RESUMO
We have utilized a polymerase chain reaction (PCR) strategy to identify a novel subunit, gamma 3, of the GABAA receptor. The gamma 3 cDNA encodes a mature protein of 450 amino acids that contains structural features typically conserved among subunits of the GABAA receptor family. The gamma 3 subunit shares approximately 66% sequence identity with the gamma 2 subunit but only 38% and 29% with alpha 1 and beta 1 subunits, respectively. Localization of the gamma 3 mRNA indicates that it is widely distributed throughout the mouse brain in a pattern similar to that observed for mRNAs encoding the gamma 2 subunits.
Assuntos
Reação em Cadeia da Polimerase , Receptores de GABA-A/química , Sequência de Aminoácidos , Animais , Química Encefálica , Substâncias Macromoleculares , Masculino , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , Receptores de GABA-A/genética , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
We have isolated and sequenced a mouse brain cDNA encoding Ca2+/calmodulin-dependent protein kinase IV. The sequence predicts an acidic protein (pI = 4.56) of 469 amino acids (Mr = 52,627) that contains kinase catalytic and calmodulin-binding domains. The carboxy region has several primary structural features that suggest it may be a readily cleaved attachment domain. This region is highly charged and hydrophilic and contains several PEST sequences, motifs associated with high turnover proteins. Of the tissues examined, expression of the CaM kinase IV gene is restricted to brain and testis, where transcripts are differentially expressed to produce a kinase in both tissues and a calmodulin-binding protein, calspermin, in testis that lacks a kinase catalytic domain.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina , DNA/genética , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Encéfalo/enzimologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas de Ligação a Calmodulina/genética , Expressão Gênica , Isoenzimas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Mensageiro/análise , Testículo/enzimologiaRESUMO
The greater sensitivity of long-sleep (LS), as compared with short-sleep (SS), mice to ethanol is due in part to differences in GABAA receptor function in specific brain regions. To determine if differences in subunit composition of GABAA receptors contribute to this differential sensitivity, we measured alpha 1 and gamma 2 subunit mRNAs with Northern analysis and in situ hybridization and gamma 2S, gamma 2L and alpha 6 subunit mRNAs with polymerase chain reaction (PCR) amplification. No differences in mRNAs in whole brain were apparent by Northern analysis. In situ hybridization revealed that alpha 1 and gamma 2 subunit mRNAs were co-localized in many brain regions but that they still had distinct patterns of hybridization. However, the few differences observed between LS and SS mice in the levels of hybridization for these subunits did not show a regional distribution consistent with ethanol sensitivity differences. Similar ratios of gamma 2L, and gamma 2S subunit mRNAs were found in LS and SS mouse cerebral cortex and hippocampus, and both mouse lines expressed essentially only gamma 2L subunit mRNA in cerebellum. mRNA for the alpha 6 subunit was detected only in cerebellum and also was qualitatively similar between LS and SS mice. Studies of muscimol-stimulated 36Cl- uptake by cortical membrane vesicles confirmed earlier findings that ethanol does not enhance function of GABAA receptors in SS mice when assayed at 30 degrees C. However, at 34 degrees C ethanol did increase this function in SS mice although the enhancement remained greater in LS mice. These functional results, together with the results showing similar levels of alpha 1, gamma 2S, gamma 2L and alpha 6 subunits in LS and SS mice, suggest that the ethanol-insensitivity of SS mouse GABAA receptors cannot be due solely to lack of subunits required for ethanol action and further suggest that differences in catalytic mechanisms affecting post-translational processing may account for some genetic differences in ethanol sensitivity of GABAA receptors.
Assuntos
Encéfalo/fisiologia , RNA Mensageiro/metabolismo , Receptores de GABA-A/fisiologia , Sono/fisiologia , Animais , Elementos Antissenso (Genética) , Autorradiografia , Sequência de Bases , Cerebelo/fisiologia , Córtex Cerebral/fisiologia , Feminino , Hipocampo/fisiologia , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Especificidade de Órgãos , Poli A/genética , Poli A/metabolismo , Reação em Cadeia da Polimerase , Sondas RNA , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptores de GABA-A/genética , Especificidade da Espécie , Radioisótopos de EnxofreRESUMO
Southern blot analysis of Chinese hamster x mouse somatic cell hybrids was used to map the gene for a serine/threonine protein kinase expressed in brain and testis. This locus, termed Camk-4, encodes Ca2+/calmodulin-dependent protein kinase IV. Progeny of an interspecific backcross were analyzed to position Camk-4 in the centromeric region of chromosome 18 near two mutations known to affect neurological function and fertility. This raises the possibility that a defect in Camk-4 may be responsible for one of these mutant phenotypes.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina , Camundongos/genética , Proteínas Quinases/genética , Animais , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Mapeamento Cromossômico , Cricetinae , Cricetulus , Cruzamentos Genéticos , Genes , Marcadores Genéticos , Células Híbridas , Infertilidade/genética , Masculino , Camundongos Mutantes/genética , Camundongos Mutantes Neurológicos/genética , Família Multigênica , Muridae/genética , Polimorfismo de Fragmento de Restrição , Proteína Quinase C/genética , Proteínas Quinases/fisiologia , Recombinação Genética , Especificidade da EspécieRESUMO
Recent studies have identified two alternatively spliced forms of the GABA(A) receptor gamma(2) subunit that differ by the presence (gamma(2L)) or absence (gamma(2S)) of an eight-amino acid segment. This insert in the gamma(2L) isoform exists in the proposed cytoplasmic loop region, between M3 and M4, and contains a consensus sequence for phosphorylation by protein kinase C. To examine the regional distribution of this novel receptor subunit in the brain, gamma(2L) subunit mRNA was detected using both in situ hybridization histochemistry and and PCR amplification methods. Hybridization histochemistry with a gamma(2L), subunit-specific oligonucleotide probe revealed that the gamma(2L), subunit mRNA is widely distributed throughout the mouse brain. The highest levels of expression are found in the cerebral cortex, hippocampus, olfactory lobe, and cerebellum. The presence of the gamma(2L), subunit in these regions was confirmed using PCR. Additionally, PCR experiments detected yes subunit mRNA in the cerebral cortex and hippocampus but not in the cerebellum. To examine the functional properties of the gamma(2) subunit isoforms, gamma(2S) and gamma(2L), subunit mRNAs were coexpressed with alpha(1)beta(1) subunit mRNAs in Xenopus oocytes. These experiments indicate that the gamma(2L) and gamma(2S) subunit variants exhibit similar pharmacological properties, including the ability of both isoforms to confer diazepam sensitivity to the receptor complex. In addition, potentiation of GABA responses by pentobarbital in oocytes expressing either subunit isoform is similar. These data indicate that the presence or absence of the additional eight amino acids in the gamma(2) subunit isoforms does not appear to alter the response of the GABA(A) receptor complex to either benzodiazepines and barbiturates at the level of protein phosphorylation present in the oocyte.