Two novel GABAA receptor subunits exist in distinct neuronal subpopulations

Two cDNAs encoding novel GABAA receptor subunits were isolated from a rat brain library. These subunits, gamma 2 and delta, share approximately 35% sequence identity with alpha and beta subunits and form functional GABA-gated chloride channels when expressed alone in vitro. The gamma 2 subunit is the rat homolog of the human gamma 2 subunit recently shown to be important for benzodiazepine pharmacology. Cellular localization of the mRNAs encoding the gamma 2 and delta subunits in rat brain revealed that largely distinct neuronal subpopulations express the two subunits. The delta subunit distribution resembles that of the high affinity GABAA receptor labeled with [3H]muscimol; the gamma 2 subunit distribution resembles that of GABAA/benzodiazepine receptors labeled with [3H]flunitrazepam. These findings have implications for the composition of two different GABAA receptor subtypes and for information processing in networks using GABA for signaling.     

Analysis of the presence and abundance of GABAA receptors containing two different types of alpha subunits in murine brain using point-mutated alpha subunits

Gamma-aminobutyric acid, type A (GABAA) receptors are pentameric proteins of which the majority is composed of two alpha subunits, two beta subunits and one gamma subunit. It is well documented that two different types of alpha subunits can exist in a singles GABAA receptor complex. However, information on the abundance of such GABAA receptors is rather limited. Here we tested whether mice containing the His to Arg point mutation in the alpha1, alpha2, or alpha3 subunit at positions 101, 101, and 126, respectively, which render the respective subunits insensitive to diazepam, would be suitable to analyze this issue. Immunodepletion studies indicated that the His to Arg point mutation solely rendered those GABAA receptors totally insensitive to diazepam binding that contain two mutated alpha subunits in the receptor complex, whereas receptors containing one mutated and one heterologous alpha subunit not carrying the mutation remained sensitive to diazepam binding. This feature permitted a quantitative analysis of native GABAA receptors containing heterologous alpha subunits by comparing the diazepam-insensitive binding sites in mutant mouse lines containing one mutated alpha subunit with those present in mouse lines containing two different mutated alpha subunits. The data indicate that the alpha1alpha1-containing receptors with 61% is the most abundant receptor subtype in brain, whereas the alpha1alpha2 (13%), alpha1alpha3 (15%), alpha2alpha2 (12%), alpha2alpha3 (2%), and alpha3alpha3 combinations (4%) are considerably less expressed. Only within the alpha1-containing receptor population does the combination of equal alpha subunits (84% alpha1alpha1, 7% alpha1alpha2, and 8% alpha1alpha3) prevail, whereas in the alpha2-containing receptor population (46% alpha2alpha2, 36% alpha2alpha1, and 19% alpha2alpha3) and particularly in the alpha3-containing receptor population (27% alpha3alpha3, 56% alpha3alpha1, and 19% alpha3alpha2), the receptors with two different types of alpha subunits predominate. This experimental approach provides the basis for a detailed analysis of the abundance of GABAA receptors containing heterologous alpha subunits on a brain regional level.     

GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs.

Cloned cDNAs encoding two new beta subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The beta 2 and beta 3 subunits share approximately 72% sequence identity with the previously characterized beta 1 polypeptide. Northern analysis showed that both beta 2 and beta 3 mRNAs are more abundant in the brain than beta 1 mRNA. All three beta subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each beta subunit, when co-expressed in Xenopus oocytes with an alpha subunit, forms functional GABAA receptors. These results, together with the known alpha subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations.     

Kainic acid-induced status epilepticus alters GABA receptor subunit mRNA and protein expression in the developing rat hippocampus

Kainic acid-induced status epilepticus leads to structural and functional changes in inhibitory GABAA receptors in the adult rat hippocampus, but whether similar changes occur in the developing rat is not known. We have used in situ hybridization to study status epilepticus-induced changes in the GABAAalpha1-alpha5, beta1-beta3, gamma1 and gamma2 subunit mRNA expression in the hippocampus of 9-day-old rats during 1 week after the treatment. Immunocytochemistry was applied to detect the alpha1, alpha2 and beta3 subunit proteins in the control and treated rats. In the saline-injected control rats, the alpha1 and alpha4 subunit mRNA expression significantly increased between the postnatal days 9-16, whereas those of alpha2, beta3 and gamma2 subunits decreased. The normal developmental changes in the expression of alpha1, alpha2, beta3 and gamma2 subunit mRNAs were altered after the treatment. The immunostainings with antibodies to alpha1, alpha2 and beta3 subunits confirmed the in situ hybridization findings. No neuronal death was detected in any hippocampal subregion in the treated rats. Our results show that status epilepticus disturbs the normal developmental expression pattern of GABAA receptor subunit in the rat hippocampus during the sensitive postnatal period of brain development. These perturbations could result in altered functional and pharmacological properties of GABAA receptors.     

The mRNAs encoding GABAA/benzodiazepine receptor subunits are localized in different cell populations of the bovine cerebellum

The expression of the mRNAs encoding the alpha and beta subunits of the GABAA/benzodiazepine receptor was examined in the bovine cerebellum by in situ hybridization histochemistry. The alpha subunit mRNA, which encodes the benzodiazepine binding site, was localized in all Purkinje and granule cells and in some cells of the molecular layer. The distribution of the beta subunit mRNA, which encodes the GABA binding site, only partially overlapped with that of the alpha subunit mRNA. While cells in the granule cell layer expressed the beta subunit mRNA, no message could be detected in other cell populations. These findings suggest that the subunit composition of the GABAA/benzodiazepine receptor is heterogeneous and that additional, as yet unidentified, beta subunits exist.     

The cDNA sequence and chromosomal location of the murine GABAA alpha 1 receptor gene.

The murine GABAA/benzodiazepine (GABAA/BZ) receptor alpha 1 subunit cDNA has been isolated from a BALB/c mouse brain library and sequenced. The cDNA is 2665 nucleotides long with an open reading frame of 455 amino acids. It shows significant homology to the GABAA receptor alpha 1 subunit cDNA sequences of other species. Excluding deletions, the murine GABAA alpha 1 receptor exhibits 96% nucleotide and 100% amino acid sequence homology to the rat alpha 1 receptor cDNA and over 91% nucleotide and 98% amino acid sequence homology to the bovine and human alpha 1 receptor cDNAs in the protein coding region. This murine cDNA was used to locate the alpha 1 receptor subunit gene, Gabra-1, to murine Chromosome 11 between Il-3 and Rel. This assignment extends proximally the segment of mouse Chromosome 11 with known homology to human chromosome 5.     

Developmental expression of the GABAA receptor alpha 1 subunit mRNA in the rat brain

Recent studies have suggested that the GABAA, receptor complex, the site of action of the inhibitory neurotransmitter gamma amino-butyric acid (GABAA) and the anxiolytic benzodiazepines, is heterogeneous. Moreover, its composition may change during development. To better understand the molecular basis of receptor heterogeneity, the levels and distribution of the mRNA encoding the alpha 1 receptor subunit were examined in the developing and adult rat brain with quantitative in situ hybridization histochemistry. Our studies demonstrate that alpha 1 subunit mRNA expression changes during ontogeny. At late embryonic stages and in the first postnatal week, low levels of the mRNA were detected in the cortex, inferior colliculus, and hippocampus. The mRNA levels in these regions increased during the second and third postnatal weeks. Furthermore, a dramatic change in the distribution of the alpha 1 subunit mRNA was seen in the second postnatal week when the message first became detectable in the cerebellar cortex. During subsequent development and in the mature brain, the alpha 1 subunit mRNA was most abundant in the cerebellum, olfactory bulb, and inferior colliculus, although the absolute levels of mRNA varied by as much as sixfold in selected brain regions. The mature distribution of alpha 1 subunit mRNA, along with its temporal appearance in the cerebellum, suggests that this subunit is a constituent of the Type 1 benzodiazepine site of the GABAA receptor complex. Furthermore, the onset of alpha 1 subunit mRNA expression in the cerebellar cortex coincides with a period of extensive synapse formation, raising the possibility that synaptic interactions modulate the appearance of this GABAA receptor subunit in the cerebellum.     

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.     

Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor alpha 4 subunit.

A cDNA of rat brain encoding the GABAA receptor alpha 4 subunit has been cloned. Recombinant receptors composed of alpha 4, beta 2 and gamma 2 subunit bind with high affinity the GABA agonist [3H]muscimol and the benzodiazepine 'alcohol antagonist' [3H]Ro 15-4513, but fail to bind benzodiazepine agonists. The alpha 4 subunit is expressed mainly in the thalamus, as assessed by in situ hybridization histochemistry, and may participate in a major population of thalamic GABAA receptors. The alpha 4 mRNA is found at lower levels in cortex and caudate putamen, and is rare in cerebellum.     

GABAA receptor subtypes immunopurified from rat brain with alpha subunit-specific antibodies have unique pharmacological properties.

The unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor were expressed in bacterial and used to produce subunit-specific polyclonal antisera. Antibodies immobilized on protein A-Sepharose were used to isolate naturally occurring alpha-specific populations of GABAA receptors from rat brain that retained the ability to bind [3H]muscimol, [3H]flunitrazepam, [3H]Ro15-1788, and [125I]iodo-clonazepam with high affinity. Pharmacological characterization of these subtypes revealed marked differences between the isolated receptor populations and was generally in agreement with the reported pharmacological profiles of GABAA receptors in cells transiently transfected with alpha 1 beta 1 gamma 2, alpha 2 beta 1 gamma 2, alpha 3 beta 1 gamma 2, and alpha 5 beta 1 gamma 2 combinations of subunits. Additional subtypes were also identified that bind [3H]muscimol but do not bind benzodiazepines with high affinity. The majority of GABAA receptor oligomers contains only a single type of alpha subunit, and we conclude that alpha 1, alpha 2, alpha 3, and alpha 5 subunits exist in vivo in combination with the beta subunit and gamma 2 subunit.     

Structural and functional characterization of the gamma 1 subunit of GABAA/benzodiazepine receptors.

The GABAA receptor gamma 1 subunit of human, rat and bovine origin was molecularly cloned and compared with the gamma 2 subunit in structure and function. Both gamma subunit variants share 74% sequence similarity and are prominently synthesized in often distinct areas of the central nervous system as documented by in situ hybridization. When co-expressed with alpha and beta subunits in Xenopus oocytes and mammalian cells, the gamma variants mediate the potentiation of GABA evoked currents by benzodiazepines and help generate high-affinity binding sites for these drugs. However, these sites show disparate pharmacological properties which, for receptors assembled from alpha 1, beta 1 and gamma 1 subunits, are characterized by the conspicuous loss in affinity for neutral antagonists (e.g. flumazenil) and negative modulators (e.g. DMCM). These findings reveal a pronounced effect of gamma subunit variants on GABAA/benzodiazepine receptor pharmacology.     

Investigation of the abundance and subunit composition of GABAA receptor subtypes in the cerebellum of alpha1-subunit-deficient mice

In cerebellum, 75% of all GABAA receptors contain alpha1 subunits. Here, we investigated compensatory changes in GABAA receptor subunit expression and composition in alpha1 subunit-knockout mice. In these mice the total number of cerebellar GABAA receptors was reduced by 46%. Whereas the number of receptors containing alpha6 subunits was unchanged, the total amount of alpha6 subunits was significantly elevated. RT-PCR showed no increase of alpha6 mRNA levels, arguing against increased biosynthesis of these subunits. Elevated levels of alpha6 subunits in alpha1 -/- mice might thus have been caused by an increased incorporation of unassembled alpha6 subunits, replacing alpha1 subunits in alpha1alpha6betagamma2 or alpha1alpha6betadelta receptors, thus rescuing alpha6 subunits from degradation. Elevated levels of alpha3 and alpha4 subunits in the cerebellum of alpha1 -/- mice possibly can be explained similarly. Finally, a small amount of receptors containing no gamma or delta subunits was identified in these mice. Results suggest a total loss of GABAA receptors in cell types where alpha1 was the only alpha subunit expressed and a partial compensation for receptor loss in cell types containing other alpha subunits. Our results do not support a significant compensatory synthesis of other GABAA receptor subunits in the cerebellum of alpha1 -/- mice.     

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.     

Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.     

Developmental changes in the expression of GABAA receptor subunits alpha1, alpha2, and alpha3 in the rat pre-Botzinger complex.

Previously, we reported that the pre-B?tzinger complex (PBC) exhibited a dramatic reduction in cytochrome oxidase activity at postnatal day (P) 12. This coincided in time with decreases in glutamate and NMDA receptor subunit 1 and increases in GABA, GABAB, glycine receptor, and glutamate receptor GluR2. To test our hypothesis that various alpha-subunits of GABAA receptors also undergo changes in their expression during postnatal development, as they do in other brain regions, we undertook an in-depth immunohistochemical study of GABAA receptor subunits alpha1, alpha2, and alpha3 in the PBC of P0 to P21 rats. We found that 1) GABAA alpha3-subunit was expressed at relatively high levels at P0, which then declined with age; 2) GABAA alpha1-subunit was expressed at relatively low levels at P0 but increased with age; 3) the developmental trends of subunits alpha1 and alpha3 intersected at P12; and 4) GABAA alpha2-subunit expression was moderate to light at P0 and remained quite constant during development, being lowest at P21. These findings suggest that the apparent switch in relative expressions of subunits alpha3 and alpha1 during development and the intersection of slopes around P12 may be associated with possible changes in GABAA receptor subtypes that would mediate different functional properties of GABA transmission, such as primarily a less efficient inhibitory transmission before P12 and a more mature inhibitory effect at P12 and thereafter, as suggested by the kinetics of distinct postsynaptic potentials. This mechanism may contribute partially to the dramatic reduction in cytochrome oxidase activity within the PBC at P12, as shown previously.     

Recombinant extracellular domain of the three major subunits of GABAA receptor show comparable secondary structure and benzodiazepine binding properties

The three most widely expressed subunits of the GABAA receptor are alpha(1), beta(2), and gamma(2) subunits, and the major isoform in the human brain is a pentameric receptor composed of 2alpha(1)2beta(2)1gamma(2). Previously, we overexpressed the extracellular domain Q28-R248 of GABAA receptor alpha(1) subunit. In the present study, the homologous extracellular domains Q25-G243 of GABAA receptor beta(2) subunit and Q40-G273 of gamma(2) subunit were also obtained through overexpression in Escherichia coli. Successful production of recombinant beta(2) and gamma(2) subunit receptor protein domains facilitates the comparison of structural and functional properties of the three subunits. To this end, the secondary structures of the three fragments were measured using CD spectroscopy and the beta-strand contents calculated to be >30%, indicating a beta-rich structure for all three fragments. In addition, the benzodiazepine (BZ)-binding affinity of the recombinant fragments were measured using fluorescence polarization to be 2.16 microM, 3.63 microM, and 1.34 microM for the alpha(1), beta(2), and gamma(2) subunit fragments, respectively, indicating that all three homomeric assemblies, including that of the beta(2) subunit, generally not associated with BZ binding, can bind BZ in the micromolar range. The finding that the BZ binding affinity of these recombinant domains was highest for the gamma(2) subunit and lowest for the beta(2) subunit is consistent with results from previous binding studies using hetero-oligomeric receptors. The present results exemplify the effective approach to characterize and compare the three major subunits of the GABAA receptor, for two of which the overexpression in E. coli is reported for the first time.     

The Voltage-Gated Calcium Channel γ Subunits: A Review of the Literature

Members of the voltage-gated calcium channel y subunit gene family (Cacng), have been rapidly discovered since the discovery of the identification of the mouse gamma2 gene (Cacng2) and its association with the stargazer mutant mouse line. The fact that this mutant mouse line exists has allowed researchers to gain insights into the function of the gamma2 subunit. For example, stargazer mice have elevated levels of neuropeptide Y production, very low cerebellar brain derived neurotrophic factor production, and diminished cerebellar GABAA alpha6 and beta3 production. Study of this mutant mouse line has also revealed that the gamma2 subunit is involved in AMPA receptor trafficking and targeting to the synaptic membrane. For the most part, the effect of gamma2 subunits on the electrophysiology of voltage-gated calcium channels is to downregulate calcium channel activity by causing a hyperpolarizing shift in the inactivation curve. This finding and the association of these subunits with AMPA receptor trafficking has led some researchers to question the actual role of the gamma subunits. This article reviews the discovery, cellular localization, tissue distribution, and function of the eight members of the Cacng family.     

Cellular distribution ofl-glutamate decarboxylase (GAD) andγ-aminobutyric acidA (GABAA) receptor mRNAs in the retina

1. Gamma-aminobutryic acid (GABA), a major inhibitory transmitter of the vertebrate retina, is synthesized from glutamate by L-glutamate decarboxylase (GAD) and mediates neuronal inhibition at GABAA receptors. GAD consists of two distinct molecular forms, GAD65 and GAD67, which have similar distribution patterns in the nervous system (Feldblum et al., 1990; Erlander and Tobin, 1991). GABAA receptors are composed of several distinct polypeptide subunits, of which the GABAA alpha 1 variant has a particularly extensive and widespread distribution in the nervous system. The aim of this study was to determine the cellular localization patterns of GAD and GABAA alpha 1 receptor mRNAs to define GABA- and GABAA receptor-synthesizing neurons in the rat retina. 2. GAD and GABAA alpha 1 mRNAs were localized in retinal neurons by in situ hybridization histochemistry with 35S-labeled antisense RNA probes complementary to GAD67 and GABAA alpha 1 mRNAs. 3. The majority of neurons expressing GAD67 mRNA is located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL). Occasional GAD67 mRNA-containing neurons are present in the inner plexiform layer. Labeled neurons are not found in the distal INL or in the outer nuclear layer (ONL). 4. GABAA alpha 1 mRNA is expressed by neurons distributed to all regions of the INL. Some discretely labeled cells are present in the GCL. Labeled cells are not observed in the ONL. 5. The distribution of GAD67 mRNA demonstrates that numerous amacrine cells (conventional, interstitial, and displaced) and perhaps interplexiform cells synthesize GABA. These cells are likely to employ GABA as a neurotransmitter. 6. The distribution of GABAA alpha 1 mRNA indicates that bipolar, amacrine, and perhaps ganglion cells express GABAA receptors having an alpha 1 polypeptide subunit, suggesting that GABA acts directly upon these cells.