Mark G. Darlison

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.

Multiple genes for neuropeptides and their receptors: co-evolution and physiology

It is now well established that neuropeptide receptors, which are present throughout the CNS and in peripheral tissues, frequently exist in a variety of different forms (called subtypes), each of which is encoded by a distinct gene. With the recent identification of new neuropeptide genes, it has become clear that families of neuropeptides also occur, which raises the possibility that specific peptide ligands activate particular receptor subtypes preferentially. This article reviews some of the recent advances in the neuropeptide field and provides evidence in support of three ideas: (1) that different receptor subtypes for a given ligand can be distinguished physiologically; (2) that neuropeptide genes probably arose before the corresponding receptor genes; and (3) that, despite the current wealth of information on neuropeptides and neuropeptide receptors, several new members are likely to be discovered before the beginning of the next millennium.

Cloning and functional pharmacology of two corticotropin-releasing factor receptors from a teleost fish

Although it is well established that fish possess corticotropin-releasing factor (CRF) and a CRF-like peptide, urotensin I, comparatively little is known about the pharmacology of their cognate receptors. Here we report the isolation and functional expression of two complementary DNAs (cDNAs), from the chum salmon Oncorhynchus keta, which encode orthologues of the mammalian and amphibian CRF type 1 (CRF(1)) and type 2 (CRF(2)) receptors. Radioligand competition binding experiments have revealed that the salmon CRF(1) and CRF(2) receptors bind urotensin I with approximately 8-fold higher affinity than rat/human CRF. These two peptides together with two related CRF-like peptides, namely, sauvagine and urocortin, were also tested in cAMP assays; for cells expressing the salmon CRF(1) receptor, EC(50) values for the stimulation of cAMP production were between 4.5+/-1.8 and 15.3+/-3.1 nM. For the salmon CRF(2) receptor, the corresponding values were: rat/human CRF, 9.4+/-0.4 nM; urotensin I, 21.2+/-2.1 nM; sauvagine, 0.7+/-0.1 nM; and urocortin, 2.2+/-0.7 nM. We have also functionally coupled the O. keta CRF(1) receptor, in Xenopus laevis oocytes, to the endogenous Ca(2+)-activated chloride conductance by co-expression with the G-protein alpha subunit, G(alpha16). The EC(50) value for channel activation by rat/human CRF (11.2+/-2.6 nM) agrees well with that obtained in cAMP assays (15.3+/-3.1 nM). We conclude that although sauvagine is 13- and 30-fold more potent than rat/human CRF and urotensin I, respectively, in activating the salmon CRF(2) receptor, neither receptor appears able to discriminate between the native ligands CRF and urotensin I.

Invertebrate GABA and glutamate receptors: molecular biology reveals predictable structures but some unusual pharmacologies.

Determination of the sequences of invertebrate gamma-aminobutyric acid (GABA)-gated and glutamate-gated receptor/ion channels, through the application of recombinant DNA methods, is not just an academic exercise to effect evolutionary comparisons with the sequences of the corresponding vertebrate receptors. The isolation of DNA clones would provide the tools to investigate the exact locations and functional properties of these neurotransmitter receptors within simple nervous systems. In addition, since GABA receptors, at least, have been suggested to be the targets of certain pesticides, the availability of invertebrate receptor cDNAs might provide the agrochemical industry with the basis for high-throughput screening methods for novel pesticidal compounds. Recently, the isolation of molluscan and Drosophila GABA receptor and glutamate receptor cDNAs, and the pharmacological properties of a GABA receptor expressed from one of these clones, have been reported. These studies should stimulate further research into the electrophysiology and pharmacology of native invertebrate ion channel proteins.

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.

Localization of the ρ1- and ρ2-subunit messenger RNAs in chick retina by in situ hybridization predicts the existence of γ-aminobutyric acid type C receptor subtypes ☆

Abstract We have amplified partial complementary DNAs for the chicken γ-aminobutyric acid type C (GABAC) receptor ρ1 and ρ2 subunits using the polymerase chain reaction. These nucleotide sequences have been utilized to design specific oligonucleotide probes for the in situ hybridization localization of the corresponding messenger RNAs (mRNAs) in the 1-day-old chick retina. Although both transcripts are found almost exclusively in the inner nuclear layer, their distributions differ markedly. From the locations of the hybridization signals, we deduce that the ρ1-subunit mRNA is present mainly in bipolar cells and that the ρ2-subunit mRNA is present in both amacrine and horizontal cells. These results suggest that the ρ1 and ρ2 subunits frequently occur in different receptor complexes and, therefore, that subtypes of the GABAC receptor exist.

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)

Genetic linkage and radiation hybrid mapping of the three human GABAC receptor ρ subunit genes: GABRR1, GABRR2 and GABRR3

GABA(C) receptors mediate rapid inhibitory neurotransmission in retina. We have mapped, in detail, the human genes which encode the three polypeptides that comprise this receptor: rho1 (GABRR1), rho2 (GABRR2) and rho3 (GABRR3). We show that GABRR1 and GABRR2 are located close together, in a region of chromosome 6q that contains loci for inherited disorders of the eye, but that GABRR3 maps to chromosome 3q11-q13.3. Our mapping data suggest that the rho polypeptide genes, which are thought to share a common ancestor with GABA(A) receptor subunit genes, diverged at an early stage in the evolution of this gene family.

Genomic mapping and evolution of human GABAA receptor subunit gene clusters

Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow G11 6NU, UK Department of Biochemistry and Molecular Genetics, Imperial College School of Medicine at St Mary’s Hospital, London W2 1PG, UK Institut für Zellbiochemie und klinische Neurobiologie, Universita ̈ts-Krankenhaus Eppendorf, Universita ̈t Hamburg, 20246 Hamburg, Germany Institut für Humangenetik, Justus-Liebig-Universita ̈t Giessen, 35392 Giessen, Germany Laboratoire de Genetique Moleculaire de la Neurotransmission et des Processus Neurodegeneratifs, Centre National de la Recherche Scientifique, 91198 Gif-sur-yvette, France Department of Cardiothoracic Surgery, Hammersmith Hospital, London W12 0HS, UK