GABAA Receptor Subtypes Expressed in Cerebellar Granule Cells: A Developmental Study

Abstract: The developmental properties of primary rat cerebellar granule cells have been characterised with respect to their expression of GABA_A receptor subtypes using both an immunological approach and radioligand binding assays. At day 1 in culture, the GABA_A receptor α1 subunit was detectable in immunoblots and increased in level up to day 9. The GABA_A receptor α6 subunit was not detectable at day 1; however, at days 3–5, a specific M_r 58,000 anti-α6 1–16 Cys immunoreactive species was present which further increased in level up to 9 days in culture. Similar qualitative results were obtained for the expression of the GABA_A receptor α6 subunit in age-matched rat cerebellar membranes. In parallel studies, it was found that although there was an overall increase in [³H]Ro 15–4513 binding sites with days in culture, the relative contributions of diazepam-sensitive and diazepam-in-sensitive [³H]Ro 15–4513 binding changed. A time-dependent enrichment of the diazepam-insensitive binding site up to a maximum of 74% of total [³H]Ro 15–4513 sites was found. This was concomitant with the appearance of the GABA_A receptor α6 subunit. These results are in agreement with the pharmacology described for α6βγ2 cloned receptors. They suggest a developmentally regulated expression of the GABA_A receptor α6 subunit gene at a time that is correlated in vivo with establishment of neuronal connections.     

The effects of zolpidem treatment on GABA(A) receptors in cultured cerebellar granule cells: changes in functional coupling

AimsHypnotic zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) action, with preferential although not exclusive binding for α1 subunit-containing GABA_A receptors. The pharmacological profile of this drug is different from that of classical benzodiazepines, although it acts through benzodiazepine binding sites at GABA_A receptors. The aim of this study was to further explore the molecular mechanisms of GABA_A receptor induction by zolpidem.Main methodsIn the present study, we explored the effects of two-day zolpidem (10 μM) treatment on GABA_A receptors on the membranes of rat cerebellar granule cells (CGCs) using [³H]flunitrazepam binding and semi-quantitative PCR analysis.Key findingsTwo-day zolpidem treatment of CGCs did not significantly affect the maximum number (B_max) of [³H]flunitrazepam binding sites or the expression of α1 subunit mRNA. However, as shown by decreased GABA [³H]flunitrazepam binding, two-day exposure of CGCs to zolpidem caused functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptor complexes.SignificanceIf functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptors is the mechanism responsible for the development of tolerance following long-term administration of classical benzodiazepines, chronic zolpidem treatment may induce tolerance.     

Obovatol isolated from Magnolia obovata enhances pentobarbital-induced sleeping time: Possible involvement of GABAA receptors/chloride channel activation

This study aimed to investigate the effects of obovatol isolated from Magnolia obovata on pentobarbital-induced sleeping behaviors and to determine whether these effects were mediated by GABA_A receptors/chloride channel activation, using a western blot technique and Cl^? sensitive fluorescence probe. GABA_A receptors subunits expression and chloride influx were investigated in cultured cerebellar granule cells. Obovatol (0.05, 0.1, and 0.2 mg/kg) prolonged the sleeping time induced by pentobarbital (42 mg/kg). In addition, obovatol (20 and 50 μM) significantly increased Cl^? influx in the primary cultured cerebellar granule cells. Moreover, obovatol increased the expression of GABA_A receptor α-, β-, and γ-subunits. However, it had no effect on the abundance of the expression of glutamic acid decarboxylase (GAD), suggesting that obovatol might not activate GAD. These results suggest that obovatol potentiates pentobarbital-induced sleeping time through the GABA_A receptors/chloride channel activation.     

Differential Expression of GABAA/Benzodiazepine Receptor Subunit mRNAs and Ligand Binding Sites in Mouse Cerebellar Neurons Following In Vivo Ethanol Administration: An Autoradiographic Analysis

Abstract: The γ-aminobutyric acid_A (GABA_A)/benzodiazepine (BZ) receptor is a pentamer composed of subunits belonging to several classes (α_1–6, β_1–4, γ_1–4, δ, and ρ₁ and ρ₂). In situ hybridization, radioligand autoradiography, and immunocytochemistry were used to examine GABA_A/BZ receptor α₁, α₆, β₂, β₃, and γ₂ subunit expression in murine Purkinje, granule, and deep cerebellar neurons after in vivo ethanol exposure. Chronic ethanol treatment resulted in decreased α₁ subunit mRNA expression in each cell type, whereas the expression of α₆ and γ₂ subunit mRNA levels increased; no changes were observed in the expression of β₂ and β₃ subunit mRNA. GABA and BZ agonist binding and antibody staining paralleled the changes in mRNA levels. Acute ethanol injection resulted in increased expression of α₁ and β₃ mRNAs, whereas levels of α₆, β₂, and γ₂ mRNAs remained stable. Our results indicate that, in cerebellar neurons, the expression of specific GABA_A/BZ receptor subunit mRNAs, polypeptides, and binding sites is independently regulated by in vivo administration of alcohol. The observed changes were not restricted to any one cerebellar cell type, because subunit expression in Purkinje, granule, and deep cerebellar cells was similarly affected.     

Modulation of Ionotropic GABA Receptors by 6-Methoxyflavanone and 6-Methoxyflavone

We evaluated the effects of 6-methoxyflavanone and 6-methoxyflavone on wild-type α1/α2β2γ2L GABA_A and ρ1 GABA_C receptors and on mutant ρ1I307S, ρ1W328 M, ρ1I307S/W328 M GABA_C receptors expressed in Xenopus oocytes using two-electrode voltage clamp and radioligand binding. 6-Methoxyflavanone and 6-methoxyflavone act as a flumazenil-insensitive positive allosteric modulator of GABA responses at human recombinant α1β2γ2L and α2β2γ2L GABA_A receptors. However, unlike 6-methoxyflavone, 6-methoxyflavanone was relatively inactive at α1β2 GABA_A receptors. 6-Methoxyflavanone inhibited [³H]-flunitrazepam binding to rat brain membranes. Both flavonoids were found to be inactive as modulators at ρ1, ρ1I307S and ρ1W328 M GABA receptors but acted as positive allosteric modulators of GABA at the benzodiazepine sensitive ρ1I307S/W328 M GABA receptors. This double mutant retains ρ1 properties of being insensitive to bicuculline and antagonised by TPMPA and THIP. Additionally, 6-methoxyflavanone was also a partial agonist at ρ1W328 M GABA receptors. The relative inactivity of 6-methoxyflavanone at α1β2 GABA_A receptors and it’s partial agonist action at ρ1W328 M GABA receptors suggest that it exhibits a unique profile not matched by other flavonoids.     

GABAA receptors on rat cerebellar granule cells are potently activated by muscimol but only slightly modulated by the benzodiazepine agonist flunitrazepam

Summary GABA_A receptors present on rat cerebellar granule cells in culture were studied by the whole cell patch clamp technique. Muscimol appeared to be more potent than GABA itself in activating Cl^– currents. A benzodiazepine, flunitrazepam, only slightly (10%) potentiated the GABA action.These results support the previous suggestion that GABA_A receptors containing the subunit, such as those in the cerebellum granule cells, are potently activated by muscimol. The present results also bear out the concept that GABA action on receptors containing the subunit is not potentiated by benzodiazepines.     

Antibodies to the Human γ2 Subunit of the γ-Aminobutyric AcidA/Benzodiazepine Receptor

Abstract: A γ-aminobutyric acid_A (GABA_A) receptor (GABA_AR) γ₂ subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage λgt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors λgt11 and pGEX-3X. Both γ-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the γ₂IL were purified, and a rabbit antibody to the LacZ–γ₂IL was made. The antibody reacted with the γ₂IL of both LacZ and GST fusion proteins and immunoprecipitated the GABA_AR/ benzodiazepine receptor (GABA_AR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABA_AR/BZDR immunoblots with a wide peptide band of 44,000–49,000 M_r. Immunoprecipitation studies with the anti-γ₂IL antibody suggest that in the cerebral cortex, 87% of the GABA_ARs with high affinity for benzodiazepines and 70% of the GABA_ARs with high affinity for muscimol contain at least a γ subunit, probably a γ₂. These results indicate that there are [³H]muscimol binding GABA_ARs that do not bind [³H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABA_AR/BZDR antibodies indicate that the most abundant combination of GABA_AR subunits in the cerebral cortex involves α₁, γ₂ (or other γ), and β₂ and/or β₃ subunits. These subunits coexist in >60% of the GABA_AR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20–25%) of the cerebellar GABA_AR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain α₆, also contain γ₂ (or other γ) subunit(s). The α₁ and β₂ or β₃ subunits are also frequently associated with γ₂ (or other γ) and α₆ in these cerebellar receptors.     

N-Acetylaspartylglutamate Stimulates Metabotropic Glutamate Receptor 3 to Regulate Expression of the GABAAα6 Subunit in Cerebellar Granule Cells

Abstract: We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 µM) results in the transient increase in content of GABA_Aα6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine and (2S)-α-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in α6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, α2-adrenergic, muscarinic, and GABA_B receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in α6 levels induced by 30 µM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the β-adrenergic receptor agonist isoproterenol. The increase in α6 mRNA content is followed by a fourfold increase in α6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional α6-containing GABA_A receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA_Aα6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA_A receptor subunit composition.     

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.     

Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

GABA_A-receptors were localized in explant cultures of rat cerebellum and in dissociated primary cultures of rat cerebellar granule cells and rat cerebellar astrocytes using the monoclonal antibody bd-17 directed against the -subunit of the GABA_A/benzodiazepine/chloride channel complex. At the light microscope level specific staining of GABA_A-receptors was localized in various types of neurones in explant cultures of rat cerebellum using the indirect peroxidase-antiperoxidase (PAP) technique, whereas no specific staining was found in astrocytes. At the electron microscope level labeling of GABA_A-receptors was observed in the plasma membrane of both the cell bodies and processes in dissociated primary cultures of cerebellar granule cells using an indirect preembedding immunogold staining technique which in contrast to the classical PAP technique allows quantitative estimations to be performed. Quantification of the labeling intensity revealed a higher concentration of GABA_A-receptors per m plasma membrane in the cell bodies than in the processes. In discrete areas an extremely high density of the GABA_A-receptors was observed. No specific labeling of GABA_A-receptors was observed in dissociated primary cultures of cerebellar astrocytes.Special issue dedicated to Dr. Eugene Roberts.     

Possible Involvement of GABAA and GABAB Receptors in the Inhibitory Action of Lindane on Transmitter Release from Cerebellar Granule Neurons

Cerebellar granule cells in culture express receptors for GABA belonging to the GABA_A and GABA_B classes. In order to characterize the ability of the insecticide lindane to interact with these receptors cells were grown in either plain culture media or media containing 150 M THIP as this is known to influence the properties of both GABA_A and GABA_B receptors. It was found that lindane regardless of the culture condition inhibited evoked (40 mM K⁺) release of neurotransmitter ([³H]D-aspartate as label for glutamate). In naive cells both GABA_A and GABA_B receptor active drugs prevented the inhibitory action of lindane but in THIP treated cultures none of the GABA_A and GABA_B receptor active drugs had any effect on the inhibitory action of lindane. This lack of effect was not due to inability of baclofen itself to inhibit transmitter release. It is concluded that lindane dependent on the state of the GABA_A and GABA_B receptors is able to indirectly interfere with both GABA_A and GABA_B receptors. In case of the latter receptors it was shown using [³H]baclofen to label the receptors that lindane could not displace the ligand confirming that lindane is likely to exert its action at a site different from the agonist binding site.     

More Than one Alpha Variant May Exist in a GabaA/benzodiazepine Receptor Complex

Abstract

G A B A_A/Benzodiazepine receptors are formed by the assembly of presumably five polypeptides with unknown stoichiometry. Six α, three β, two λ, and one δ subunit have been characterized on the molecular level. In analogy to the nicotinic acetylcholine receptor, and supported by functional analysis of recombinantly expressed GABA_A receptor subunits, a structure containing at least three different polypeptides has been proposed for the functional GABA_A and benzodiazepine regulated Cl^?-channel. Using an α₁ subunit specific antiserum we could show that additional α variants are present in α₁ subunit containing GABA_A/Benzodiazepine receptor complexes. This suggests that the diversity of GABA_A/Benzodiazepine receptors may be larger than previously thought.     

Conservation of γ-Aminobutyric Acid Type A Receptor α6 Subunit Gene Expression in Cerebellar Granule Cells

Abstract: The γ-aminobutyric acid type A receptor cDNAs encoding the α6 subunit homologues from chicken and goldfish have been cloned and sequenced. These proteins exhibit 83 and 75% identity, respectively, to the rat α6 polypeptide. In situ hybridization has demonstrated that, as in mammals, the avian and teleost fish α6 subunit genes are predominately expressed in cerebellar granule cells. Correspondingly, flunitrazepam-nondisplaceable binding of [³H]Ro 15-4513 (a benzodiazepine partial inverse agonist), which is a major characteristic of γ-aminobutyric acid type A receptors that contain the α6 polypeptide, is also mainly found for cerebellar granule cells of fish and chick. The conservation of this expression pattern suggests that γ-aminobutyric acid type A receptors possessing the α6 subunit are of fundamental importance for cerebellar function and that the corresponding gene regulatory elements, e.g., granule cell-specific enhancers, have also been conserved.     

Musciomol and flunitrazepam binding sites in a subcellular fraction enriched in rat cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of GABA_A and of benzodiazepine binding sites between membranes derived from this fraction (fraction G) and from a total cerebellar homogenate (fraction T) was studied. The benzodiazepine and GABA binding sites were measured by the binding of agonists [³H]flunitrazepam and [³H]muscimol, respectively. The results indicate that both binding sites are present, but only slightly enriched, in the glomerular synapses. We found a muscimol/flunitrazepam binding site ratio of two, which is consistent with the enrichement of muscimol binding sites in the granular layer shown by both autoradiographic with radioactive glutamatergic ligands and in situ hybridization experiments respectively.     

Short and Long Form γ2 Subunits of the GABAA/Benzodiazepine Receptors

Abstract: Three novel antisera to the γ₂ subunit of the γ-aminobutyric acid_A (GABA_A) receptor/benzodiazepine receptor (GABA_AR/BZDR) complex have been made. Anti-γ_2S and anti-γ_2L are specific antibodies to synthetic peptides that recognize the γ_2S (short) and γ_2L (long) forms, respectively, of the γ₂ subunit. An antibody (anti-γ₂IL2) to staphylococcal protein A fusion protein of the large intracellular loop (γ₂IL) located between the putative transmembrane segments M3 and M4 of γ_2S recognizes both γ_2S and γ_2L subunits. The antibodies immunoprecipitated both the solubilized and affinity-purified GABA_AR/BZDR from rat and bovine brain. Immunoblots with membranes from rat brain cerebral cortex as well as with affinity-purified receptor from bovine cortex show that anti-γ_2S and anti-γ_2L recognize peptides of 45,000 and 47,000 M_r, respectively. Immunoprecipitation experiments indicate that γ_2S is more prevalent in hippocampus, whereas γ_2L is more abundant in cerebellum. Intermediate values for each form are found in the cerebral cortex. The results suggest that in the rat brain there is a considerable amount of colocalization of γ_2S and γ_2L in the same receptor complex. In the cerebral cortex, 15% of the BZDRs contain both γ_2S and γ_2L subunits and 41–48% of the γ_2L subunit coexists with γ_2S in the same receptor complex. In cerebellum, in 27% of the clonazepam-sensitive and 39% of the clonazepam-insensitive BZDRs the γ_2S and γ_2L coexist in the same receptor complex. The latter are presumably localized in granule cells and also contain α₆. In addition, almost all (93%) the clonazepam-insensitive BZDRs that contain γ_2L also contain a γ_2S subunit in the same receptor complex. The most likely interpretation of the results is that there is an important population of granule cell receptors that contain α₆, γ_2S, and γ_2L coexisting in the same receptor complex. Nevertheless, 31% of the cerebellar receptors that contain α₆ subunit(s) have neither γ_2S nor γ_2L subunits. There are also species differences with respect to the relative abundance of γ_2S and γ_2L. These results might be relevant for understanding the molecular mechanisms underlying some of the GABA_AR/BZDR-mediated effects of ethanol intoxication involving cerebellar granule cells.     

Potentiating effect of glabridin from Glycyrrhiza glabra on GABAA receptors

Extracts from Glycyrrhiza are traditionally used for the treatment of insomnia and anxiety. Glabridin is one of the main flavonoid compounds from Glycyrrhiza glabra and displays a broad range of biological properties. In the present work, we investigated the effect of glabridin on GABA_A receptors. For this purpose, we employed the two-electrode voltage-clamp technique on Xenopus laevis oocytes expressing recombinant GABA_A receptors. Through this approach, we observed that glabridin presents a strong potentiating effect on GABA_A α1β(1?3)γ2 receptors. The potentiation was slightly dependent on the β subunit and was most pronounced at the α1β2γ2 subunit combination, which forms the most abundant GABA_A receptor in the CNS. Glabridin potentiated with an EC₅₀ of 6.3±1.7 µM and decreased the EC₅₀ of the receptor for GABA by approximately 12-fold. The potentiating effect of glabridin is flumazenil-insensitive and does not require the benzodiazepine binding site. Glabridin acts on the β subunit of GABA_A receptors by a mechanism involving the M286 residue, which is a key amino acid at the binding site for general anesthetics, such as propofol and etomidate. Our results demonstrate that GABA_A receptors are strongly potentiated by one of the main flavonoid compounds from Glycyrrhiza glabra and suggest that glabridin could contribute to the reported hypnotic effect of Glycyrrhiza extracts.     

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.     

Defined concatenated α6α1β3γ2 GABAA receptor constructs reveal dual action of pyrazoloquinolinone allosteric modulators

Pyrazoloquinolinones (PQs) have been extensively studied as modulators of GABA_A receptors with different subunit composition, exerting modulatory effects by binding at α+/β- interfaces of GABA_A receptors. PQs with a substituent in position R7 have been reported to preferentially modulate α6- subunit containing GABA_A receptors which are mostly expressed in the cerebellum but were also found in the olfactory bulb, in the cochlear nucleus, in the hippocampus and in the trigeminal sensory pathway. They are considered potentially interesting in the context of sensori-motor gating deficits, depressive-like behavior, migraine and orofacial pain. Here we explored the option to modify the lead ligands’ R7 position. In the compound series we observed two different patterns of allosteric modulation in recombinantly expressed α6β3γ2 receptors, namely monophasic and biphasic positive modulation. In the latter case the additional phase occurred in the nanomolar range, while all compounds displayed robust modulation in the micromolar range. Nanomolar, near silent binding has been reported to occur at benzodiazepine binding sites, but was not investigated at the diazepam insensitive α6+/γ2- interface. To clarify the mechanism underlying the biphasic effect we tested one of the compounds in concatenated receptors. In these constructs the subunits are covalently linked, allowing to form either the α6+/γ2- interface, or the α6+/β3- interface, to study the resulting modulation. With this approach we were able to ascribe the nanomolar modulation to the α6+/γ2- interface. While not all compounds display the nanomolar phase, the strong modulation at the α6+/β3 interface proved to be tolerant for all tested R7 groups. This provides the future option to introduce e.g. isotope labelled or fluorescent moieties or substituents that enhance solubility and bioavailability.     

Alcohol Selectivity of β3-Containing GABAA Receptors: Evidence for a Unique Extracellular Alcohol/Imidazobenzodiazepine Ro15-4513 Binding Site at the α+β- Subunit Interface in αβ3δ GABAA Receptors

GABA_A receptors (GABARs) have long been the focus for acute alcohol actions with evidence for behaviorally relevant low millimolar alcohol actions on tonic GABA currents and extrasynaptic α4/6, δ, and β3 subunit-containing GABARs. Using recombinant expression in oocytes combined with two electrode voltage clamp, we show with chimeric β2/β3 subunits that differences in alcohol sensitivity among β subunits are determined by the extracellular N-terminal part of the protein. Furthermore, by using point mutations, we show that the β3 alcohol selectivity is determined by a single amino acid residue in the N-terminus that differs between GABAR β subunits (β3Y66, β2A66, β1S66). The β3Y66 residue is located in a region called “loop D” which in γ subunits contributes to the imidazobenzodiazepine (iBZ) binding site at the classical α+γ2- subunit interface. In structural homology models β3Y66 is the equivalent of γ2T81 which is one of three critical residues lining the benzodiazepine binding site in the γ2 subunit loop D, opposite to the “100H/R-site” benzodiazepine binding residue in GABAR α subunits. We have shown that the α6R100Q mutation at this site leads to increased alcohol-induced motor in-coordination in alcohol non-tolerant rats carrying the α6R100Q mutated allele. Based on the identification of these two amino acid residues α6R100 and β66 we propose a model in which β3 and δ containing GABA receptors contain a unique ethanol site at the α4/6+β3- subunit interface. This site is homologous to the classical benzodiazepine binding site and we propose that it not only binds ethanol at relevant concentrations (EC₅₀–17 mM), but also has high affinity for a few selected benzodiazepine site ligands including alcohol antagonistic iBZs (Ro15-4513, RY023, RY024, RY80) which have in common a large moiety at the C7 position of the benzodiazepine ring. We suggest that large moieties at the C7-BZ ring compete with alcohol for its binding pocket at a α4/6+β3- EtOH/Ro15-4513 site. This model reconciles many years of alcohol research on GABARs and provides a plausible explanation for the competitive relationship between ethanol and iBZ alcohol antagonists in which bulky moieties at the C7 position compete with ethanol for its binding site. We conclude with a critical discussion to suggest that much of the controversy surrounding this issue might be due to fundamental species differences in alcohol and alcohol antagonist responses in rats and mice.