Structural, developmental and functional heterogeneity of rat GABAA receptors.
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Amino Acid Sequence↗
Biomedical subjects
A J Tobin
Publications and source records attributed to A J Tobin.
The structural and functional heterogeneity of glutamic acid decarboxylase: a review.
Studies of the GABA-synthetic enzyme glutamate decarboxylase (glutamic acid decarboxylase; GAD; E.C.4.1.1.15) began in 1951 with the work of Roberts and his colleagues. Since then, many investigators have demonstrated the structural and functional heterogeneity of brain GAD. At least part of this heterogeneity derives from the existence of two GAD genes.
Animals↗
Postnatal expression of glutamate decarboxylases in developing rat cerebellum.
The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD67, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD67 mRNA predominates in the cerebellum. The mRNA for GAD65, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD65 is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD67 mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.
Aging↗
Distribution of glutamic acid decarboxylase (Mr 67,000) in the basal ganglia of the rat: an immunohistochemical study with a selective cDNA-generated polyclonal antibody.
Distinct isoforms of glutamic acid decarboxylase, the synthetic enzyme for GABA, exist in brain. Their distribution at the cellular level is not known, because previous studies have been confounded by the lack of monospecificity of available antibodies. We have examined the distribution of glutamic acid decarboxylase (Mr 67,000; GAD67) in the basal ganglia of the rat with a polyclonal antibody generated against the protein expressed in bacteria transformed with the corresponding cDNA. This antibody, which is directed against a portion of GAD67 non homologous to other known glutamic acid decarboxylase isoforms, selectively recognizes GAD67 on western blots. We show that GAD67 is present to various degree in all types of GABAergic neurons previously described in these regions. In contrast with results obtained with non-selective antibodies for glutamic acid decarboxylase, GAD67-positive neuronal cell bodies were readily detected in sections of the striatum, pallidum and substantia nigra in the absence of colchicine treatment. Modifications in the immunohistochemical procedure favoured staining of glutamic acid decarboxylase-positive fibres with the same antibody, indicating that GAD67 is also present in axon terminals of GABAergic neurons. The results suggest that GAD67 may be involved in GABA synthesis in both cell bodies and axon terminals of all GABAergic neurons of the basal ganglia, but is particularly abundant or accessible in their cell bodies.
Animals↗
Calcium binding protein (calbindin-D28k) and glutamate decarboxylase gene expression after kindling induced seizures.
In order to determine whether calcium binding protein (calbindin-D28k or CaBP) and glutamate decarboxylase (GAD) may be involved in the process underlying the generation of seizure activity, changes in CaBP protein and mRNA and in GAD mRNA were examined in the kindling model of epilepsy. Following amygdaloid (AK) and commissure (CK) kindling significant decreases in the concentration of CaBP of 20% and 30%, respectively, were specifically observed in the hippocampal formation. However, using a cDNA specific to mammalian CaBP, Northern analysis of poly(A+) RNA and slot blot analysis of total RNA revealed no changes in the levels of CaBP mRNA in hippocampus, subcortical area (including amygdala, substantia nigra and striatum) or cerebellum of rats sacrificed 30 min, 1 h, 6 h or 24 h after the last kindled seizure. Similarly when these blots were reprobed with a cDNA specific to mammalian GAD, no changes in GAD gene expression were observed. However, fos gene expression was markedly enhanced at 1 h after seizure. We also tested whether changes in CaBP or GAD mRNA could be detected at any of the various stages of the kindling process. Slot blot analysis of cortex, subcortical structures and hippocampus revealed no changes in CaBP or GAD mRNA during the course of commissure kindling. In situ hybridization studies with GAD and CaBP 35S-labeled antisense probes also indicated no obvious changes upon visual analysis of autoradiographs. However, when silver grains were counted, significant changes in GAD mRNA in individual cells in hippocampus and substantia nigra were noted after kindling induced epilepsy. Our results indicate that, unlike fos gene expression, prominent alterations in GAD and CaBP mRNA in gross brain regions (as measured by slot blot and Northern blot analyses) are not observed in the kindling process. However, our in situ hybridization studies suggest that changes in GAD mRNA in individual cells may be involved in the process underlying kindling induced seizure activity.
Animals↗
Independent cellular and ontogenetic expression of mRNAs encoding three alpha polypeptides of the rat GABAA receptor.
Previous studies have shown that several distinct but related polypeptides can serve as alpha subunits of functional GABAA receptors. Furthermore, the diversity of these polypeptides at least partially accounts for the functional heterogeneity of GABAA receptors. In this paper, we report the results of in situ hybridization studies using probes derived from our recently reported cDNAs for alpha 1, alpha 2, and alpha 4 GABAA receptor polypeptides. We show that the mRNAs that encode these isoforms have distinct regional and cellular distributions and are present at widely varying levels within the rat brain. In addition, our Northern blot analyses indicate that each of these three alpha mRNAs has a distinct pattern of ontogenetic regulation. Differential regulation of alpha polypeptide isoforms may lead to changes in GABAA receptor function during ontogeny as well as to distinct cellular responses to GABA and GABA-related drugs.
Animals↗
Two genes encode distinct glutamate decarboxylases.
gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.
Amino Acid Sequence↗
Sequence and regional distribution of the mRNA encoding the alpha 2 polypeptide of rat gamma-aminobutyric acidA receptors.
A cDNA from a rat hippocampal cDNA library encodes an isoform of the alpha polypeptide of the gamma-aminobutyric acid (GABA)/benzodiazepine (BZ) receptor. Its deduced amino acid sequence is 96% identical to that of the alpha 2 polypeptide of the bovine GABAA receptor. The polypeptide has features shared by all previously reported GABAA receptor alpha polypeptides and shares 71-76% identity with previously described rat alpha polypeptides. Most of the differences lie in the presumed extracellular and intracellular domains. On Northern blots, the alpha 2 cDNA detects two mRNAs, which are found in cortex, hippocampus, and striatum, brain regions enriched in pharmacologically defined "BZ type II" receptors. Other workers have previously shown that the alpha polypeptides of the GABAA receptor largely determine the BZ binding properties of reconstituted receptors. The distribution of alpha 2 mRNAs in rat brain suggests that the alpha 2 subunit may indeed be involved in the BZ type II receptors.
Animals↗
Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions.
Glutamate decarboxylase (GAD) catalyzes the production of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter. The mammalian brain contains two forms of GAD, with Mrs of 67,000 and 65,000 (GAD67 and GAD65). Using a new antiserum specific for GAD67 and a monoclonal antibody specific for GAD65, we show that the two forms of GAD differ in their intraneuronal distributions: GAD67 is widely distributed throughout the neuron, whereas GAD65 lies primarily in axon terminals. In brain extracts, almost all GAD67 is in an active holoenzyme form, saturated with its cofactor, pyridoxal phosphate. In contrast, only about half of GAD65 (which is found in synaptic terminals) exists as active holoenzyme. We suggest that the relative levels of apo-GAD65 and holo-GAD65 in synaptic terminals may couple GABA production to neuronal activity.
Animals↗
Parallel increases in striatal glutamic acid decarboxylase activity and mRNA levels in rats with lesions of the nigrostriatal pathway.
Adult male rats were lesioned with 6-hydroxydopamine in order to destroy the nigrostriatal dopaminergic projections. In rats with such a lesion, we found a parallel increase in glutamic acid decarboxylase (GAD) activity and GAD mRNA in the striatum ipsilateral to the lesion at 4 weeks and 4 months after the lesion. These observations support the proposal that nigral dopaminergic neurons exert a tonic inhibitory control over the striatal GABAergic neurons. Our observations also suggest that the dopaminergic neurons can inhibit gene expression in striatal GABAergic neurons and that the enhanced striatal GAD activity following lesions of the dopaminergic projections is due to 'de novo' synthesis of the enzyme.
Animals↗
Lurcher Purkinje cells express glutamic acid decarboxylase and calbindin mRNAs.
Purkinje neurons in immature Lurcher (Lc/+) mice are destined to die as a result of a defect intrinsic to the dying cells. We have used in situ hybridization to determine whether the Lc allele interferes with the normal program of gene expression in the doomed Purkinje cells. In P21 mice, degeneration of Purkinje cells is well underway, but the surviving Purkinje cells continue to express the mRNAs for both glutamate decarboxylase and calbindin D28K, two proteins whose expression is characteristic of normal Purkinje neurons. We conclude that the Lc allele probably does not interfere with the developmental program but acts to cause cell death in already differentiated Purkinje neurons.
Alleles↗
Detection of point mutations associated with genetic diseases by an exon scanning technique.
A major challenge in genetics is identifying the basis of human heritable disease. We describe an "exon scanning" technique which surveys exons in genomic DNA for sequence alterations. By hybridizing genomic DNA to RNA probes derived from cDNAs, we can use RNase A to survey entire coding regions, comprising exons spread across extensive regions of genomic DNA, for mutations associated with genetic disease. Exon scanning of the beta-globin locus in the DNA of patients with 12 different hemoglobinopathies detected all of the culpable single base substitutions and deletions, but not single base insertions. Our analysis also revealed unsuspected polymorphisms and corrected a diagnosis originally based on hemoglobin electrophoresis. Exon scanning of the ornithine aminotransferase gene in a gyrate atrophy patient detected and localized a mutation in the sixth exon. Subsequent PCR amplification and sequencing characterized this as a missense mutation (proline----glutamine). Exon scanning of genomic DNA for sequence alterations, in combination with PCR amplification and sequencing, should be a generally useful strategy for evaluating suspect genes in disorders of unknown etiology, as well as for clinical diagnosis.
Base Sequence↗
Long-term increase of glutamate decarboxylase mRNA in a rat model of temporal lobe epilepsy.
Behavioral changes following injury, neural degeneration, and aging partly reflect the synaptic plasticity of the nervous system. Such long-term plastic changes are likely to depend on alterations in the production of proteins involved in synaptic structures and neurotransmission. We have studied the regulation of the mRNA encoding one such protein, glutamate decarboxylase (GAD), the rate limiting enzyme of GABA synthesis, after a unilateral lesion in the hippocampus that leads to increased seizure susceptibility. Quantitative in situ hybridization reveals a long-term increase in GAD mRNA in several bilateral structures, as well as in specific neurons in the ipsilateral dentate gyrus. Our data do not support the often stated hypothesis that seizure susceptibility depends on the malfunction of GABA neurons.
Animals↗
Molecular biology of GABAA receptors.
The major type of receptor for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), called the GABAA receptor, is a member of a gene superfamily of ligand-gated ion channels. This receptor is a hetero-oligomeric protein composed of several distinct polypeptide types (alpha, beta, gamma, and delta). Molecular cloning of these polypeptides reveals that they show 20-40% identity with each other, and 10-20% identity with polypeptides of the nicotinic acetylcholine receptors and strychnine-sensitive glycine receptor. Each polypeptide type is also represented by a family of genes whose members have 60-80% amino acid sequence identity. Regions of conserved and variable amino acid sequence suggest structural and functional domains within each polypeptide. All of the polypeptides when expressed in heterologous cells produce GABA-activated chloride channels, and the different subtypes express different pharmacological properties. The distributions of mRNAs for the different GABAA receptor polypeptides and their subtypes show significant brain regional variation consistent with pharmacological and biochemical evidence for receptor heterogeneity. Subpopulations of GABAA receptors with different cellular and regional locations show differential sensitivity to GABA, to modulators like steroids, to physiological regulation, to disease processes, and to pharmacological manipulation by drugs such as benzodiazepines. The properties of the different subpopulations of GABAA receptors are determined by which one or more of the different polypeptides and their subtypes are expressed in a given cell to produce a variety of different oligomeric protein structures. Molecular cloning techniques have produced rapid advances in understanding the GABAA receptor protein family.
Amino Acid Sequence↗
Isolation of pharmacologically distinct GABA-benzodiazepine receptors by protein chemistry and molecular cloning.
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Affinity Labels↗
Splicing defect at the ornithine aminotransferase (OAT) locus in gyrate atrophy.
Gyrate atrophy (GA), a recessive eye disease involving progressive vision loss due to chorioretinal degeneration, is associated with the deficiency of the mitochondrial enzyme ornithine aminotransferase (OAT), with consequent hyperornithinemia. We and others have reported a number of missense mutations at the OAT locus which result in GA. Here we report a GA patient of Danish/Swedish ancestry in whom one OAT allele produces an mRNA that is missing a single 96-bp exon relative to the normal mRNA. Polymerase-chain-reaction amplification and sequencing revealed a 9-bp deletion covering the splice acceptor region of exon 5, resulting in the absence of exon 5 sequences from the mRNA with no disruption to the reading frame. This mutation, which was not present in 15 other independent GA patients, adds to the array of allelic heterogeneity observed in GA and represents the first example of a splicing mutation associated with this disorder.
Adult↗
A novel alpha subunit in rat brain GABAA receptors.
Two cDNAs (alpha 1 and alpha 4) from rat brain cDNA libraries encode isoforms of the alpha subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that alpha 1 and alpha 4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the alpha 1 mRNA is detected in the cerebellum. We injected RNA transcribed from alpha 1 and alpha 4 cDNAs into Xenopus oocytes, together with an RNA for a rat beta subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the alpha and beta polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.
Amino Acid Sequence↗
The GABA receptor-chloride ion channel protein complex.
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Animals↗