RESUMO
Calmodulin-dependent kinase II (CaMKII) is key for long-term potentiation of synaptic AMPA receptors. Whether CaMKII is involved in activity-dependent plasticity of other ionotropic glutamate receptors is unknown. We show that repeated pairing of pre- and postsynaptic stimulation at hippocampal mossy fibre synapses induces long-term depression of kainate receptor (KAR)-mediated responses, which depends on Ca(2+) influx, activation of CaMKII, and on the GluK5 subunit of KARs. CaMKII phosphorylation of three residues in the C-terminal domain of GluK5 subunit markedly increases lateral mobility of KARs, possibly by decreasing the binding of GluK5 to PSD-95. CaMKII activation also promotes surface expression of KARs at extrasynaptic sites, but concomitantly decreases its synaptic content. Using a molecular replacement strategy, we demonstrate that the direct phosphorylation of GluK5 by CaMKII is necessary for KAR-LTD. We propose that CaMKII-dependent phosphorylation of GluK5 is responsible for synaptic depression by untrapping of KARs from the PSD and increased diffusion away from synaptic sites.
Assuntos
Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Fibras Musgosas Hipocampais/metabolismo , Receptores de Ácido Caínico/metabolismo , Sinapses/metabolismo , Animais , Células COS , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Chlorocebus aethiops , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Receptores de Ácido Caínico/genética , Sinapses/genéticaRESUMO
Changes in the number of synaptic AMPA receptors underlie many forms of synaptic plasticity. These variations are controlled by an interplay between their intracellular transport (IT), export to the plasma membrane (PM), stabilization at synapses, and recycling. The cytosolic C-terminal domain of the AMPAR GluA1 subunit is specifically associated with 4.1 N and SAP97. We analyze how interactions between GluA1 and 4.1N or SAP97 regulate IT and exocytosis in basal conditions and after cLTP induction. The down-regulation of 4.1N or SAP97 decreases GluA1 IT properties and export to the PM. The total deletion of its C-terminal fully suppresses its IT. Our results demonstrate that during basal transmission, the binding of 4.1N to GluA1 allows their exocytosis whereas the interaction with SAP97 is essential for GluA1 IT. During cLTP, the interaction of 4.1N with GluA1 allows its IT and exocytosis. Our results identify the differential roles of 4.1N and SAP97 in the control of various phases of GluA1 IT.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Receptores de AMPA , Receptores de AMPA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sinapses/fisiologia , Plasticidade Neuronal/fisiologia , Membrana Celular/metabolismo , Hipocampo/metabolismoRESUMO
Intracellular trafficking of AMPA receptors is a tightly regulated process which involves several adaptor proteins, and is crucial for the activity of excitatory synapses both in basal conditions and during synaptic plasticity. We found that, in rat hippocampal neurons, an intracellular pool of the tetraspanin TSPAN5 promotes exocytosis of AMPA receptors without affecting their internalisation. TSPAN5 mediates this function by interacting with the adaptor protein complex AP4 and Stargazin and possibly using recycling endosomes as a delivery route. This work highlights TSPAN5 as a new adaptor regulating AMPA receptor trafficking.
Assuntos
Receptores de AMPA , Sinapses , Tetraspaninas , Animais , Ratos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Exocitose , Hipocampo/metabolismo , Plasticidade Neuronal/fisiologia , Transporte Proteico/fisiologia , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/fisiologia , Tetraspaninas/genéticaRESUMO
Kainate receptors (KARs) are widely expressed in the brain and are present at both presynaptic and postsynaptic sites. GluK3-containing KARs are thought to compose presynaptic autoreceptors that facilitate hippocampal mossy fiber synaptic transmission. Here we identify molecular mechanisms that underlie the polarized trafficking of KARs composed of the GluK3b splice variant. Endocytosis followed by degradation is driven by a dileucine motif on the cytoplasmic C-terminal domain of GluK3b in heterologous cells, in cultured hippocampal neurons, and in dentate granule cells from organotypic slice cultures. The internalization of GluK3b is clathrin and dynamin2 dependent. GluK3b is differentially endocytosed in dendrites as compared to the axons. These data suggest that the polarized trafficking of KARs in neurons could be controlled by the regulation of receptor endocytosis.
Assuntos
Polaridade Celular/genética , Endocitose/genética , Subunidades Proteicas/metabolismo , Receptores de Ácido Caínico/metabolismo , Animais , Animais Recém-Nascidos , Células COS , Células Cultivadas , Chlorocebus aethiops , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Subunidades Proteicas/fisiologia , Transporte Proteico/genética , Splicing de RNA/genética , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/fisiologia , Receptor de GluK3 CainatoRESUMO
The trafficking of ionotropic glutamate receptors to and from synaptic sites is regulated by proteins that interact with their cytoplasmic C-terminal domain. Profilin IIa (PfnIIa), an actin-binding protein expressed in the brain and recruited to synapses in an activity-dependent manner, was shown previously to interact with the C-terminal domain of the GluK2b subunit splice variant of kainate receptors (KARs). Here, we characterize this interaction and examine the role of PfnIIa in the regulation of KAR trafficking. PfnIIa directly and specifically binds to the C-terminal domain of GluK2b through a diproline motif. Expression of PfnIIa in transfected COS-7 cells and in cultured hippocampal neurons from PfnII-deficient mice decreases the level of extracellular of homomeric GluK2b as well as heteromeric GluK2a/GluK2b KARs. Our data suggest a novel mechanism by which PfnIIa exerts a dual role on the trafficking of KARs, by a generic inhibition of clathrin-mediated endocytosis through its interaction with dynamin-1, and by controlling KARs exocytosis through a direct and specific interaction with GluK2b.
Assuntos
Exocitose/fisiologia , Hipocampo/metabolismo , Neurônios/metabolismo , Profilinas/metabolismo , Receptores de Ácido Caínico/metabolismo , Sinapses/metabolismo , Animais , Células COS , Chlorocebus aethiops , Clatrina/genética , Clatrina/metabolismo , Dinamina I/genética , Dinamina I/metabolismo , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Profilinas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Receptores de Ácido Caínico/genética , Sinapses/genética , Receptor de GluK2 CainatoRESUMO
The properties of synaptic receptors determine their mode of action at presynaptic and postsynaptic loci. Here, we investigated the atypical biophysical properties of GluK3-containing kainate receptors, which contribute to presynaptic facilitation at hippocampal mossy fiber synapses. We show, using fast glutamate applications on outside-out patches and kinetic modeling, that the low sensitivity of GluK3 receptors for glutamate is attributable to fast desensitization of partially bound receptors. Consequently, these receptors can only be activated by fast transients of high glutamate concentration. In addition, GluK3 receptors are very sensitive to voltage-dependent block by intracellular spermine that precludes activation of substantial currents at potentials positive to -50 mV. Two specific residues within the channel pore define this high-affinity site. Finally, GluK3 are calcium permeable in the same way as unedited GluK2 receptors. These receptors present unique properties among AMPA/kainate receptors that could reflect a specialized presynaptic function.
Assuntos
Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Linhagem Celular , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Hipocampo/química , Hipocampo/fisiologia , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Cinética , Potenciais da Membrana/fisiologia , Modelos Neurológicos , Dados de Sequência Molecular , Mutação , Neurônios/química , Neurônios/fisiologia , Técnicas de Patch-Clamp , Receptores de Ácido Caínico/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espermina/química , Espermina/metabolismo , Receptor de GluK2 Cainato , Receptor de GluK3 CainatoRESUMO
Kainate receptors (KAR) are composed of several distinct subunits and splice variants, but the functional relevance of this diversity remains largely unclear. Here we show that two splice variants of the GluR6 subunit, GluR6a and GluR6b, which differ in their C-terminal domains, do not show distinct functional properties, but coassemble as heteromers in vitro and in vivo. Using a proteomic approach combining affinity purification and MALDI-TOF mass spectrometry, we found that GluR6a and GluR6b interact with two distinct subsets of cytosolic proteins mainly involved in Ca(2+) regulation of channel function and intracellular trafficking. Guided by these results, we provide evidence that the regulation of native KAR function by NMDA receptors depends on the heteromerization of GluR6a and GluR6b and interaction of calcineurin with GluR6b. Thus, GluR6a and GluR6b bring in close proximity two separate subsets of interacting proteins that contribute to the fine regulation of KAR trafficking and function.
Assuntos
Processamento Alternativo/genética , Sistema Nervoso Central/metabolismo , Receptores de Ácido Caínico/metabolismo , Sinapses/metabolismo , Animais , Animais Recém-Nascidos , Células COS , Calcineurina/metabolismo , Células Cultivadas , Chlorocebus aethiops , Citosol/metabolismo , Humanos , Canais Iônicos/metabolismo , Substâncias Macromoleculares , Espectrometria de Massas , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/genética , Receptor de GluK2 CainatoRESUMO
We identified four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, as interactors with the kainate receptor (KAR) subunits GluR5(2b,) GluR5(2c), and GluR6. Of these, we show that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKC alpha can phosphorylate ct-GluR5(2b) at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. We propose that PICK1 targets PKC alpha to phosphorylate KARs, causing their stabilization at the synapse by an interaction with GRIP. Importantly, this mechanism is not involved in the constitutive recycling of AMPA receptors since blockade of PDZ interactions can simultaneously increase AMPAR- and decrease KAR-mediated synaptic transmission at the same population of synapses.
Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Fibras Musgosas Hipocampais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Química Encefálica , Proteínas do Citoesqueleto , Proteína 4 Homóloga a Disks-Large , Potenciais Pós-Sinápticos Excitadores/fisiologia , Guanilato Quinases , Hipocampo/citologia , Hipocampo/metabolismo , Técnicas In Vitro , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Fosforilação , Ligação Proteica/fisiologia , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Subunidades Proteicas/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sinteninas , Técnicas do Sistema de Duplo-HíbridoRESUMO
Using single-molecule microscopy, we present a method to quantify the number of single autofluorescent proteins when they cannot be optically resolved. This method relies on the measurement of the total intensity emitted by each aggregate until it photobleaches. This strategy overcomes the inherent problem of blinking of green fluorescent proteins. In the case of small protein aggregates, our method permits us to describe the mean composition with a precision of one protein. For aggregates containing a large number of proteins, it gives access to the average number of proteins gathered and a signature of the inhomogeneity of the aggregates' population. We applied this methodology to the quantification of small purified citrine multimers.
Assuntos
Algoritmos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/química , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Técnicas de Sonda Molecular , Espectrometria de Fluorescência/métodos , FótonsRESUMO
Regulation of AMPA receptor (AMPAR) trafficking is a key modulator of excitatory synaptic transmission; however, intracellular vesicular transport of newly synthesized AMPARs has been little studied due to technical limitations. By combining molecular tools with imaging strategies in cultured rat hippocampal neurons, we found that vesicles containing newly synthesized, GluA1-subunit-containing AMPARs are transported antero- and retrogradely at a mean speed of 1.5 µm.s-1. Synaptic activity and variations in intracellular calcium levels bidirectionally modulate GluA1 transport. Chemical long-term potentiation (cLTP) initially induces a halt in GluA1 transport, followed by a sustained increase, while acute glutamate uncaging on synaptic spines arrests vesicular movements. GluA1 phosphomimetic mutants preferentially travel to the dendritic tip, probably to replenish extrasynaptic pools, distal to the soma. Our findings indicate that AMPAR intracellular transport is highly regulated during synaptic plasticity and likely controls AMPAR numbers at the plasma membrane.
Assuntos
Cálcio/metabolismo , Neurônios/metabolismo , Receptores de Glutamato/genética , Animais , Transporte Proteico , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
Impaired hippocampal synaptic plasticity contributes to cognitive impairment in Huntington's disease (HD). However, the molecular basis of such synaptic plasticity defects is not fully understood. Combining live-cell nanoparticle tracking and super-resolution imaging, we show that AMPAR surface diffusion, a key player in synaptic plasticity, is disturbed in various rodent models of HD. We demonstrate that defects in the brain-derived neurotrophic factor (BDNF)-tyrosine receptor kinase B (TrkB) signaling pathway contribute to the deregulated AMPAR trafficking by reducing the interaction between transmembrane AMPA receptor regulatory proteins (TARPs) and the PDZ-domain scaffold protein PSD95. The disturbed AMPAR surface diffusion is rescued by the antidepressant drug tianeptine via the BDNF signaling pathway. Tianeptine also restores the impaired LTP and hippocampus-dependent memory in different HD mouse models. These findings unravel a mechanism underlying hippocampal synaptic and memory dysfunction in HD, and highlight AMPAR surface diffusion as a promising therapeutic target.
Assuntos
Hipocampo/fisiopatologia , Doença de Huntington/fisiopatologia , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Receptores de AMPA/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Difusão , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Potenciação de Longa Duração/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptor trkB/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Tiazepinas/farmacologiaRESUMO
Alzheimer's disease (AD) is emerging as a synaptopathology driven by metaplasticity. Indeed, reminiscent of metaplasticity, oligomeric forms of the amyloid-ß peptide (oAß) prevent induction of long-term potentiation (LTP) via the prior activation of GluN2B-containing NMDA receptors (NMDARs). However, the downstream Ca2+-dependent signaling molecules that mediate aberrant metaplasticity are unknown. In this study, we show that oAß promotes the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) via GluN2B-containing NMDARs. Importantly, we find that CaMKII inhibition rescues both the LTP impairment and the dendritic spine loss mediated by oAß. Mechanistically resembling metaplasticity, oAß prevents subsequent rounds of plasticity from inducing CaMKII T286 autophosphorylation, as well as the associated anchoring and accumulation of synaptic AMPA receptors (AMPARs). Finally, prolonged oAß treatment-induced CaMKII misactivation leads to dendritic spine loss via the destabilization of surface AMPARs. Thus, our study demonstrates that oAß engages synaptic metaplasticity via aberrant CaMKII activation.
Assuntos
Peptídeos beta-Amiloides/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Fragmentos de Peptídeos/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Células Cultivadas , Espinhas Dendríticas/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/química , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Heteromeric kainate receptors (KARs) containing both glutamate receptor 6 (GluR6) and KA2 subunits are involved in KAR-mediated EPSCs at mossy fiber synapses in CA3 pyramidal cells. We report that endogenous glutamate, by activating KARs, reversibly inhibits the slow Ca2+-activated K+ current I(sAHP) and increases neuronal excitability through a G-protein-coupled mechanism. Using KAR knockout mice, we show that KA2 is essential for the inhibition of I(sAHP) in CA3 pyramidal cells by low nanomolar concentrations of kainate, in addition to GluR6. In GluR6(-/-) mice, both ionotropic synaptic transmission and inhibition of I(sAHP) by endogenous glutamate released from mossy fibers was lost. In contrast, inhibition of I(sAHP) was absent in KA2(-/-) mice despite the preservation of KAR-mediated EPSCs. These data indicate that the metabotropic action of KARs did not rely on the activation of a KAR-mediated inward current. Biochemical analysis of knock-out mice revealed that KA2 was required for the interaction of KARs with Galpha(q/11)-proteins known to be involved in I(sAHP) modulation. Finally, the ionotropic and metabotropic actions of KARs at mossy fiber synapses were differentially sensitive to the competitive glutamate receptor ligands kainate (5 nM) and kynurenate (1 mM). We propose a model in which KARs could operate in two modes at mossy fiber synapses: through a direct ionotropic action of GluR6, and through an indirect G-protein-coupled mechanism requiring the binding of glutamate to KA2.
Assuntos
Fibras Musgosas Hipocampais/fisiologia , Subunidades Proteicas/fisiologia , Receptores de Ácido Caínico/fisiologia , Sinapses/fisiologia , Animais , Ácido Caínico/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musgosas Hipocampais/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Receptores de Ácido Caínico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/fisiologia , Sinapses/efeitos dos fármacosRESUMO
Glutamate receptors of the kainate type have been identified recently as key players in the modulation of neuronal-network activity. The role of kainate receptors depends on their precise subcellular localization in presynaptic, postsynaptic and extrasynaptic domains. Subcellular localization of kainate receptors has been inferred mainly from electrophysiological studies with the help of selective pharmacological tools and kainate receptor mutant mice. These studies, combined with recent ultrastructural data, highlight the diversity of subcellular localizations of kainate receptors. It is important to understand the molecular mechanisms that underlie the polarized trafficking of kainate receptors in distinct neuronal domains. In this article, we review recent data that shed light on the trafficking and membrane delivery of kainate receptor isoforms, and on the identification of proteins that interact with kainate receptors and might regulate this trafficking.
Assuntos
Transporte Proteico/fisiologia , Receptores de Ácido Caínico/fisiologia , Receptores de Ácido Caínico/ultraestrutura , Frações Subcelulares/fisiologia , Frações Subcelulares/ultraestrutura , Animais , Eletrofisiologia/métodos , Modelos Moleculares , Isoformas de Proteínas , Receptores de Ácido Caínico/química , Frações Subcelulares/químicaRESUMO
Trafficking and biophysical properties of AMPA receptors (AMPARs) in the brain depend on interactions with associated proteins. We identify Shisa6, a single transmembrane protein, as a stable and directly interacting bona fide AMPAR auxiliary subunit. Shisa6 is enriched at hippocampal postsynaptic membranes and co-localizes with AMPARs. The Shisa6 C-terminus harbours a PDZ domain ligand that binds to PSD-95, constraining mobility of AMPARs in the plasma membrane and confining them to postsynaptic densities. Shisa6 expressed in HEK293 cells alters GluA1- and GluA2-mediated currents by prolonging decay times and decreasing the extent of AMPAR desensitization, while slowing the rate of recovery from desensitization. Using gene deletion, we show that Shisa6 increases rise and decay times of hippocampal CA1 miniature excitatory postsynaptic currents (mEPSCs). Shisa6-containing AMPARs show prominent sustained currents, indicating protection from full desensitization. Accordingly, Shisa6 prevents synaptically trapped AMPARs from depression at high-frequency synaptic transmission.
Assuntos
Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/fisiologia , Receptores de AMPA/metabolismo , Animais , Células Cultivadas , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Hipocampo/citologia , Humanos , Proteínas de Membrana/genética , Camundongos , Neurônios/citologia , Ratos , Receptores de AMPA/genética , Sinapses , Técnicas do Sistema de Duplo-HíbridoRESUMO
Kainate receptors (KARs) are heteromeric ionotropic glutamate receptors (GluRs) that play various roles in the regulation of synaptic transmission. The KAR subunits GluR5 and GluR6 exist under different splice variant isoforms in the C-terminal domain (GluR5a, GluR5b, GluR5c, GluR6a, GluR6b). The differential role of KAR subunit splice variants is presently unknown. In transfected COS-7 cells and neurons from wild-type and GluR5 x GluR6 mice, we have found that the subcellular localization and membrane delivery differed between these splice variants. GluR6a was highly expressed at the plasma membrane. GluR6b, GluR5a, and GluR5b were detected at lower levels in the plasma membrane and mainly colocalized with calreticulin in the endoplasmic reticulum (ER). GluR5c was strongly retained in the ER by an RXR motif. GluR6a acted as a key subunit splice variant promoting surface expression of ER-retained subunit splice variants when assembled in heteromeric KARs. Surface expression of GluR6a was independent of its PDZ (postsynaptic density-95/discs large/zona occludens-1) binding motif and was promoted by a stretch of four basic amino acid residues at its C terminus. Overall, splice variants and subunit composition of KARs regulate receptor trafficking from the endoplasmic reticulum to the plasma membrane.
Assuntos
Processamento Alternativo/genética , Membrana Celular/metabolismo , Receptores de Ácido Caínico/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neurônios/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico/fisiologia , RNA Mensageiro/biossíntese , Ratos , Receptores de Ácido Caínico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor de GluK2 CainatoRESUMO
Kainate receptors modulate synaptic transmission by acting either at presynaptic or at postsynaptic sites. The precise localization of kainate receptors as well as the mechanisms of targeting and stabilization of these receptors in neurons are largely unknown. We have generated transgenic mice expressing the kainate receptor subunit glutamate receptor 6 (GluR6) bearing an extracellular myc epitope (myc-GluR6), in forebrain neurons, in which it assembles with endogenous kainate receptor subunits. In transgenic mice crossed with GluR6-deficient mice, myc-GluR6 efficiently rescues the missing subunit. Immunoprecipitation of transgenic brain extracts with anti-myc antibodies demonstrates an interaction with cadherins, beta-catenin, and p120 catenin, as well as with the associated proteins calcium calmodulin-dependent serine kinase and Velis, but not with alpha-catenin. In glutathione S-transferase-pulldown experiments, beta-catenin interacts, although indirectly, with the last 14 aa of GluR6. Transfected myc-GluR6 colocalizes with beta-catenin at cell-cell junctions in non-neuronal cells. Finally, activation of N-cadherins by ligand-covered latex beads recruits GluR6 to cadherin/catenin complexes. These results suggest an important role for cadherin/catenin complexes in the stabilization of kainate receptors at the synaptic membrane during synapse formation and remodeling.
Assuntos
Caderinas/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Receptores de Ácido Caínico/metabolismo , Transativadores/metabolismo , Animais , Química Encefálica , Proteínas de Transporte/metabolismo , Cateninas , Células Cultivadas , Cruzamentos Genéticos , Feminino , Guanilato Quinases , Humanos , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Núcleosídeo-Fosfato Quinase/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica/fisiologia , Subunidades Proteicas , Proteínas Proto-Oncogênicas c-myc/genética , Células Piramidais/metabolismo , Ratos , Receptores de Glutamato/metabolismo , Receptores de Ácido Caínico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , beta Catenina , delta Catenina , Receptor de GluK2 CainatoRESUMO
Short-term plasticity of AMPAR currents during high-frequency stimulation depends not only on presynaptic transmitter release and postsynaptic AMPAR recovery from desensitization, but also on fast AMPAR diffusion. How AMPAR diffusion within the synapse regulates synaptic transmission on the millisecond scale remains mysterious. Using single-molecule tracking, we found that, upon glutamate binding, synaptic AMPAR diffuse faster. Using AMPAR stabilized in different conformational states by point mutations and pharmacology, we show that desensitized receptors bind less stargazin and are less stabilized at the synapse than receptors in opened or closed-resting states. AMPAR mobility-mediated regulation of short-term plasticity is abrogated when the glutamate-dependent loss in AMPAR-stargazin interaction is prevented. We propose that transition from the activated to the desensitized state leads to partial loss in AMPAR-stargazin interaction that increases AMPAR mobility and allows faster recovery from desensitization-mediated synaptic depression, without affecting the overall nano-organization of AMPAR in synapses.
Assuntos
Canais de Cálcio/metabolismo , Ácido Glutâmico/farmacologia , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Animais , Canais de Cálcio/genética , Células Cultivadas , Embrião de Mamíferos , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Potenciais Sinápticos/efeitos dos fármacos , Potenciais Sinápticos/genética , Transmissão Sináptica/fisiologiaRESUMO
PSD-95 is a prominent organizer of the postsynaptic density (PSD) that can present a filamentous orientation perpendicular to the plasma membrane. Interactions between PSD-95 and transmembrane proteins might be particularly sensitive to this orientation, as "long" cytoplasmic tails might be required to reach deeper PSD-95 domains. Extension/retraction of transmembrane protein C-tails offer a new way of regulating binding to PSD-95. Using stargazin as a model, we found that enhancing the apparent length of stargazin C-tail through phosphorylation or by an artificial linker was sufficient to potentiate binding to PSD-95, AMPAR anchoring, and synaptic transmission. A linear extension of stargazin C-tail facilitates binding to PSD-95 by preferentially engaging interaction with the farthest located PDZ domains regarding to the plasma membrane, which present a greater affinity for the stargazin PDZ-domain-binding motif. Our study reveals that the concerted orientation of the stargazin C-tail and PSD-95 is a major determinant of synaptic strength.