists were ineffective in activating this response, in both the presence and absence of UK 14,304. Synergistic interactions were not observed for alpha 1-mediated responses in slices of rat cerebral cortex, either for formation of 3H-InsPs or potentiation of isoproterenol- or adenosine-stimulated cyclic AMP accumulation. Both UK 14,304 and phenylephrine inhibited NE-stimulated 3H-InsP formation in concentrations similar to those necessary to activate this response directly. These results suggest that NE activates 3H-InsP formation in primary glial cultures by synergistic actions on both alpha 1- and alpha 2-adrenergic receptors. The agonists UK 14,304 and phenylephrine also can act to inhibit the response to NE competitively.     

Immunochemical Characterization of the β2 Subunit of the GABAA Receptor

To date three β subunits of the GABA_A receptor have been identified in rat brain as a result of cDNA library screening. The β2 subunit has been reported to have a wide distribution in rat brain based on in situ hybridization studies quantifying β2 mRNA. To study the β2 subunit more directly, we have raised a polyclonal antibody to a synthetic peptide representing residues 315–334 of the intracellular loop of the β2 subunit. The antibody, which had been affinity-purified, recognized the β2 peptide but did not immunolabel homologous β1 and β3 subunit peptides, indicating that this antibody is specific for the β2 subunit of the receptor. In western blots of the purified receptor, the antibody recognized a major diffuse band of 54–58 kDa arid exhibited minor labeling of lower-molecular-mass polypeptides. In western blots of cortex homogenate, the antibody exhibited nervous system-specific labeling of a 55-kDa band that comigrated with the 55-kDa band of the purified receptor. Quantitative immunolabeling of this 55-kDa polypeptide permitted direct determination of the relative amounts of the β2 subunit in different brain regions. The brainstem contained the highest relative specific activity of the β2 subunit, followed by the inferior colliculus, olfactory lobe, and cerebellum. Lower levels of immunolabeling were seen in hypothalamus, hippocampus, thalamus, and cortex.     

Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain

A human cDNA clone containing the 5' coding region of the GABAA/benzodiazepine receptor alpha subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the alpha subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus much greater than pons = striatum = medulla. In situ hybridization revealed high levels of alpha subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor alpha subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.     

Proteins Associated with α1-Subunit-Containing GABAA Receptors from Bovine Brain

Abstract: In contrast to some other ion channels, there are at present no proteins known that bind specifically to mature GABA_A receptor channels. Such proteins may be important for the structural organization and cytoskeletal anchoring of GABA_A receptors and could also be expected to have channel modulatory effects. To identify proteins that are associated with naturally occurring GABA_A receptors we immunoprecipitated these receptors from detergent-solubilized bovine brain membranes by an antibody directed against the α1-subunit. Tubulin and actin were observed to coprecipitate specifically with the receptors. Nine additional proteins were detected, hinting at a complex protein network associated with α1-subunit-containing GABA_A receptors. Results of a biochemical characterization of these G ABA_A receptor- t ubulin complex- a ssociated p roteins (GTAPs) are presented here. Peptide mass fingerprinting analysis and microsequencing of tryptic peptides indicated that at least three GTAPs have not been described until the present.