Subject(s)
Isoenzymes/genetics , Neurons/enzymology , Parkinsonian Disorders/genetics , Protein Kinase C/genetics , Animals , Base Sequence , Codon, Nonsense , Codon, Terminator , Crosses, Genetic , Dopamine/metabolism , Genetic Linkage , Humans , Immunohistochemistry , Isoenzymes/chemistry , Isoenzymes/metabolism , Molecular Sequence Data , Mutation , Parkinsonian Disorders/enzymology , Parkinsonian Disorders/physiopathology , Protein Kinase C/chemistry , Protein Kinase C/metabolism , Purkinje Cells/enzymology , RatsABSTRACT
The quantitative measurement of steady-state mRNA levels is fundamental to the analysis of gene expression. A variety of techniques are widely used to achieve this including Northern blotting, RNase protection, and S1 nuclease protection. We describe here in detail a relatively recent extension of the S1 nuclease protection technique (1) in which radiolabeled oligonucleotides are used as probes in a solution hybridization assay (2). The principle advantage of this technique is that it allows, in a single RNA sample, the simultaneous measurement of the relative levels of at least six mRNA species, including that of a control mRNA. Further, a large number of RNA samples can be analyzed at one time.
Subject(s)
Oligonucleotide Probes , RNA, Messenger/analysis , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Nucleic Acid HybridizationABSTRACT
The 5-HT3 receptor is a ligand-gated ion channel with significant structural similarity to the nicotinic acetylcholine receptor. Several regions that form the ligand binding site in the nicotinic acetylcholine receptor are partially conserved in the 5-HT3 receptor, presumably reflecting the conserved signal transduction mechanism. Specific amino acid differences in these regions may account for their distinct ligand recognition properties. Using site-directed mutagenesis, we have replaced one of these residues, glutamate 106 (E106), with aspartate (D), asparagine (N), alanine (A) or glutamine (Q) and characterized the ligand-binding and electrophysiological properties of the mutant receptors after transient expression in HEK-293 cells. The affinity for the selective 5-HT3 receptor antagonist [3H]GR65630 was decreased 14-fold in the mutant E106D (Kd = 3.69 +/- 0.32 nM) when compared to wildtype (WT, E106) 5-HT3 receptor (0.27 +/- 0.03 nM), while the affinity for E106N was unchanged (0.42 +/- 0.07 nM, means +/- SEM, n = 3-10). Decreased affinities for both E106D and E106N were observed for the antagonists granisetron, ondansetron and renzapride and for the agonists 5-HT (130- and 30-fold) and 2-methyl-5-HT (250- and 20-fold), respectively. Both mutants still formed 5-HT-activatable ion channels, but the high Hill coefficient of the concentration effect curves in wildtype (2.0) was decreased to unity in both cases. The EC50 of 5-HT was increased seven-fold in E106N (8.7 microM) when compared to wildtype (1.2 microM), but unchanged in E106D, and the potency of the antagonist ondansetron for both mutants was decreased. E106A and E106Q expressed poorly preventing a detailed characterization. These data suggest that E106 contributes to the ligand-binding site of the 5-HT3 receptor and may form an ionic or hydrogen bond interaction with the primary ammonium group of 5-HT.
Subject(s)
Glutamic Acid/physiology , Mutagenesis, Site-Directed/drug effects , Receptors, Serotonin/genetics , Receptors, Serotonin/metabolism , Animals , Binding Sites , Binding, Competitive , Cell Line , DNA, Complementary/biosynthesis , Electrophysiology , Humans , Kidney/metabolism , Ligands , Mice , Patch-Clamp Techniques , Radioligand Assay , Receptors, Serotonin/biosynthesis , Serotonin/metabolismABSTRACT
Mental illness, such as panic disorder and depression, display comorbidity as well as common therapeutic treatments. These features point toward a common etiology and/or therapeutic pathway. There is evidence to suggest that some antipanic agents may mediate their effects by altering gamma-aminobutyric acid (GABA) levels or by modulating the activity of the GABAA receptor. Chronic stimulation of GABAA receptors by agonists or modulators results in changes in the pharmacological properties of the receptor concomitant with alterations in the expression of specific GABAA receptor subunits. Therefore, we investigated the hypothesis that long-term exposure to three antidepressant/antipanic drugs (imipramine, phenelzine and alprazolam) would produce changes in the steady-state levels of those subunit mRNAs that are believed to encode the major GABAA receptor subtype. Further, these changes in gene expression would be different to those produced by the non-antipanic anxiolytic (buspirone). We report here that, following a 21 day treatment, imipramine, phenelzine, alprazolam and buspirone differentially altered rat brainstem levels of GABAA receptor alpha 1-, beta 2- and gamma 2-subunit RNAs. These results demonstrate novel actions of antidepressant/antipanic drugs on GABAergic neurotransmission.
Subject(s)
Anti-Anxiety Agents/pharmacology , GABA Agonists/pharmacology , GABA-A Receptor Agonists , Panic Disorder/drug therapy , RNA, Messenger/biosynthesis , Receptors, GABA-A/biosynthesis , Animals , Brain Stem/drug effects , Brain Stem/metabolism , Buspirone/pharmacology , Imipramine/pharmacology , Monoamine Oxidase Inhibitors/pharmacology , Neurotransmitter Uptake Inhibitors/pharmacology , Phenelzine/pharmacology , RatsABSTRACT
cDNAs have been cloned that encode the chicken (Gallus domesticus) gamma-aminobutyric acidA receptor gamma 1 subunit, the mature sequence of which shares 90, 79, and 69% identity with those of the rat gamma 1, gamma 2, and gamma 3 subunits, respectively. In situ hybridization reveals that there are pronounced differences in the regional and cellular localizations of the corresponding gamma-aminobutyric acidA receptor gamma-subunit mRNA compared with that of the gamma 2-subunit mRNA in 1-day-old chick brain. The absence of the gamma 1-subunit transcript in certain chick brain nuclei of visual and auditory pathways, in which gamma 2-subunit mRNA is present, points to differences in the functional roles of receptors containing one or other of these polypeptides. Certain cells in other brain regions appear to contain both gamma 1- and gamma 2-subunit mRNAs, suggesting that they either have two gamma-aminobutyric acidA receptor subtypes or possess receptors incorporating two different gamma subunits. We have also found contrasts in the distribution patterns, in homologous brain regions, of the chicken gamma 1-subunit mRNA and the rat gamma 1-subunit mRNA. These data may reflect different functional roles of the chicken and rat gamma 1 subunits.
Subject(s)
Brain/metabolism , Chickens/genetics , RNA, Messenger/biosynthesis , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , In Situ Hybridization , Macromolecular Substances , Mammals/genetics , Molecular Sequence Data , Oligonucleotides, Antisense , Organ Specificity , RNA, Messenger/analysis , Rats/genetics , Sequence Homology, Amino Acid , Transcription, GeneticSubject(s)
Receptors, GABA-A/genetics , Animals , Gene Expression , Genome , Humans , Receptors, GABA-A/biosynthesisABSTRACT
We have used sequence-specific oligonucleotide probes and in situ hybridisation histochemistry to examine the distribution of the GABAA receptor alpha 1- and gamma 2-subunit mRNAs in serial sections of 1-day-old chick brain. Both transcripts are present together, at high levels, in many brain regions. Differences are found, however, in the relative amounts of these mRNAs in two isthmic nuclei of the optic lobe, the deep cerebellar nuclei, and the dorsal thalamus. We therefore conclude that while the alpha 1 and gamma 2 subunits predominantly occur together in the same receptor complex, they may also be found separately in other GABAA receptor subtypes.
Subject(s)
Brain Chemistry/physiology , RNA, Messenger/metabolism , Receptors, GABA-A/biosynthesis , Animals , Autoradiography , Base Sequence , Chickens , Gene Expression , Molecular Sequence Data , Nucleic Acid Hybridization , Oligonucleotides/analysis , Sulfur RadioisotopesABSTRACT
We report the sequence of a complementary DNA (cDNA) that encodes the chicken GABAA receptor alpha 1 subunit, which is extremely homologous to mammalian alpha 1 subunits. The distribution of alpha 1 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 alpha 1 subunit may not necessarily be restricted to receptors having BZI pharmacology.
Subject(s)
Brain/physiology , DNA/genetics , RNA, Messenger/genetics , Receptors, GABA-A/genetics , Amino Acid Sequence , Animals , Autoradiography , Base Sequence , Chick Embryo , Gene Library , Macromolecular Substances , Molecular Sequence Data , Oligonucleotide Probes , Poly A/genetics , Poly A/isolation & purification , RNA/genetics , RNA/isolation & purification , RNA, Messenger/analysis , Sulfur RadioisotopesABSTRACT
Amino-acid sequences derived from complementary DNAs encoding the alpha- and beta-subunits of the GABA/benzodiazepine receptor from bovine brain show homology with other ligand-gated receptor subunits, suggesting that there is a super-family of ion-channel-containing receptors. Co-expression of the in vitro-generated alpha-subunit and beta-subunit RNAs in Xenopus oocytes produces a functional receptor and ion channel with the pharmacological properties characteristic of the GABAA receptor.
