Robert J. Harvey

Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid

Combinations of cloned GABAA receptor subtypes, having the subunit combinations alpha i + beta 1 or alpha i + beta 1 + gamma 2 (i = 1, 2, 3), were expressed in Xenopus oocytes. The endogenous steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiates GABA currents induced therein by GABA. This potentiation was greater in the alpha 1 + beta and alpha 3 + beta 1 than in the alpha 2 + beta 1 combinations. The presence of the gamma 2-subunit increased the steroid potency in alpha 1 + beta 1 and alpha 2 + beta 1, but the combination alpha 3 + beta 1 + gamma 2 became much less steroid-sensitive. It is concluded that the steroid modification of the GABAA receptor is strongly influenced by the alpha- and the gamma 2-subunit types.

Sequence of a functional invertebrate GABAA receptor subunit which can form a chimeric receptor with a vertebrate alpha subunit.

The sequence of an invertebrate GABAA receptor subunit is described. This was deduced from a cDNA which was isolated from the mollusc Lymnaea stagnalis and which corresponds to a transcript of extremely low abundance. The cDNA was isolated using short exonic sequences from part of the corresponding gene in combination with a variant of the polymerase chain reaction (PCR) known as RACE (rapid amplification of cDNA ends). The mature polypeptide has a predicted molecular weight of 54,569 Daltons and exhibits approximately 50% identity to vertebrate GABAA receptor beta subunits. The six intron‐exon boundaries determined to date in the molluscan gene occur at the same relative positions as those found in vertebrate GABAA receptor genes. Functional expression, in Xenopus oocytes, of the molluscan cDNA alone results in the formation of GABA‐activated chloride ion channels that have a finite open probability even in the absence of agonist. These GABA‐evoked currents can be reversibly blocked by the vertebrate GABAA receptor antagonist bicuculline. Surprisingly, the molluscan beta subunit is capable of replacing vertebrate beta subunits in co‐expression experiments with the bovine GABAA receptor alpha 1 subunit. These findings suggest that invertebrate GABAA receptors exist in vivo as hetero‐oligomeric complexes.

Molecular cloning reveals the existence of a fourth γ subunit of the vertebrate brain GABAA receptor

We have isolated a cDNA, from the chicken, that encodes a fourth type of γ subunit of the vertebrate brain GABAA receptor. The mature polypeptide (which we name γ4) displays 67%, 69% and 70% identity, respectively, to the rat γ1, γ2 and γ3 subunits. In the developing chicken brain, the γ4‐subunit mRNA is first detected at embryonic day 13; the transcript level then increases progressively during embryogenesis. In situ hybridization reveals that the γ4‐subunit mRNA is abundant in several brain regions, including the ectostriatum, nucleus rotundus and hyperstriatum ventrale, which are involved in visual processing and learning.

Alternative Splicing of a 51-Nucleotide Exon that Encodes a Putative Protein Kinase C Phosphorylation Site Generates Two Forms of the Chicken γ-Aminobutyric AcidAReceptor β2 Subunit

Abstract: Complementary DNAs that encode two forms of the chicken ‐γ‐aminobutyric acid type A (GABAA) receptor β2 subunit have been isolated. These polypeptides differ by the presence (β2L) or absence (β2S) of 17 amino acids, which contain a possible target for phosphorylation by protein kinase C, in the large intracellular loop between the third and fourth membrane‐spanning domains. The extra sequence in the chicken β2L subunit is not found in previously published GABAA receptor β2‐subunit sequences. Analysis of genomic DNA has revealed that the two β2‐subunit mRNAs arise by alternative splicing of a novel 51‐nucleotide exon. Although the two β2‐subunit transcripts appear to be present in 1 ‐day‐old chick brain at similar steady‐state levels, we have been unable to detect an mRNA for the long form of the β2 subunit in either the bovine or the rat. Because the various GABAA receptor genes are thought to have arisen by duplication of a common ancestor, our data, taken together with that on the γ2 subunit, which occurs in two forms that arise by alternative splicing of a 24‐nucleotide exon, suggest that the coding region of the primordial gene or one of its very early descendants contained 10 exons, not nine as previously thought.

Sequence of a Drosophila Ligand‐Gated Ion‐Channel Polypeptide with an Unusual Amino‐Terminal Extracellular Domain

Abstract: We report the isolation of a full‐length clone from a Drosophila melanogaster head cDNA library that encodes a 614‐residue polypeptide that exhibits all of the features of a ligand‐gated chloride‐channel/receptor subunit. This polypeptide, which has been named GRD (denoting that the polypeptide is a GABAA and glycine receptor‐like subunit of Drosophila), displays between 33 and 44% identity to vertebrate GABAA and glycine receptor subunits and 32–37% identity to the GABAA receptor‐like polypeptides from Drosophila and Lymnaea. It is interesting that the large amino‐terminal, presumed extracellular domain of the GRD protein contains an insertion, between the dicysteine loop and the first putative membrane‐spanning domain, of 75 amino acids that is not found in any other ligand‐gated chloride‐channel subunit. Analysis of cDNA and genomic DMA reveals that these residues are encoded by an extension of an exon that is equivalent to exon 6 of vertebrate GABAA and glycine receptor genes. The gene (named Grd) that encodes the Drosophila polypeptide has been mapped, by in situ hybridization, to position 75A on the left arm of chromosome 3.

The chicken GABAA receptor α1 subunit: cDNA sequence and localization of the corresponding mRNA

Abstract We report the sequence of a complementary DNA (cDNA) that encodes the chicken GABA A receptor α1 subunit, which is extremely homologous to mammalian α1 subunits. The distribution of α1 subunit transcripts is shown to correlate mainly, but not completely, with the previously-reported pattern of benzodiazepine type I (BZI) binding sites in the avian brain. These results suggest that the α1 subunit may not necessarily be restricted to receptors having BZI pharmacology.

Developmental up-regulation and agonist-dependent down-regulation of GABAA receptor subunit mRNAs in chick cortical neurons

We have used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression of GABAA receptor subunit genes in cultured neurons from the chick embryo cerebral cortex. During maturation of the neurons between day 2 and day 8 in culture, levels of the alpha 1 subunit transcript (per ng total RNA) increased 3.8 +/- 0.3 fold, while those for the beta 2S and beta 4S subunits increased 2.4 +/- 0.4 and 1.8 +/- 0.2 fold, respectively. The accumulation of the beta 4 S subunit mRNA was more rapid than those encoding either the alpha 1 or beta 2S polypeptides. After 4 days in culture the beta 4S subunit transcript level reached 105 +/- 7.7% of that found after 8 days, while the corresponding amounts for the alpha 1 and beta 2S subunit mRNAs were 50 +/- 7.1% and 44 +/- 10.7%, respectively. On the other hand, no significant differences were observed in the level of either the gamma 1 or the gamma 2S subunit mRNA during development in vitro. Likewise, the ratios of the large/small splice variants (beta 2 = 0.16 +/- 0.02; beta 4 = 0.57 +/- 0.02; gamma 2 = 0.30 +/- 0.06) did not show detectable changes during this period. To study the down-regulation of the mRNAs, a single dose of 100 microM GABA was added to the culture medium. After 7 days of exposure to GABA, the levels of transcripts for the alpha 1, beta 2, beta 4, gamma 1, and gamma 2 subunits and their splice variants (where present) were all reduced by 47-65% compared to untreated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of a cDNA that encodes an invertebrate glutamate receptor subunit

A full‐length cDNA which encodes a putative glutamate receptor polypeptide was isolated from the pond snailLymnaea stagnalis, using a short stretch of exonic sequence and two variants of the polymerase chain reaction. In this first comparison of invertebrate and vertebrate glutamate receptor sequences, the mature molluscan polypeptide, which comprises 898 amino acids and has a predictedM r of 100 913, displays between 37% and 46% amino‐acid identity to the rat ionotropic glutamate receptor subunits, GluR1 to GluR6.

In situ hybridization and reverse transcription--polymerase chain reaction studies on the expression of the GABA(C) receptor rho1- and rho2-subunit genes in avian and rat brain.

The pharmacological properties of homo‐oligomeric channels formed by the GABA type A receptor‐like ρl and ρ2 polypeptides are very reminiscent of those of the GABA type C receptors that have been extensively characterized in the retina. Similar receptors have been reported to occur in certain brain regions of a variety of vertebrate species. We have used in situ hybridization to investigate the expression patterns of the ρ1‐ and ρ ‐polypeptide genes in the brain of the 1‐day‐old chick (Gallus domesticus) and the adult rat (Rattus norvegicus). Our results show that in the chick both the ρ1‐ and ρ2‐subunit transcripts are present in the cerebellum, the optic tectum, the epithalamus and the nucleus pretectalis. However, the two messenger RNAs are often found in different populations of cells. Thus, only the ρ1‐subunit gene is expressed in the deep cerebellar nuclei, the dorsal thalamus, the ectostriatum and the tractus vestibulomesencephalicus, while only the ρ2‐subunit gene is transcribed in the nucleus habenularis lateralis and the nucleus isthmo‐opticus. In contrast, neither of the ρ‐polypeptide messenger RNAs can be detected by in situ hybridization in the rat central nervous system. Reverse transcription‐polymerase chain reaction amplification has been used to confirm the expression of the two ρ‐subunit genes in the chicken brain. Surprisingly, this highly sensitive technique also revealed transcription of these genes in the rat brain. We conclude that the ρl‐ and ρ2‐subunit genes are expressed at a much higher level in the avian brain than in the rat brain and that, at least in birds, subtypes of the GABC receptor exist.

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.