ABSTRACT
We generated mouse mutants with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at different levels and deficient in Q/R-site editing. All mutant lines had increased AMPAR calcium permeabilities in pyramidal neurons, and one showed elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx induced NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium-triggered neuronal death was not observed, but mutants had mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlated with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons.
Subject(s)
Gene Expression/physiology , Nervous System Diseases/genetics , Receptors, Glutamate/genetics , Alleles , Animals , Brain/pathology , Calcium/metabolism , Calcium/physiology , Electric Conductivity , Hippocampus/physiopathology , Long-Term Potentiation/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic/genetics , Neural Pathways/physiopathology , Phenotype , Receptors, AMPA/physiologyABSTRACT
Recently, GBR1, a seven-transmembrane domain protein with high affinity for gamma-aminobutyric acid (GABA)B receptor antagonists, was identified. Here, a GBR1-related protein, GBR2, was shown to be coexpressed with GBR1 in many brain regions and to interact with it through a short domain in the carboxyl-terminal cytoplasmic tail. Heterologously expressed GBR2 mediated inhibition of adenylyl cyclase; however, inwardly rectifying potassium channels were activated by GABAB receptor agonists only upon coexpression with GBR1 and GBR2. Thus, the interaction of these receptors appears to be crucial for important physiological effects of GABA and provides a mechanism in receptor signaling pathways that involve a heterotrimeric GTP-binding protein.
Subject(s)
Brain/metabolism , Potassium Channels, Inwardly Rectifying , Receptors, GABA-B/chemistry , Receptors, GABA-B/metabolism , Receptors, GABA/chemistry , Receptors, GABA/metabolism , Adenylyl Cyclase Inhibitors , Amino Acid Sequence , Animals , Cell Line , Cyclic AMP/metabolism , Dimerization , G Protein-Coupled Inwardly-Rectifying Potassium Channels , GABA-B Receptor Agonists , Humans , In Situ Hybridization , Molecular Sequence Data , Neurons/metabolism , Potassium/metabolism , Potassium Channels/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence AlignmentABSTRACT
The complex anatomy of neurons demands a high degree of functional organization. Therefore, membrane receptors and ion channels are often localized to selected subcellular sites and coupled to specific signal transduction machineries. PDZ domains have come into focus as protein interaction modules that mediate the binding of a class of submembraneous proteins to membrane receptors and ion channels and thus subserve these organizational aspects. The structures of two PDZ domains have been resolved, which has led to a structural understanding of the specificity of interactions of various PDZ domains with their respective partners. The functional implications of PDZ domain interactions are now being addressed in vitro and in vivo.
Subject(s)
Ion Channels/metabolism , Ion Channels/physiology , Membrane Proteins/genetics , Models, Genetic , Receptors, Glutamate/physiology , Animals , Membrane Proteins/metabolismABSTRACT
The N-methyl-D-aspartate (NMDA) receptor subserves synaptic glutamate-induced transmission and plasticity in central neurons. The yeast two-hybrid system was used to show that the cytoplasmic tails of NMDA receptor subunits interact with a prominent postsynaptic density protein PSD-95. The second PDZ domain in PSD-95 binds to the seven-amino acid, COOH-terminal domain containing the terminal tSXV motif (where S is serine, X is any amino acid, and V is valine) common to NR2 subunits and certain NR1 splice forms. Transcripts encoding PSD-95 are expressed in a pattern similar to that of NMDA receptors, and the NR2B subunit co-localizes with PSD-95 in cultured rat hippocampal neurons. The interaction of these proteins may affect the plasticity of excitatory synapses.
Subject(s)
Hippocampus/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Cytoplasm/chemistry , Disks Large Homolog 4 Protein , Genes, Reporter , Intracellular Signaling Peptides and Proteins , Membrane Proteins , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Neuronal Plasticity , RNA Splicing , Rats , Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/genetics , Recombinant Fusion Proteins/metabolism , Signal TransductionABSTRACT
The murine gene encoding the GluR-B subunit of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors was characterized with respect to exon-intron organization, transcriptional start site, alternatively spliced transcripts, and adenosine to guanosine substitutions between gene and complementary DNA sequence. The GluR-B gene spans > 90 kilobase pairs and harbors 17 exons. Transcription appears to initiate approximately 430 nucleotides upstream of the translational start codon, with no intron in the 5'-untranslated region of the gene. Four alternatively spliced mRNAs are generated from the primary GluR-B transcript, two containing the modules Flip and Flop, and another two with alternate C-terminal coding sequence. The major GluR-B mRNAs in murine brain, 4 and 6 kilobase differ in the length of their 3'-untranslated region.
Subject(s)
Receptors, AMPA/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary , Mice , Molecular Sequence Data , RNA, Messenger/genetics , Rats , Receptors, AMPA/chemistry , Transcription, GeneticABSTRACT
The in vivo expression of mutations constructed within helix 34 of 16S rRNA has been examined together with a nonsense tRNA suppressor for their action at stop codons. The data revealed two novel results: in contrast to previous findings, some of the rRNA mutations affected suppression at UAA and UAG nonsense codons. Secondly, both an increase and a decrease in the efficiency of the suppressor tRNA were induced by the mutations. This is the first report that rRNA mutations decreased the efficiency of a suppressor tRNA. The data are interpreted as there being competition between the two release factors (RF-1 and RF-2) for an overlapping domain and that helix 34 influences this interaction.
