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.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

Bidirectional Alterations of GABAA Receptor Subunit Peptide Levels in Rat Cortex During Chronic Ethanol Consumption and Withdrawal

Abstract: The pharmacological properties of γ-aminobutyric acid_A (GABA_A) receptors are altered by prolonged exposure to ethanol both in vivo and in vitro. We have shown previously that prolonged ethanol exposure elicits selective alterations in various GABA_A receptor subunit mRNA levels in rat cerebral cortex. Some of these effects are rapidly reversed during ethanol withdrawal. The present study was conducted to determine the effects of prolonged ethanol exposure (dependence) and ethanol withdrawal on cerebral cortical peptide expression for several subunits. GABA_A receptor α1 subunit peptide levels were decreased by nearly 40%, whereas α4 subunit peptide levels were increased by 27% in both ethanol-dependent and withdrawn rats. These changes correlate well with observed alterations in mRNA levels following prolonged ethanol exposure in dependent rats, but do not match the effects on mRNA levels during ethanol withdrawal. β2/3 subunit peptide levels increased by ～32% in both ethanol-dependent rats and rats undergoing ethanol withdrawal. We observed a 30–60% increase in γ1 subunit peptide levels in both dependent rats and those undergoing withdrawal, also correlating with the previous report on ethanol-induced alterations in mRNA levels. Peptide levels for γ2 subunits did not differ from control values in either condition. These findings show that specific alterations in GABA_A receptor subunit peptide levels are associated with ethanol dependence in rats. GABA_A receptor subunit peptide expression is more stable than mRNA expression, and mRNA levels are not representative of peptide expression during ethanol withdrawal. These findings are consistent with the suggestion that alterations in GABA_A receptor gene expression underlie the functional properties of GABA_A receptors in ethanol-dependent rats and those undergoing ethanol withdrawal.     

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.     

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.     

Complex Role of Collybistin and Gephyrin in GABAA Receptor Clustering

Gephyrin and collybistin are key components of GABA_A receptor (GABA_AR) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA_AR subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA_AR α2 and α3 subunit intracellular M3–M4 domain (but not α1, α4, α5, α6, β1–3, or γ1–3) with gephyrin. Curiously, GABA_AR α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA_AR α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA_AR α3 interacts with gephyrin, GABA_AR α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA_AR α2 and collybistin or GABA_AR α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA_AR α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA_AR α2 is capable of “activating ” collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA_AR α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA_AR and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABA_ARs, but not GlyRs or other GABA_AR subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA_ARs containing the α2 subunit.     

Assembly of GABAA receptors (Review)

GABA_A receptors are the major inhibitory transmitter receptors in the central nervous system. They are chloride ion channels that can be opened by γ-aminobutyric acid (GABA) and are the targets of action of a variety of pharmacologically and clinically important drugs. GABA_A receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to the formation of a large variety of distinct GABA_A receptor subtypes in the brain. The majority of GABA_A receptors seems to be composed of two α, two β and one γ subunit and the occurrence of a defined subunit stoichiometry and arrangement in αβγ receptors strongly indicates that assembly of GABA_A receptors proceeds via defined pathways. Based on the differential ability of subunits to interact with each other, a variety of studies have been performed to identify amino acid sequences or residues important for assembly. Such residues might be involved in direct protein-protein interactions, or in stabilizing direct contact sites in other regions of the subunit. Several homo-oligomeric or hetero-oligomeric assembly intermediates could be the starting point of GABA_A receptor assembly but so far no unequivocal assembly mechanism has been identified. Possible mechanisms of assembly of GABA_A receptors are discussed in the light of recent publications.     

The diversity of GABAA receptors

The amino acid γ-aminobutyric-acid (GABA) prevails in the CNS as an inhibitory neurotrans-mitter that mediates most of its effects through fast GABA-gated Cl^?-channels (GABA_AR). Molecular biology uncovered the complex subunit architecture of this receptor channel, in which a pentameric assembly derived from five of at least 17 mammalian subunits, grouped in the six classes α, β, γ, δ, ε, and ρ, permits a vast number of putative receptor isoforms. The subunit composition of a particular receptor determines the specific effects of allosterical modulators of the GABA_ARs like benzodiazepines (BZs), barbiturates, steroids, some convulsants, polyvalent cations, and ethanol. To understand the physiology and diversity of GABA_ARs, the native isoforms have to be identified by their localization in the brain and by their pharmacology. In heterologous expression systems, channels require the presence of α, β, and γ subunits in order to mimic the full repertoire of native receptor responses to drugs, with the BZ pharmacology being determined by the particular α and γ subunit variants. Little is known about the functional properties of the β, δ, and ε subunit classes and only a few receptor subtype-specific substances like loreclezole and furosemide are known that enable the identification of defined receptor subtypes. We will summarize the pharmacology of putative receptor isoforms and emphasize the characteristics of functional channels. Knowledge of the complex pharmacology of GABA_ARs might eventually enable site-directed drug design to further our understanding of GABA-related disorders and of the complex interaction of excitatory and inhibitory mechanisms in neuronal processing.     

Molecular and pharmacological properties of GABA-ρ subunits from white perch retina

Five γ-aminobutyric acid (GABA)-ρ subunits were cloned from a white perch retinal cDNA library and expressed in Xenopus oocytes. The deduced amino acid sequences indicated that all are highly homologous to the GABA-ρ subunits cloned from mammalian retinas; two clones (perch-ρ1A and perch-ρ1B) were in the ρ1 family, two (perch-ρ2A and perch-ρ2B) were in the ρ2 family, and one clone has been tentatively identified as a perch-ρ3 subunit. When expressed in Xenopus oocytes, all but one of the subunits (ρ3) formed functional homooligomeric receptors. However, the receptors expressed by each of the GABA-ρ subunits displayed unique response properties that distinguished one from the other. For example, receptors formed by perch-ρ1B subunits were more sensitive to GABA than the receptors formed by other GABA-ρ subunits, the dose–response curves for the various receptors revealed different Hill coefficients, and there were differences in the kinetics of the GABA-induced currents. In addition, the GABA-mediated current–voltage curve for ρ2 receptors was approximately linear, whereas the responses from ρ1 receptors showed outward rectification. A further division in the properties of the GABA-ρ subunits was revealed in their responses to imidazole-4-acetic acid (I4AA); the drug behaved as an antagonist on A-type ρ receptors and a partial agonist on the B-type ρ receptors. These results suggest that there is a large diversity of GABA_c receptors in the vertebrate retina, probably formed by homooligomeric and heterooligomeric combinations of GABA ρ subunits, that exhibit different functional properties. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 305–320, 1998     

Subunit Composition and Function of GABAA Receptors of Rat Spermatozoa

GABA triggers mammalian sperm acrosome reaction (AR). Here, evidence is presented, showing that rat spermatozoa contain GABA_A receptors, composed of ₅, ₁ and ₃ subunits. The effects of GABA_A receptor agonist and antagonist on the induction of AR in rat spermatozoa were assessed using the chlortetracycline assay. Muscimol, a GABA_A receptor agonist, triggered AR; whereas bicuculline, a GABA_A receptor antagonist and picrotoxin, a GABA_A receptor/Cl^– channel blocker, inhibited the ability of GABA or progesterone to induce AR. In conclusion, GABA_A receptors appear to mediate the action of progesterone in inducing AR in rat spermatozoa.     

Differential Regulation of GABAA Receptor Gene Expression by Ethanol in the Rat Hippocampus Versus Cerebral Cortex

Abstract: Previous research has shown that chronic ethanol consumption dramatically alters GABA_A receptor α₁ and α₄ subunit gene expression in the cerebral cortex and GABA_A receptor α₁ and α₆ subunit gene expression in the cerebellum. However, it is not yet known if chronic ethanol consumption produces similar alterations in GABA_A receptor gene expression in other brain regions. One brain region of interest is the hippocampus because it has recently been shown that a subset of GABA_A receptors in the hippocampus is responsive to pharmacologically relevant concentrations of ethanol. Therefore, we directly compared the effects of chronic ethanol consumption on GABA_A receptor subunit gene expression in the hippocampus and cerebral cortex. Furthermore, we investigated whether the duration of ethanol consumption (14 or 40 days) would influence regulation of GABA_A receptor gene expression in these two brain regions. Chronic ethanol consumption produced a significant increase in the level of GABA_A receptor α₄ subunit peptide in the hippocampus following 40 days but not 14 days. The relative expression of hippocampal GABA_A receptor α₁, α₂, α₃, α_2/3, or γ₂ was not altered by either period of chronic ethanol exposure. In marked contrast, chronic ethanol consumption for 40 days significantly increased the relative expression of cerebral cortical GABA_A receptor α₄ subunits and significantly decreased the relative expression of cerebral cortical GABA_A receptor α₁ subunits. This finding is consistent with previous results following 14 days of chronic ethanol consumption. Hence, chronic ethanol consumption alters GABA_A receptor gene expression in the hippocampus but in a different manner from that in either the cerebral cortex or the cerebellum. Furthermore, these alterations are dependent on the duration of ethanol exposure.     

GABAC receptors in the vertebrate retina

In the central nervous system (CNS), the inhibitory transmitter GABA interacts with three subtypes of GABA receptors, type A, type B, and type C. Historically, GABA receptors have been classified as either the inotropic GABA_A receptors or the metabotropic GABA_B receptors. Over the past 10 yr, studies have shown that a third class, called the GABA_C receptor, also exists. GABA_C receptors are found primarily in the vertebrate retina and to some extent in other parts of the CNS. Although GABA_A and GABA_C receptors both gate chloride channels, they are pharmacologically, molecularly, and functionally distinct. The ρ subunit of the GABA_C receptor, which has about 35% amino acid homology to GABA_A receptor subunits, was cloned from the retina and, when expressed inXenopus oocytes, has properties similar to retinal GABA_C receptors. There are probably distinct roles for GABA_C receptors in the retina, because they are found on only a subset of neurons, whereas GABA_A receptors are ubiquitous. This article reviews recent electrophysiological and molecular studies that have characterized the unique properties of GABA_C receptors and describes the roles that these receptors may play in visual information processing in the retina.     

Brain GABAA receptors studied with subunit-specific antibodies

Brain GABA_A/benzodiazepine receptors are highly heterogeneous. This heterogeneity is largely derived from the existence of many pentameric combinations of at least 16 different subunits that are differentially expressed in various brain regions and cell types. This molecular heterogeneity leads to binding differences for various ligands, such as GABA agonists and antagonists, benzodiazepine agonists, antagonists, and inverse agonists, steroids, barbiturates, ethanol, and Cl⁻ channel blockers. Different subunit composition also leads to heterogeneity in the properties of the Cl⁻ channel (such as conductance and open time); the allosteric interactions among subunits; and signal transduction efficacy between ligand binding and Cl⁻ channel opening. The study of recombinant receptors expressed in heterologous systems has been very useful for understanding the functional roles of the different GABA_A receptor subunits and the relationships between subunit composition, ligand binding, and Cl⁻ channel properties. Nevertheless, little is known about the complete subunit composition of the native GABA_A receptors expressed in various brain regions and cell types. Several laboratories, including ours, are using subunit-specific antibodies for dissecting the heterogeneity and subunit composition of native (not reconstituted) brain GABA_A receptors and for revealing the cellular and subcellular distribution of these subunits in the nervous system. These studies are also aimed at understanding the ligand-binding, transduction mechanisms, and channel properties of the various brain GABA_A receptors in relation to synaptic mechanisms and brain function. These studies could be relevant for the discovery and design of new drugs that are selective for some GABA_A receptors and that have fewer side effects.     

Revisiting the TRAK Family of Proteins as Mediators of GABAA Receptor Trafficking

γ-Aminobutyric acid type A (GABA_A) receptor interacting factor-1 (GRIF-1) was originally discovered as a result of studies aiming to find the elusive GABA_A receptor clustering protein. It was identified as a GABA_A receptor associated protein by virtue of its specific interaction with the GABA_A receptor β2 subunit intracellular loop in a yeast two-hybrid screen of a rat brain cDNA library. Further work however, established that GRIF-1, now known as trafficking kinesin protein 2 (TRAK2), is a member of the TRAK family of kinesin adaptor proteins. A pivotal role for TRAK1 and TRAK2 in the transport of mitochondria is well recognized. Notwithstanding this progress, there is a body of evidence that still supports a role for TRAKs in the intracellular transport of GABA_A receptors. This is critically reviewed in this article.     

GABAA receptor subunit polypeptides increase in parallel but exhibit distinct distributions in the developing rat cerebellum

The GABA_A receptor, a multisubunit ligand-gated ion channel, plays a central role in cell–cell communication in the developing and adult nervous system. Although the developmental expression of mRNAs encoding many subunit isoforms has been extensively characterized throughout the central nervous system, little is known concerning the relationship between subunit mRNA and polypeptide expression. To address this issue, we examined the developmental expression of the α1, β2/3, and γ2 subunit polypeptides, subunits that are thought to coassemble in many brain regions. Western blot analysis using subunit-specific antibodies revealed that the levels of these polypeptides in both the cerebral cortex and cerebellum increased severalfold during the second postnatal week. Whereas polypeptide expression in the cerebellum paralleled that of the corresponding subunit mRNAs, increase in β2/3 and γ2 polypeptide expression in the cerebral cortex occurred in the absence of detectable changes in the mRNA levels. To determine whether the increases in subunit polypeptide expression in the cerebellum were accompanied by changes in distribution, immunohistochemistry was performed. These studies demonstrated that the subunits exhibited different but partially overlapping distributions that remained constant throughout postnatal development. Our findings suggest that although GABA_A receptor subunit polypeptide expression may be regulated primarily at the level of the mRNA, additional regulatory mechanisms may play role. Furthermore, the observation that subunit distribution remains constant in the cell bodies of cerebellar Purkinje neurons, which express the α1, β2, β3, and γ2 subunit mRNAs exclusively, suggests that GABA_A receptor subunit composition in this cell population does not change during postnatal maturation. 1994 John Wiley & Sons, Inc.     

Altered Cortical GABAA Receptor Composition,Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome

Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABA_A receptors (GABA_ARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Het_α1KO) of the human epilepsy gene, the GABA_AR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Het_α1KO on the expression and physiology of GABA_ARs in the mouse cortex. We found that Het_α1KO caused modest reductions in the total and surface expression of the β2 subunit but did not alter β1 or β3 subunit expression, results consistent with a small reduction of GABA_ARs. Cortices partially compensated for Het_α1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Het_α1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3βγ receptors. Patch clamp electrophysiology studies showed that Het_α1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Het_α1KO reduced base-line GABA_AR endocytosis, an effect that probably contributes to the observed changes in GABA_AR expression. These findings demonstrate that Het_α1KO exerts two principle disinhibitory effects on cortical GABA_AR-mediated inhibitory neurotransmission: 1) a modest reduction of GABA_AR number and 2) a partial compensation with GABA_AR isoforms that possess physiological properties different from those of the otherwise predominant α1βγ GABA_ARs.     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.     

High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors

The human neuronal Cys‐loop ligand‐gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high‐level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high‐level heterologous production of pure receptors in a state that supports agonist‐induced allosteric conformational changes. In a tetracycline‐inducible stable human embryonic kidney cells (HEK293S) cell line, GABA_A receptors containing α1 and β3 subunits could be expressed with specific activities of 29–34 pmol/mg corresponding to 140–170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5‐HT_3A) receptors were 49–63 pmol/mg and 245–315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3–3.0 L. Both receptor constructs had a FLAG epitope inserted at the N‐terminus and could be purified in one step after solubilization using ANTI‐FLAG affinity chromatography with yields of 30–40%. Purified receptors were functional. Binding of the agonist [³H]muscimol to the purified GABA_AR was enhanced allosterically by the general anesthetic etomidate, and purified 5‐hydroxytryptamine‐3A receptor supported serotonin‐stimulated cation flux when reconstituted into lipid vesicles.