RESUMO
This article is part of a Special Issue "Estradiol and Cognition". In estrogen-induced synaptic plasticity, a correlation of structure, function and behavior in the hippocampus has been widely established. 17ß-estradiol has been shown to increase dendritic spine density on hippocampal neurons and is accompanied by enhanced long-term potentiation and improved performance of animals in hippocampus-dependent memory tests. After inhibition of aromatase, the final enzyme of estradiol synthesis, with letrozole we consistently found a strong and significant impairment of long-term potentiation (LTP) in female mice as early as after six hours of treatment. LTP impairment was followed by loss of hippocampal spine synapses in the hippocampal CA1 area. Interestingly, these effects were not found in male animals. In the Morris water maze test, chronic administration of letrozole did not alter spatial learning and memory in either female or male mice. In humans, analogous effects of estradiol on hippocampal morphology and physiology were observed using neuroimaging techniques. However, similar to our findings in mice, an effect of estradiol on memory performance has not been consistently observed.
Assuntos
Estradiol/fisiologia , Hipocampo/fisiologia , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Animais , Aromatase/metabolismo , Inibidores da Aromatase/farmacologia , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Cognição/efeitos dos fármacos , Cognição/fisiologia , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/fisiologia , Espinhas Dendríticas/ultraestrutura , Estradiol/farmacologia , Feminino , Hipocampo/efeitos dos fármacos , Humanos , Letrozol , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nitrilas/farmacologia , Ratos , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Triazóis/farmacologiaRESUMO
Calumenin is a Ca(2+)-binding protein that belongs to the CREC superfamily. It contains six EF-hand domains that exhibit a low affinity for Ca(2+) as well as an endoplasmic reticulum retention signal. Calumenin exhibits a broad and relatively high expression in various brain regions during development as demonstrated by in situ hybridization. Signal intensity of calumenin is highest during the early development and then declines over time to reach a relatively low expression in adult animals. Immunohistochemistry indicates that at the P0 stage, calumenin expression is most abundant in migrating neurons in the zones around the lateral ventricle. In the brain of adult animals, it is expressed in various glial and neuronal cell types, including immature neurons in subgranular zone of hippocampal dentate gyrus. At the subcellular level, calumenin is identified in punctuate and diffuse distribution mostly in somatic regions where it co-localizes with endoplasmic reticulum (ER) and partially Golgi apparatus. Upon subcellular fractionation, calumenin is enriched in fractions containing membranes and is only weakly present in soluble fractions. This study points to a possible important role of calumenin in migration and differentiation of neurons, and/or in Ca(2+) signaling between glial cells and neurons.
Assuntos
Química Encefálica/fisiologia , Proteínas de Ligação ao Cálcio/biossíntese , Animais , Animais Recém-Nascidos , Western Blotting , Encéfalo/crescimento & desenvolvimento , Química Encefálica/genética , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Centrifugação , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Membranas/metabolismo , Camundongos , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Frações Subcelulares/metabolismo , Distribuição TecidualRESUMO
Active neuronal transport along microtubules participates in the targeting of mRNAs, proteins and organelles to their sites of action. Cytoplasmic dynein represents a minus-end-directed microtubule-dependent motor protein. Due to the polarity of microtubules in axonal and distal dendritic compartments, with microtubule minus-ends pointing toward the inside of the cell, dyneins mainly mediate retrograde transport pathways in neurons. Since dyneins transport synaptic proteins, we asked whether changes in neuronal activity would in general influence dynein transport. KCl-induced depolarization, a condition that mimics the effects of neuronal activity, or pharmacological blockade of neuronal action potentials, respectively, was combined with neuronal live cell imaging, using an autofluorescent dynein intermediate chain fusion (monomeric red fluorescent protein [mRFP]-dynein intermediate chain [DIC]) as a model protein. Notably, we found that induced activity significantly reduced dynein particle mobility, as well as both the total distance and velocity of movements in mouse cultured hippocampal neurons. In contrast, blockade of neuronal action potentials through TTX did not alter any of the parameters analyzed. Neuronal depolarization processes therefore represent candidate mechanisms to regulate intracellular transport of neuronal cargoes.
Assuntos
Dineínas/metabolismo , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Células Cultivadas , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Proteínas Luminescentes , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurotransmissores/administração & dosagem , Cloreto de Potássio/administração & dosagem , Bloqueadores dos Canais de Sódio/administração & dosagem , Tetrodotoxina/administração & dosagem , Gravação em Vídeo , Proteína Vermelha FluorescenteRESUMO
The brain-specific GDP/GTP exchange factor collybistin interacts with the receptor-anchoring protein gephyrin and activates the Rho-like GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with hetero-oligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pull-down assays and in the yeast two-hybrid system. Site-directed mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyR beta-subunit binding motif. Our data are consistent with collybistin-gephyrin interactions occuring during inhibitory postsynaptic membrane formation.
Assuntos
Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Conformação Proteica , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Fatores de Troca de Nucleotídeo Guanina RhoRESUMO
Gephyrin has been shown to be essential for the synaptic localization of the inhibitory glycine receptor and major GABA(A) receptor (GABA(A)R) subtypes. However, in retina certain GABA(A)R subunits are found at synaptic sites in the absence of gephyrin. Here, we quantitatively analyzed GABA(A)R alpha1, alpha2, alpha3, alpha5, beta2/3, and gamma2 subunit immunoreactivities in spinal cord sections derived from wild-type and gephyrin-deficient (geph -/-) mice. The punctate staining of GABA(A)R alpha1 and alpha5 subunits was unaltered in geph -/- mice, whereas the numbers of alpha2-, alpha3-, beta2/3-, and gamma2-subunit-immunoreactive synaptic sites were significantly or even strikingly reduced in the mutant animals. Immunostaining with an antibody specific for the vesicular inhibitory amino acid transporter revealed that the number of inhibitory presynaptic terminals is unaltered upon gephyrin deficiency. These data show that in addition to gephyrin other clustering proteins must exist that mediate the synaptic localization of selected GABA(A)R subtypes.
Assuntos
Sistemas de Transporte de Aminoácidos , Diferenciação Celular/genética , Proteínas de Membrana/deficiência , Inibição Neural/genética , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Medula Espinal/embriologia , Membranas Sinápticas/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Feto , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imuno-Histoquímica , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Plasticidade Neuronal/genética , Neurônios/citologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Medula Espinal/citologia , Medula Espinal/metabolismo , Membranas Sinápticas/ultraestrutura , Proteínas Vesiculares de Transporte de Aminoácidos InibidoresRESUMO
Gephyrin is a ubiquitously expressed protein that, in the central nervous system, forms a submembraneous scaffold for anchoring inhibitory neurotransmitter receptors in the postsynaptic membrane. The N- and C-terminal domains of gephyrin are homologous to the Escherichia coli enzymes MogA and MoeA, respectively, both of which are involved in molybdenum cofactor biosynthesis. This enzymatic pathway is highly conserved from bacteria to mammals, as underlined by the ability of gephyrin to rescue molybdenum cofactor deficiencies in different organisms. Here we report the x-ray crystal structure of the N-terminal domain (amino acids 2-188) of rat gephyrin at 1.9-A resolution. Gephyrin-(2-188) forms trimers in solution, and a sequence motif thought to be involved in molybdopterin binding is highly conserved between gephyrin and the E. coli protein. The atomic structure of gephyrin-(2-188) resembles MogA, albeit with two major differences. The path of the C-terminal ends of gephyrin-(2-188) indicates that the central and C-terminal domains, absent in this structure, should follow a similar 3-fold arrangement as the N-terminal region. In addition, a central beta-hairpin loop found in MogA is lacking in gephyrin-(2-188). Despite these differences, both structures show a high degree of surface charge conservation, which is consistent with their common catalytic function.
Assuntos
Proteínas de Transporte/química , Coenzimas , Proteínas de Escherichia coli , Proteínas de Membrana/química , Sequência de Aminoácidos , Animais , Arabidopsis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Caenorhabditis elegans , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Drosophila melanogaster , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloproteínas/metabolismo , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Cofatores de Molibdênio , Conformação Proteica , Pteridinas/metabolismo , Ratos , Alinhamento de Sequência , Eletricidade EstáticaRESUMO
The neurotransmitter receptor anchoring protein gephyrin is encoded by a highly mosaic gene whose primary transcript is subject to extensive alternative splicing. Gephyrin mRNAs are widely expressed in various mammalian tissues, and gephyrin has been implicated in neuron-specific and general metabolic functions. Using a novel affinity isolation procedure, we report the identification of different gephyrin variants in various organs of the adult rat. In particular, polypeptides of 52, 56, 60, and 91 kDa were detected in addition to the previously characterized 93-kDa protein. Our results suggest tissue-specific functional differences between gephyrin variants.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Western Blotting , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Cromatografia de Afinidade , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , RNA Mensageiro/genética , RatosRESUMO
The dbl-like GDP/GTP exchange factor, collybistin, binds to the receptor anchoring protein gephyrin and activates the Rho-like GTPase Cdc42. Collybistin was found in two splice variants I and II, both of which share a tandem Dbl homology/pleckstrin homology (DH/PH) domain. In heterologous expression systems, collybistin II induces the formation of submembraneous gephyrin aggregates and therefore has been implicated in inhibitory synapse formation. Expression of collybistin is restricted to neuronal tissues and is predominantly found in brain. Here, we investigated the spatio-temporal distribution of collybistin transcripts in the embryonic mouse brain and compared it to gephyrin and glycine receptor mRNA patterns. Our data show that collybistin expression is upregulated when neurons become postmitotic and start to differentiate.
Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/genética , Animais , Encéfalo/citologia , Proteínas de Transporte/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Cerebelo/citologia , Cerebelo/embriologia , Cerebelo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/embriologia , Gânglios Espinais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/metabolismo , Hibridização In Situ , Proteínas de Membrana/genética , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/embriologia , Bulbo Olfatório/metabolismo , RNA Mensageiro/análise , Receptores de GABA-A/metabolismo , Receptores de Glicina/genética , Fatores de Troca de Nucleotídeo Guanina Rho , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Células-Tronco/citologia , Sinapses/metabolismo , Domínios de Homologia de src/fisiologiaRESUMO
Clustering of neurotransmitter receptors in postsynaptic densities involves proteins that aggregate the receptors and link them to the cytoskeleton. In the case of glycine and GABA(A) receptors, gephyrin has been shown to serve this function. However, it is unknown whether gephyrin is involved in the clustering of all glycine and GABA(A) receptors or whether it interacts only with specific isoforms. This was studied in the retinae of mice, whose gephyrin gene was disrupted, with immunocytochemistry and antibodies that recognize specific subunits of glycine and GABA(A) receptors. Because homozygous (geph -/-) mutants die around birth, an organotypic culture system of the mouse retina was established to study the clustering of gephyrin and the receptors in vitro. We found that all gephyrin and all glycine receptor clusters (hot spots) were abolished in the geph (-/-) mouse retina. In the case of GABA(A) receptors, there was a significant reduction of clusters incorporating the gamma2, alpha2, and alpha3 subunits; however, a substantial number of hot spots was still present in geph (-/-) mutant retinae. This shows that gephyrin interacts with all glycine receptor isoforms but with only certain forms of GABA(A) receptors. In heterozygous geph (+/-) mutants, no reduction of hot spots was observed in the retina in vivo, but a significant reduction was found in the organotypic cultures. This suggests that mechanisms may exist in vivo that allow for the compensation of a partial gephyrin deficit.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Receptores de GABA/metabolismo , Receptores de Glicina/metabolismo , Retina/metabolismo , Sinapses/metabolismo , Animais , Proteínas de Transporte/genética , Feminino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Técnicas de Cultura de Órgãos , Gravidez , Receptores de GABA/genética , Receptores de Glicina/genética , Sinapses/genéticaRESUMO
G protein-coupled receptors regulate gene expression by cellular signaling cascades that target transcription factors and their recognition by specific DNA sequences. In the central nervous system, heteromeric metabotropic gamma-aminobutyric acid type B (GABA(B)) receptors through adenylyl cyclase regulate cAMP levels, which may control transcription factor binding to the cAMP response element. Using yeast-two hybrid screens of rat brain libraries, we now demonstrate that GABA(B) receptors are engaged in a direct and specific interaction with the activating transcription factor 4 (ATF-4), a member of the cAMP response element-binding protein /ATF family. As confirmed by pull-down assays, ATF-4 associates via its conserved basic leucine zipper domain with the C termini of both GABA(B) receptor (GABA(B)R) 1 and GABA(B)R2 at a site which serves to assemble these receptor subunits in heterodimeric complexes. Confocal fluorescence microscopy shows that GABA(B)R and ATF-4 are strongly coclustered in the soma and at the dendritic membrane surface of both cultured hippocampal neurons as well as retinal amacrine cells in vivo. In oocyte coexpression assays short term signaling of GABA(B)Rs via G proteins was only marginally affected by the presence of the transcription factor, but ATF-4 was moderately stimulated in response to receptor activation in in vivo reporter assays. Thus, inhibitory metabotropic GABA(B)Rs may regulate activity-dependent gene expression via a direct interaction with ATF-4.
Assuntos
Receptores de GABA-B/metabolismo , Fatores de Transcrição/metabolismo , Fator 4 Ativador da Transcrição , Sequência de Aminoácidos , Animais , Western Blotting , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Clonagem Molecular , AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Eletrofisiologia , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Glutationa Transferase/metabolismo , Imuno-Histoquímica , Luciferases/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Neurônios/metabolismo , Oócitos/metabolismo , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Retina/metabolismo , Vírus da Floresta de Semliki/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Xenopus laevisRESUMO
Recent studies indicate an important role of cytoskeleton-associated and lipid-anchored proteins in the formation of inhibitory postsynaptic membrane specializations. Membrane apposition of the tubulin-binding protein gephyrin is essential for the recruitment of inhibitory glycine receptors and GABAA receptors to developing postsynaptic sites. Newly disclosed interactions between gephyrin, exchange factors for G proteins of the Rho and Rac families, the translational regulator RAFT1, and actin-binding proteins like profilin might integrate activity-dependent and trophic-factor-mediated signals at developing postsynaptic sites. A model of inhibitory neurotransmitter receptor clustering, is proposed, in which this process is initiated by receptor-driven activation of phosphatidylinositol 3-kinase.
Assuntos
Inibição Neural/fisiologia , Neurônios/metabolismo , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais/fisiologia , Sinapses/metabolismo , AnimaisRESUMO
gamma-Aminobutyric acid type A receptors (GABA(A)Rs) are ligand-gated chloride channels that exist in numerous distinct subunit combinations. At postsynaptic membrane specializations, different GABA(A)R isoforms colocalize with the tubulin-binding protein gephyrin. However, direct interactions of GABA(A)R subunits with gephyrin have not been reported. Recently, the GABA(A)R-associated protein GABARAP was found to bind to the gamma2 subunit of GABA(A)Rs. Here we show that GABARAP interacts with gephyrin in both biochemical assays and transfected cells. Confocal analysis of neurons derived from wild-type and gephyrin-knockout mice revealed that GABARAP is highly enriched in intracellular compartments, but not at gephyrin-positive postsynaptic membrane specializations. Our data indicate that GABARAP-gephyrin interactions are not important for postsynaptic GABA(A)R anchoring but may be implicated in receptor sorting and/or targeting mechanisms. Consistent with this idea, a close homolog of GABARAP, p16, has been found to function as a late-acting intra-Golgi transport factor.
Assuntos
Proteínas de Transporte/metabolismo , Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Transporte Biológico , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Humanos , Invertebrados/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Neurônios/metabolismo , Células PC12 , Ratos , Retina/metabolismo , Homologia de Sequência de Aminoácidos , Frações SubcelularesRESUMO
The synaptic localization of ion channel receptors is essential for efficient synaptic trans-mission and the precise regulation of diverse neuronal functions, such as signal integration and synaptic plasticity. Emerging evidence points to an important role of cytoskeleton-associated proteins that assemble receptors and components of the subsynaptic machinery at postsynaptic membrane specializations. This article reviews interactions of inhibitory postsynaptic neurotransmitter receptors with the receptor anchoring protein gephyrin and intracellular components involved in downstream signalling and/or control of signal transduction processes. The presently available data suggest a central synaptic organizer function for gephyrin in inhibitory postsynaptic membrane assembly and stabilization.
Assuntos
Proteínas de Transporte/metabolismo , Coenzimas , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Transmissão Sináptica/fisiologia , Animais , Metaloproteínas/biossíntese , Metaloproteínas/metabolismo , Cofatores de Molibdênio , Neurotransmissores/metabolismo , Ligação Proteica , Pteridinas/metabolismo , Receptores de GABA/metabolismo , Receptores de Glicina/metabolismo , Transdução de SinaisRESUMO
The tubulin-binding protein gephyrin, which anchors the inhibitory glycine receptor (GlyR) at postsynaptic sites, decorates GABAergic postsynaptic membranes in various brain regions, and postsynaptic gephyrin clusters are absent from cortical cultures of mice deficient for the GABA(A) receptor gamma2 subunit. Here, we investigated the postsynaptic clustering of GABA(A) receptors in gephyrin knock-out (geph -/-) mice. Both in brain sections and cultured hippocampal neurons derived from geph -/- mice, synaptic GABA(A) receptor clusters containing either the gamma2 or the alpha2 subunit were absent, whereas glutamate receptor subunits were normally localized at postsynaptic sites. Western blot analysis and electrophysiological recording revealed that normal levels of functional GABA(A) receptors are expressed in geph -/- neurons, however the pool size of intracellular GABA(A) receptors appeared increased in the mutant cells. Thus, gephyrin is required for the synaptic localization of GlyRs and GABA(A) receptors containing the gamma2 and/or alpha2 subunits but not for the targeting of these receptors to the neuronal plasma membrane. In addition, gephyrin may be important for efficient membrane insertion and/or metabolic stabilization of inhibitory receptors at developing postsynaptic sites.
Assuntos
Proteínas de Transporte/fisiologia , Hipocampo/fisiologia , Proteínas de Membrana/fisiologia , Neurônios/fisiologia , Receptores de GABA-A/fisiologia , Medula Espinal/fisiologia , Sinapses/fisiologia , Animais , Astrócitos/citologia , Astrócitos/fisiologia , Proteínas de Transporte/genética , Células Cultivadas , Meios de Cultivo Condicionados , Dendritos/fisiologia , Glicina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , N-Metilaspartato/farmacologia , Proteínas do Tecido Nervoso/análise , Neurônios/citologia , Neurônios/efeitos dos fármacos , Receptores de AMPA/análise , Receptores de AMPA/fisiologia , Receptores de GABA-A/análise , Receptores de GABA-A/química , Receptores de N-Metil-D-Aspartato/fisiologia , Proteínas Associadas SAP90-PSD95RESUMO
Glycine receptors (GlyRs) are ligand-gated chloride channel proteins composed of alpha- and beta-subunits. GlyRs are located to and anchored at postsynaptic sites by the receptor-associated protein gephyrin. Previous work from our laboratory has identified a core motif for gephyrin binding in the cytoplasmic loop of the GlyR beta-subunit. Here, we localized amino acid residues implicated in gephyrin binding by site-directed mutagenesis. In a novel transfection assay, a green fluorescent protein-gephyrin binding motif fusion protein was used to monitor the consequences of amino acid substitutions for beta-subunit interaction with gephyrin. Only multiple, but not single, replacements of hydrophobic side chains abolished the interaction between the two proteins. Our data are consistent with gephyrin binding being mediated by the hydrophobic side of an imperfect amphipathic helix.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Glicina/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Linhagem Celular , Proteínas de Fluorescência Verde , Humanos , Rim/citologia , Rim/metabolismo , Proteínas Luminescentes/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Receptores de Glicina/química , Receptores de Glicina/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
The anion exchange protein AE1 is the most abundant membrane protein in human erythrocytes mediating the electroneutral chloride/bicarbonate exchange. We identified a promoter region in the 5' flanking region of the human AE1 gene which controls transcription in a cell type independent manner. In addition a second, distal promoter element mediates gene expression only in erythroid cells and in dependence upon differentiation. Within this distal promoter region we defined a 44 bp sequence containing a novel CT-rich motif with very strong promoter activity whereas a second 28 bp segment suppresses gene expression.