RESUMO
The formation of postsynaptic clusters of various ligand-gated ion channels is regulated by receptor activity. Here we describe the developmental- and activity-dependent modification of N-methyl-D-aspartate (NMDA) receptor clustering in spinal cord neurons in vitro detected by immunofluorescence analysis using subunit and splice variant specific antibodies. NMDA receptors form synaptic and extrasynaptic clusters with sequential changes in subunit composition during in vitro development. During the first week of in vitro culture, a NR1 splice variant containing the C2-carboxy terminus and lacking the N1-cassette and the NR2B subunit are the prevailing components of receptor clusters at synaptic and extrasynaptic sites. After 3 weeks in culture (days in vitro [DIV] 22), the numbers of postsynaptic receptor clusters with N1-containing NR1 splice variants and NR2A subunits are upregulated. At DIV22, C2-specific clusters are abundant and are predominantly localized at postsynaptic sites, whereas the total number of C2'-clusters in dendrites is much lower and these clusters are localized mostly extrasynaptically. However, upon chronic inhibition of NMDA receptor activity in DIV8 and DIV22 cultures with MK801, the number of postsynaptic NR1-C2' subunit clusters is strongly upregulated. In contrast, numbers of NR1-C2 clusters are only modestly increased in DIV8 and not changed in DIV22 cultures upon MK801 treatment, suggesting a specific role of NR1 carboxy-terminal sequences in the activity-dependent synaptic targeting of NMDA receptor clusters of spinal cord neurons.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Receptores de N-Metil-D-Aspartato/genética , Sinapses/fisiologia , Animais , Desenvolvimento Embrionário , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/fisiologia , Medula Espinal/embriologia , Medula Espinal/fisiologiaRESUMO
The gene encoding the beta subunit of the inhibitory glycine receptor (GlyR) is widely expressed throughout the mammalian central nervous system. To unravel the elements regulating its transcription, we isolated its 5' non-coding and upstream flanking regions from mouse. Sequence analysis revealed significant differences between the 5' region of the beta subunit gene and the corresponding regions of the homologous GlyR alpha subunit genes; it also identified a novel exon (exon 0) that encodes most of the 5'-untranslated portion of the GlyR beta mRNA. Primer extension experiments disclosed multiple transcriptional start sites. Transfection experiments with luciferase reporter gene constructs showed that sequences encompassing 1.58 kb of upstream flanking region and 180 bp of exon 0 displayed high promoter activity in two neuroblastoma cell lines but not in non-neural cells. Analysis of various deletion constructs showed that the 5' flanking region preceding the transcriptional start sites silences expression in non-neural cells but is not essential for general promoter activity. In contrast, the deletion of sequences within exon 0 drastically decreased or abolished transcription; the removal of sequences harbouring Sp1 consensus sequences within exon 0 decreased expression specifically in a neuroblastoma cell line. Band-shift assays confirmed the binding of Sp1 to sites within the deleted sequence. Our results indicate that neural-specific expression of the GlyR beta subunit gene might depend on a direct interaction of Sp1 transcription factors with cis elements located downstream from transcription initiation sites.
Assuntos
Éxons , Neurônios/metabolismo , Receptores de Glicina/genética , Fator de Transcrição Sp1/metabolismo , Células 3T3 , Animais , Sequência de Bases , Hibridização In Situ , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Mensageiro/genéticaRESUMO
Startle disease or hereditary hyperekplexia has been shown to result from mutations in the alpha1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR alpha or beta-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR beta transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR beta-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR beta transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at approximately 3-4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease.
Assuntos
Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Reflexo de Sobressalto/genética , Medula Espinal/metabolismo , Tremor/genética , Animais , Membrana Celular/metabolismo , Cruzamentos Genéticos , Feminino , Genótipo , Homozigoto , Humanos , Hipercinese/genética , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , RNA Mensageiro/genética , Ratos , Transcrição Gênica , Tremor/fisiopatologiaRESUMO
1. The effects of Zn2+ on glycine receptor (GlyR) currents were analysed in Xenopus oocytes and human embryonic kidney cells expressing homomeric human wild-type and mutant alpha1 subunit GlyRs. 2. Low concentrations (10 microM) of extracellular Zn2+ converted the partial agonist taurine into a high-efficacy agonist. Concentration-response analysis showed that the EC50 for taurine decreased whereas the Hill coefficient increased under these conditions. In contrast, 50-500 microM Zn2+ showed an increased EC50 value and reduced maximal inducible taurine currents. The potency of competitive antagonists was not affected in the presence of Zn2+. 3. Single-channel recording from outside-out patches revealed different kinetics of glycine- and taurine-gated currents. With both agonists, Zn2+ altered the open probability of the alpha1 GlyR without changing its unitary conductance. Low Zn2+ concentrations (5 microM) increased both the opening frequency and mean burst duration, whereas higher Zn2+ concentrations (> 50 microM) reduced GlyR open probability mainly by decreasing the open frequency and the relative contribution of the longest burst of the single-channel events. 4. Site-directed mutagenesis of the GlyR alpha1 subunit identified aspartate 80 and threonine 112 as important determinants of Zn2+ potentiation and inhibition, respectively, without affecting potentiation by ethanol. 5. Our data support the view that Zn2+ modulates different steps of the receptor binding and gating cycle via specific allosteric high- and low-affinity binding sites in the extracellular N-terminal region of the GlyR alpha1 subunit.
Assuntos
Receptores de Glicina/efeitos dos fármacos , Receptores de Glicina/genética , Zinco/farmacologia , Algoritmos , Animais , Linhagem Celular , Análise Mutacional de DNA , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Rim/metabolismo , Cinética , Modelos Biológicos , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , RNA Complementar/genética , Receptores de Glicina/agonistas , Receptores de Glicina/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Taurina/farmacologia , Transfecção , Xenopus laevisRESUMO
The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous alpha and beta subunits. Single expression of alpha subunits generates functional homo-oligomeric GlyRs, whereas the beta subunit requires a co-expressed alpha subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (alpha(3)beta(2)). Here, we identified eight amino acid residues within the N-terminal region of the alpha1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the alpha1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR alpha and beta subunits.
Assuntos
Receptores de Glicina/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Humanos , Microscopia Confocal , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA Complementar/farmacologia , Transfecção , Xenopus/embriologiaRESUMO
Different amino side chains in the N-terminal extracellular region of the inhibitory glycine receptor (GlyR) have been shown to be crucial for ligand recognition. Here we describe a novel domain of the GlyRalpha1 subunit that constitutes an important determinant of antagonist activity. The antagonists strychnine, nipecotic acid, and isobutyric acid displayed reduced potencies at recombinant GlyRs formed from alpha1 subunits, in which lysine 104, phenylalanine 108, or threonine 112 were replaced by alanine. Agonist affinities, in contrast, were slightly increased at these mutant receptors. Taurine and beta-aminoisobutyric acid, which are partial agonists at the wild-type GlyR, behaved as full agonists at the mutant GlyRs and failed to inhibit glycine-induced currents. This is consistent with apolar residues at positions 104, 108, and 112 of the alpha1 subunit reducing the antagonistic, but not the agonistic, binding of beta-amino acids. Our data support a model in which the partial agonism of beta-amino acids results from their self-inhibitory activity.
Assuntos
Receptores de Glicina/agonistas , Receptores de Glicina/antagonistas & inibidores , Substituição de Aminoácidos , Aminobutiratos/farmacologia , Ácidos Aminoisobutíricos/farmacologia , Animais , Sítios de Ligação , Humanos , Modelos Biológicos , Mutagênese , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Conformação Proteica , Receptores de Glicina/química , Receptores de Glicina/genética , Xenopus laevis , Ácido gama-Aminobutírico/farmacologiaRESUMO
The strychnine-sensitive glycine receptor (GlyR) is a pentameric chloride channel protein that exists in several developmentally and regionally regulated isoforms in the CNS. These result from the differential expression of four genes encoding different variants (alpha 1-alpha 4) of the ligand-binding subunit of the GlyR. Their assembly with the structural beta subunit is governed by "assembly cassettes" within the extracellular domains of these proteins and creates chloride channels of distinct conductance properties. GlyR gating is potentiated by Zn2+, a metal ion co-released with different neurotransmitters. Site-directed mutagenesis has unraveled major determinants of agonist binding and Zn2+ potentiation. During development, glycine receptors mediate excitation that results in Ca2+ influx and neurotransmitter release. Ca2+ influx triggered by the activation of embryonic GlyRs is required for the synaptic localization of the GlyR and its anchoring protein gepyhrin. In the adult, mutations in GlyR-subunit genes result in motor disorders. The spastic and spasmodic phenotypes in mouse as well as human hereditary startle disease will be discussed.
Assuntos
Receptores de Glicina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Canais de Cloreto/metabolismo , Modelos Animais de Doenças , Ligação Genética , Humanos , Proteínas de Membrana/metabolismo , Mutação , Receptores de Glicina/genética , Reflexo de Sobressalto/genética , Estricnina/farmacologia , Transmissão Sináptica , Zinco/metabolismoRESUMO
The peripheral membrane protein gephyrin is essential for the postsynaptic localization of inhibitory glycine receptors (GlyRs). Binding of gephyrin to the GlyR beta subunit is mediated by a sequence motif located in the intracellular loop region connecting transmembrane segments 3 and 4. Here, insertion of this binding motif is shown to alter the subcellular distribution of an excitatory neurotransmitter receptor in transfected mammalian cells. Upon coexpression with gephyrin, a mutant N-methyl-D-aspartate (NMDA) receptor containing NMDA receptor 1 (NR1) subunits which harboured a gephyrin-binding motif within its cytoplasmic tail region, was targeted to intracellular gephyrin-rich domains, as previously observed for the GlyR beta subunit. Our data indicate that a gephyrin-binding motif located in a cytoplasmic domain of an integral membrane protein suffices for routing to gephyrin-rich domains.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , Proteínas de Transporte/química , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , DNA Complementar , Imunofluorescência , Humanos , Rim/citologia , Proteínas de Membrana/química , Mutagênese Sítio-Dirigida , Oócitos/fisiologia , Técnicas de Patch-Clamp , Mutação Puntual , Estrutura Terciária de Proteína , Receptores de N-Metil-D-Aspartato/químicaRESUMO
The cytoplasmic C-terminal domains (CTs) of the NR1 and NR2 subunits of the NMDA receptor have been implicated in its anchoring to the subsynaptic cytoskeleton. Here, we used affinity chromatography with glutathione S-transferase-NR1-CT and -NR2B-CT fusion proteins to identify novel binding partner(s) of these NMDA receptor subunits. Upon incubation with rat brain cytosolic protein fraction, both NR1-CT and NR2B-CT, but not glutathione S-transferase, specifically bound tubulin. The respective fusion proteins also bound tubulin purified from brain, suggesting a direct interaction between the two binding partners. In tubulin polymerization assays, NR1-CT and NR2B-CT significantly decreased the rate of microtubule formation without destabilizing preformed microtubules. Moreover, only minor fractions of either fusion protein coprecipitated with the newly formed microtubules. Consistent with these findings, ultrastructural analysis of the newly formed microtubules revealed a limited association only with the CTs of the NR1 and NR2B. These data suggest a direct interaction of the NMDA receptor channel subunit CTs and tubulin dimers or soluble forms of tubulin. The efficient modulation of microtubule dynamics by the NR1 and NR2 cytoplasmic domains suggests a functional interaction of the receptor and the subsynaptic cytoskeletal network that may play a role during morphological adaptations, as observed during synaptogenesis and in adult CNS plasticity.
Assuntos
Glutationa Transferase/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Cromatografia de Afinidade , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Glutationa Transferase/genética , Técnicas In Vitro , Microscopia Eletrônica , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Ratos , Receptores de N-Metil-D-Aspartato/genética , Solubilidade , Tubulina (Proteína)/biossínteseRESUMO
Glycine receptors are anchored at inhibitory chemical synapses by a cytoplasmic protein, gephyrin. Molecular cloning revealed the similarity of gephyrin to prokaryotic and invertebrate proteins essential for synthesizing a cofactor required for activity of molybdoenzymes. Gene targeting in mice showed that gephyrin is required both for synaptic clustering of glycine receptors in spinal cord and for molybdoenzyme activity in nonneural tissues. The mutant phenotype resembled that of humans with hereditary molybdenum cofactor deficiency and hyperekplexia (a failure of inhibitory neurotransmission), suggesting that gephyrin function may be impaired in both diseases.
Assuntos
Proteínas de Transporte/fisiologia , Coenzimas , Proteínas de Membrana/fisiologia , Metaloproteínas/metabolismo , Molibdênio/metabolismo , Pteridinas/metabolismo , Agregação de Receptores , Receptores de Glicina/fisiologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/fisiologia , Quimera , Marcação de Genes , Glicina/fisiologia , Humanos , Camundongos , Cofatores de Molibdênio , Neurônios Motores/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Fenótipo , Medula Espinal/citologia , Medula Espinal/fisiologia , Células-Tronco , Transmissão Sináptica , Xantina Desidrogenase/metabolismoRESUMO
Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor, a subtype of the ionotropic glutamate receptor family. The glycine binding site of this hetero-oligomeric ion channel protein is formed by two distinct extracellular regions, S1 and S2, of the NR1 subunit, whereas the homologous domains of the NR2 subunit mediate glutamate binding. Here, segments S1 and S2 of the NR1 polypeptide were fused via a linker peptide followed by N- and C-terminally tagging with Flag and His6 epitopes, respectively. Infection of High Five insect cells with a recombinant baculovirus containing this glycine binding site construct resulted in efficient secretion of a soluble fusion protein of about 53 kDa. After affinity purification to near-homogeneity, the fusion protein bound the competitive glycine site antagonist [3H]MDL105,519 with high affinity (Kd = 5.22 +/- 0. 13 nM) similar to that determined with rat brain membrane fractions. This high affinity binding could be competed by the glycine site antagonist 7-chlorokynurenic acid as well as the agonists glycine and D-serine but not by L-glutamate. This indicates that the S1 and S2 domains of the NR1 subunit are sufficient for the formation of a glycine binding site that displays pharmacological properties similar to those of the NMDA receptor in vivo.
Assuntos
Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/química , Animais , Baculoviridae/genética , Sítios de Ligação/fisiologia , Ligação Competitiva/fisiologia , Antagonistas de Aminoácidos Excitatórios/metabolismo , Ácido Glutâmico/metabolismo , Indóis/farmacologia , Ácido Cinurênico/análogos & derivados , Ácido Cinurênico/metabolismo , Ligação Proteica/fisiologia , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Spodoptera/genéticaRESUMO
The binding site for the co-agonist glycine on N-methyl-D-aspartate (NMDA) receptors has been mapped to the NR1 subunit whereas binding of the principal agonist glutamate is mediated by the NR2 subunits. Using the novel glycine site antagonist and photoaffinity label CGP 61594, distinct contributions of the NR2 subunit variants to the glycine antagonist binding domains of NMDA receptor subtypes are demonstrated. High affinity sites for CGP 61594 were exclusively displayed by NR1/2B receptors, as shown by their co-distribution with the NR2B subunit, by subunit-selective immunoprecipitation and by functional analysis of NR1/2B receptors expressed in Xenopus oocytes (inhibitory potency, IC50 = 45 +/- 11 nM). Other NMDA receptor subtypes are clearly distinguished by reduced inhibitory potencies for CGP 61594, being low for NR1/2A and NR1/2D receptors (IC50 = 430 +/- 105 nM and 340 +/- 61 nM, respectively) and intermediate for NR1/2C receptors (IC50 = 164 +/- 27 nM). Glycine antagonist sites with low and intermediate affinity for [3H]CGP 61594 were detected also in situ by radioligand binding in brain areas predominantly expressing the NR2A and NR2C subunits, respectively. Thus, [3H]CGP 61594 is the first antagonist radioligand that reliably distinguishes the glycine site of NMDA receptor subtypes. [3H]CGP 61594 is a promising tool to identify the NR2 subunit domains that contribute to differential glycine antagonist sites of NMDA receptor subtypes.
Assuntos
Azidas/metabolismo , Glicina/metabolismo , Quinolinas/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Regulação Alostérica , Azidas/farmacologia , Sítios de Ligação , Linhagem Celular , Antagonistas de Aminoácidos Excitatórios/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Testes de Precipitina , Quinolinas/farmacologia , Receptores de N-Metil-D-Aspartato/classificação , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes/metabolismo , TrítioRESUMO
The amino acids L-glutamate and glycine are essential agonists of the excitatory NMDA receptor, a subtype of the ionotropic glutamate receptor family. The native NMDA receptor is composed of two types of homologous membrane-spanning subunits, NR1 and NR2. Here, the numbers of glycine-binding NR1 and glutamate-binding NR2 subunits in the NMDA receptor hetero-oligomer were determined by coexpressing the wild-type (wt) NR1 with the low-affinity mutant NR1(Q387K), and the wt NR2B with the low-affinity mutant NR2BE387A, subunits in Xenopus oocytes. In both cases, analysis of the resulting dose-response curves revealed three independent components of glycine and glutamate sensitivity. These correspond to the respective wild-type and mutant affinities and an additional intermediate hybrid affinity, indicating the existence of three discrete receptor populations. Binomial analysis of these data indicates the presence of two glycine and two glutamate binding subunits in the functional receptor. In addition, we analyzed the inhibitory effects of the negative dominant NR1(R505K) and NR2BR493K mutants on maximal inducible whole-cell currents of wt NR1/NR2B receptors. The inhibition profiles obtained on expression of increasing amounts of these mutant proteins again were fitted best by assuming an incorporation of two NR1 and two NR2 subunits into the receptor hetero-oligomer. Our data are consistent with NMDA receptors being tetrameric proteins that are composed of four homologous subunits.
Assuntos
Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Animais , Relação Dose-Resposta a Droga , Eletrofisiologia , Expressão Gênica , Ácido Glutâmico/farmacologia , Glicina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Microinjeções , Mutagênese/fisiologia , Oócitos/fisiologia , Conformação Proteica , RNA Complementar/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , XenopusRESUMO
NMDA receptors require both L-glutamate and the coagonist glycine for efficient channel activation. The glycine binding site of these heteromeric receptor proteins is formed by regions of the NMDAR1 (NR1) subunit that display sequence similarity to bacterial amino acid binding proteins. Here, we demonstrate that the glutamate binding site is located on the homologous regions of the NR2B subunit. Mutation of residues within the N-terminal domain and the loop region between membrane segments M3 and M4 significantly reduced the efficacy of glutamate, but not glycine, in channel gating. Some of the mutations also decreased inhibition by the glutamate antagonists, D-AP5 and R-CPP. Homology-based molecular modeling of the glutamate and glycine binding domains indicates that the NR2 and NR1 subunits use similar residues to ligate the agonists' alpha-aminocarboxylic acid groups, whereas differences in side chain interactions and size of aromatic residues determine ligand selectivity.
Assuntos
Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/agonistas , 2-Amino-5-fosfonovalerato/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Ácido Glutâmico/farmacologia , Glicina/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Piperazinas/farmacologia , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits. Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis. Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity. These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins. Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly. These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.
Assuntos
Proteínas de Bactérias , Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Epitopos/química , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Cinurênico/análogos & derivados , Ácido Cinurênico/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligopeptídeos/genética , Conformação Proteica , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Mutations in inhibitory glycine receptor (GlyR) subunit genes are associated with neuromotor diseases in man and mouse. To use the potential of the mouse mutants as animal models of human disease, we altered GlyR levels in mutant mice and studied their phenotype. A transgene coding for the beta subunit of the rat GlyR was introduced into the genetic background of the spa mutation, which is characterized by low endogenous expression levels of the beta subunit and a dramatic neuromotor phenotype. The resulting transgenic mice expressed the beta subunit mRNA at intermediate levels, and their phenotype was rescued. This provides formal proof for the casual relationship between GlyR beta gene mutation and motor disease, and indicates that a low level of beta gene expression (25% of normal) is sufficient for proper functioning of glycinergic synapses.
Assuntos
Modelos Animais de Doenças , Doenças Neuromusculares/genética , Receptores de Glicina/biossíntese , Animais , Sequência de Bases , Encéfalo/anatomia & histologia , Química Encefálica , Glicinérgicos/metabolismo , Hibridização In Situ , Membranas/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Doenças Neuromusculares/metabolismo , Linhagem , Fenótipo , RNA Mensageiro/biossíntese , Receptores de Glicina/genética , Medula Espinal/metabolismo , Estricnina/metabolismoRESUMO
In adult spinal neurons inhibitory glycine receptors (GlyR) are localized at postsynaptic membrane specializations underlying glycinergic nerve terminals. The peripheral membrane protein gephyrin has been shown to be essential for the formation of postsynaptic GlyR clusters. Here, we coexpressed GlyR polypeptides and gephyrin in 293 cells and observed rerouting of hetero-oligomeric GlyR and its beta, but not of alpha subunits to intracellular gephyrin aggregates. A GlyR chimeric alpha 1/beta protein was also accumulated at these gephyrin aggregates, indicating that colocalization with gephyrin depends on cytoplasmic domains of the beta subunit. gamma-Aminobutyric acid type-A receptor (GABAAR) subunits were not targeted to intracellular gephyrin aggregates with the exception of the GABAAR beta 3 subunit, which partially colocalized with gephyrin. These data show that gephyrin alters the subcellular localization of the GlyR beta and, to some extent, GABAAR beta 3 subunits. Thus, gephyrin-binding subunits might target hetero-oligomeric ion channels to a gephyrin matrix underlying the differentiating postsynaptic membrane.
Assuntos
Proteínas de Transporte/metabolismo , Rim/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Glicina/classificação , Receptores de Glicina/imunologia , Animais , Células Cultivadas , Humanos , Imuno-Histoquímica , Rim/embriologia , Ratos , Receptores de GABA-A/metabolismoRESUMO
Significant progress has been made towards the identification of functional domains of the inhibitory glycine receptor. Several residues crucial for ligand binding, ion-channel properties and stoichiometric subunit assembly have been identified. A major recent advance has been the finding that the biogenesis of postsynaptic glycine receptor clusters requires the tubulin-binding protein, gephyrin. Another area of exciting research has focused on mutations of glycine receptor alpha and beta subunit genes, which have been found to be causal for different hereditary motor disorders.
Assuntos
Canais Iônicos/química , Receptores de Glicina/química , Receptores de Glicina/genética , Receptores de Glicina/fisiologia , Animais , Modelos Biológicos , Estrutura Molecular , Terminações Pré-Sinápticas/fisiologiaRESUMO
1. The effect of the divalent cation Zn2+ on inhibitory glycine receptor (GlyR) currents was investigated in rat embryonic spinal cord neurons and Xenopus oocytes expressing recombinant GlyRs. 2. In cultured spinal neurons, Zn2+ potentiated glycine-induced whole-cell currents about 3-fold when applied extracellularly at concentrations of 0.5-10 microM. In contrast, higher concentrations (> 100 microM) of Zn2+ decreased the glycine response. 3. A similar biphasic modulation of glycine-induced currents by Zn2+ was also found with recombinant homo- and hetero-oligomeric GlyRs generated in Xenopus oocytes. Dose-response analysis showed that both the potentiating and inhibitory effects of Zn2+ result from changes in apparent agonist affinity. 4. Analysis of chimeric constructs of the GlyR alpha 1- and beta-subunits revealed that the positive and negative modulatory effects of Zn2+ are mediated by different regions of the alpha 1-subunit. 5. Our data indicate the existence of distinct high- and low-affinity Zn2+ binding sites on the ligand-binding alpha-subunits of the GlyR. These sites may be implicated in the regulation of synaptic efficacy within glycinergic pathways.
Assuntos
Receptores de Glicina/efeitos dos fármacos , Zinco/farmacologia , Sequência de Aminoácidos , Animais , Quimera , Condutividade Elétrica , Eletrofisiologia , Espaço Extracelular/metabolismo , Glicina/metabolismo , Glicina/farmacologia , Humanos , Íons , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Oócitos/metabolismo , Ratos , Ratos Wistar , Receptores de Glicina/genética , Receptores de Glicina/fisiologia , Proteínas Recombinantes , Medula Espinal/citologia , Xenopus , Zinco/metabolismoRESUMO
Mice homozygous for the spastic mutation (spa) suffer from a complex motor disorder resulting from reduced CNS levels of the adult glycine receptor isoform GlyRA, which is composed of ligand-binding alpha 1 and structural beta polypeptides. The beta subunit-encoding gene (Glyrb) was mapped near the spa locus on mouse chromosome 3. In spa/spa mice, aberrant splicing of the beta subunit pre-mRNA strikingly diminishes the CNS contents of full-length transcripts, whereas truncated beta subunit mRNAs accumulate. This is a result of exon skipping, which causes translational frameshifts and premature stop codons. Intron 5 of the spa Glyrb gene contains an L1 transposable element that apparently is causal for the aberrant splicing of beta subunit transcripts.