Glutamate-gated ion channels (ionotropic glutamate receptors, iGluRs) sense the extracellular milieu via an extensive extracellular portion, comprised of two clamshell-shaped segments. The distal, N-terminal domain (NTD) has allosteric potential in NMDA-type iGluRs, which has not been ascribed to the analogous domain in AMPA receptors (AMPARs). In this study, we present new structural data uncovering dynamic properties of the GluA2 and GluA3 AMPAR NTDs. GluA3 features a zipped-open dimer interface with unconstrained lower clamshell lobes, reminiscent of metabotropic GluRs (mGluRs). The resulting labile interface supports interprotomer rotations, which can be transmitted to downstream receptor segments. Normal mode analysis reveals two dominant mechanisms of AMPAR NTD motion: intraprotomer clamshell motions and interprotomer counter-rotations, as well as accessible interconversion between AMPAR and mGluR conformations. In addition, we detect electron density for a potential ligand in the GluA2 interlobe cleft, which may trigger lobe motions. Together, these data support a dynamic role for the AMPAR NTDs, which widens the allosteric landscape of the receptor and could provide a novel target for ligand development. 相似文献
Long-term potentiation (LTP) and long-term depression (LTD) of excitatory neurotransmission are believed to be the neuronal basis of learning and memory. Both processes are primarily mediated by neuronal activity–induced transport of postsynaptic AMPA-type glutamate receptors (AMPARs). While AMPAR subunits and their specific phosphorylation sites mediate differential AMPAR trafficking, LTP and LTD could also occur in a subunit-independent manner. Thus, it remains unclear whether and how certain AMPAR subunits with phosphorylation sites are preferentially recruited to or removed from synapses during LTP and LTD. Using immunoblot and immunocytochemical analysis, we show that phosphomimetic mutations of the membrane-proximal region (MPR) in GluA1 AMPAR subunits affect the subunit-dependent endosomal transport of AMPARs during chemical LTD. AP-2 and AP-3, adaptor protein complexes necessary for clathrin-mediated endocytosis and late endosomal/lysosomal trafficking, respectively, are reported to be recruited to AMPARs by binding to the AMPAR auxiliary subunit, stargazin (STG), in an AMPAR subunit–independent manner. However, the association of AP-3, but not AP-2, with STG was indirectly inhibited by the phosphomimetic mutation in the MPR of GluA1. Thus, although AMPARs containing the phosphomimetic mutation at the MPR of GluA1 were endocytosed by a chemical LTD-inducing stimulus, they were quickly recycled back to the cell surface in hippocampal neurons. These results could explain how the phosphorylation status of GluA1-MPR plays a dominant role in subunit-independent STG-mediated AMPAR trafficking during LTD. 相似文献
Scaffolding proteins are involved in the incorporation, anchoring, maintenance, and removal of AMPA receptors (AMPARs) at synapses, either through a direct interaction with AMPARs or via indirect association through auxiliary subunits of transmembrane AMPAR regulatory proteins (TARPs). Synaptic scaffolding molecule (S-SCAM) is a newly characterized member of the scaffolding proteins critical for the regulation and maintenance of AMPAR levels at synapses, and directly binds to TARPs through a PDZ interaction. However, the functional significance of S-SCAM–TARP interaction in the regulation of AMPARs has not been tested. Here we show that overexpression of the C-terminal peptide of TARP-γ2 fused to EGFP abolished the S-SCAM-mediated enhancement of surface GluA2 expression. Conversely, the deletion of the PDZ-5 domain of S-SCAM that binds TARPs greatly attenuated the S-SCAM-induced increase of surface GluA2 expression. In contrast, the deletion of the guanylate kinase domain of S-SCAM did not show a significant effect on the regulation of AMPARs. Together, these results suggest that S-SCAM is regulating AMPARs through TARPs. 相似文献
AMPA-type glutamate receptors (AMPARs) play a critical role in mediating fast excitatory synaptic transmission in the brain. Alterations in receptor expression, distribution, and trafficking have been shown to underlie synaptic plasticity and higher brain functions, including learning and memory, as well as brain dysfunctions such as drug addiction and psychological disorders. Therefore, it is essential to elucidate the molecular mechanisms that regulate AMPAR dynamics. We have shown previously that mammalian AMPARs are subject to posttranslational modification by ubiquitin, with AMPAR ubiquitination enhancing receptor internalization and reducing AMPAR cell surface expression. Here we report a crucial role for epidermal growth factor receptor substrate 15 (Eps15), an endocytic adaptor, in ubiquitination-dependent AMPAR internalization. We find that suppression or overexpression of Eps15 results in changes in AMPAR surface expression. Eps15 interacts with AMPARs, which requires Nedd4-mediated GluA1 ubiquitination and the ubiquitin-interacting motif of Eps15. Importantly, we find that Eps15 plays an important role in AMPAR internalization. Knockdown of Eps15 suppresses the internalization of GluA1 but not the mutant GluA1 that lacks ubiquitination sites, indicating a role of Eps15 for the internalization of ubiquitinated AMPARs. These results reveal a novel molecular mechanism employed specifically for the trafficking of the ubiquitin-modified AMPARs. 相似文献
AMPA-type glutamate receptors (AMPARs) play a major role in excitatory synaptic transmission and plasticity. Channel properties are largely dictated by their composition of the four subunits, GluR1-4 (or A-D). Here we show that AMPAR assembly and subunit stoichiometry are determined by RNA editing in the pore loop. We demonstrate that editing at the GluR2 Q/R site regulates AMPAR assembly at the step of tetramerization. Specifically, edited R subunits are largely unassembled and ER retained, whereas unedited Q subunits readily tetramerize and traffic to synapses. This assembly mechanism restricts the number of the functionally critical R subunits in AMPAR tetramers. Therefore, a single amino acid residue affects channel composition and, in turn, controls ion conduction through the majority of AMPARs in the brain. 相似文献
α-Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs) are the primary mediators of excitatory synaptic transmission in the brain. Alterations in AMPAR localization and turnover have been considered critical mechanisms underpinning synaptic plasticity and higher brain functions, but the molecular processes that control AMPAR trafficking and stability are still not fully understood. Here, we report that mammalian AMPARs are subject to ubiquitination in neurons and in transfected heterologous cells. Ubiquitination facilitates AMPAR endocytosis, leading to a reduction in AMPAR cell-surface localization and total receptor abundance. Mutation of lysine residues to arginine residues at the glutamate receptor subunit 1 (GluA1) C-terminus dramatically reduces GluA1 ubiquitination and abolishes ubiquitin-dependent GluA1 internalization and degradation, indicating that the lysine residues, particularly K868, are sites of ubiquitination. We also find that the E3 ligase neural precursor cell expressed, developmentally down-regulated 4 (Nedd4) is enriched in synaptosomes and co-localizes and associates with AMPARs in neurons. Nedd4 expression leads to AMPAR ubiquitination, leading to reduced AMPAR surface expression and suppressed excitatory synaptic transmission. Conversely, knockdown of Nedd4 by specific siRNAs abolishes AMPAR ubiquitination. These data indicate that Nedd4 is the E3 ubiquitin ligase responsible for AMPAR ubiquitination, a modification that regulates multiple aspects of AMPAR molecular biology including trafficking, localization and stability. 相似文献
Ion channel biogenesis involves an intricate interplay between subunit folding and assembly. Channel stoichiometries vary and give rise to diverse functions, which impacts on neuronal signalling. AMPA glutamate receptor (AMPAR) assembly is modulated by RNA processing. Here, we provide mechanistic insight into this process. First, we show that a single alternatively spliced residue within the ligand-binding domain alters AMPAR secretion from the ER. Local contacts differ between Leu758 of the GluR2-flop splice form as compared with the flip-specific Val758, which is transmitted globally to alter resensitization kinetics. Detailed biochemical and functional analysis of mutants suggest that AMPARs sample the gating cascade prior to ER export. Irreversibly locking the receptor within various states of the cascade severely attenuates ER transit. Alternative RNA processing by contrast, shifts equilibria between transition states reversibly and thereby modulates secretion kinetics. These data reveal how RNA processing tunes AMPAR biogenesis, and imply that gating transitions in the ER determine iGluR secretory traffic. 相似文献
The AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subfamily of iGluRs (ionotropic glutamate receptors) is essential for fast excitatory neurotransmission in the central nervous system. The malfunction of AMPARs (AMPA receptors) has been implicated in many neurological diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The active channels of AMPARs and other iGluR subfamilies are tetramers formed exclusively by assembly of subunits within the same subfamily. It has been proposed that the assembly process is controlled mainly by the extracellular ATD (N-terminal domain) of iGluR. In addition, ATD has also been implicated in synaptogenesis, iGluR trafficking and trans-synaptic signalling, through unknown mechanisms. We report in the present study a 2.5 ? (1 ?=0.1 nm) resolution crystal structure of the ATD of GluA1. Comparative analyses of the structure of GluA1-ATD and other subunits sheds light on our understanding of how ATD drives subfamily-specific assembly of AMPARs. In addition, analysis of the crystal lattice of GluA1-ATD suggests a novel mechanism by which the ATD might participate in inter-tetramer AMPAR clustering, as well as in trans-synaptic protein-protein interactions. 相似文献
A hallmark of ischemic/reperfusion injury is a change in subunit composition of synaptic 2‐amino‐3‐(3‐hydroxy‐5‐methylisoazol‐4‐yl)propionic acid receptors (AMPARs). This change in AMPAR subunit composition leads to an increase in surface expression of GluA2‐lacking Ca2+/Zn2+ permeable AMPARs. These GluA2‐lacking AMPARs play a key role in promoting delayed neuronal death following ischemic injury. At present, the mechanism(s) responsible for the ischemia/reperfusion‐induced subunit composition switch and degradation of the GluA2 subunit remain unclear. In this study, we investigated the role of NADPH oxidase, and its importance in mediating endocytosis and subsequent degradation of the GluA2 AMPAR subunit in adult rat hippocampal slices subjected to oxygen–glucose deprivation/reperfusion (OGD/R) injury. In hippocampal slices pre‐treated with the NADPH oxidase inhibitor apocynin attenuated OGD/R‐mediated sequestration of GluA2 and GluA1 as well as prevent the degradation of GluA2. We provide compelling evidence that NADPH oxidase mediated sequestration of GluA1‐ and GluA2‐ involved activation of p38 MAPK. Furthermore, we demonstrate that inhibition of NADPH oxidase blunts the OGD/R‐induced association of GluA2 with protein interacting with C kinase‐1. In summary, this study identifies a novel mechanism that may underlie the ischemia/reperfusion‐induced AMPAR subunit composition switch and a potential therapeutic target.
Newly formed glutamatergic synapses often lack postsynaptic AMPA-type glutamate receptors (AMPARs). Aside from 'unsilencing' the postsynaptic site, however, the significance of postsynaptic AMPAR insertion during synapse maturation remains unclear. To investigate the role of AMPAR in synapse maturation, we used RNA interference (RNAi) to knockdown AMPARs in cultured hippocampal neurons. Surprisingly, loss of postsynaptic AMPARs increased the occurrence of presynaptically inactive synapses without changing the release probability of the remaining active synapses. Additionally, heterologous synapses formed between axons and AMPAR-expressing HEK cells develop significantly fewer inactive presynaptic terminals. The extracellular domain of the AMPAR subunit GluA2 was sufficient to reproduce this effect at heterologous synapses. Indeed, the retrograde signalling by AMPARs is independent of their channel function as RNAi-resistant AMPARs restore synaptic transmission in neurons lacking AMPARs despite chronic receptor antagonist treatment. Our findings suggest that postsynaptic AMPARs perform an organizational function at synapses that exceeds their standard role as ionotropic receptors by conveying a retrograde trans-synaptic signal that increases the transmission efficacy at a synapse. 相似文献
Multiple signaling pathways are involved in AMPAR trafficking to synapses during synaptic plasticity and learning. The mechanisms for how these pathways are coordinated in parallel but maintain their functional specificity involves subcellular compartmentalization of kinase function by scaffolding proteins, but how this is accomplished is not well understood. Here, we focused on characterizing the molecular machinery that functions in the sequential synaptic delivery of GluA1- and GluA4-containing AMPARs using an in vitro model of eyeblink classical conditioning. We show that conditioning induces the interaction of selective protein complexes with the key structural protein SAP97, which tightly regulates the synaptic delivery of GluA1 and GluA4 AMPAR subunits. The results demonstrate that in the early stages of conditioning the initial activation of PKA stimulates the formation of a SAP97-AKAP/PKA-GluA1 protein complex leading to synaptic delivery of GluA1-containing AMPARs through a SAP97-PSD95 interaction. This is followed shortly thereafter by generation of a SAP97-KSR1/PKC-GluA4 complex for GluA4 AMPAR subunit delivery again through a SAP97-PSD95 interaction. These data suggest that SAP97 forms the molecular backbone of a protein scaffold critical for delivery of AMPARs to the PSD during conditioning. Together, the findings reveal a cooperative interaction of multiple scaffolding proteins for appropriately timed delivery of subunit-specific AMPARs to synapses and support a sequential two-stage model of AMPAR synaptic delivery during classical conditioning. 相似文献
Direct phosphorylation of GluA1 by PKC controls α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid (AMPA) receptor (AMPAR) incorporation into active synapses during long‐term potentiation (LTP). Numerous signalling molecules that involved in AMPAR incorporation have been identified, but the specific PKC isoform(s) participating in GluA1 phosphorylation and the molecule triggering PKC activation remain largely unknown. Here, we report that the atypical isoform of PKC, PKCλ, is a critical molecule that acts downstream of phosphatidylinositol 3‐kinase (PI3K) and is essential for LTP expression. PKCλ activation is required for both GluA1 phosphorylation and increased surface expression of AMPARs during LTP. Moreover, p62 interacts with both PKCλ and GluA1 during LTP and may serve as a scaffolding protein to place PKCλ in close proximity to facilitate GluA1 phosphorylation by PKCλ. Thus, we conclude that PKCλ is the critical signalling molecule responsible for GluA1‐containing AMPAR phosphorylation and synaptic incorporation at activated synapses during LTP expression. 相似文献
AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain. 相似文献
The perisynaptic extracellular matrix (ECM) contributes to the control of the lateral mobility of AMPA-type glutamate receptors (AMPARs) at spine synapses of principal hippocampal neurons. Here, we have studied the effect of the ECM on the lateral mobility of AMPARs at shaft synapses of aspiny interneurons. Single particle tracking experiments revealed that the removal of the hyaluronan-based ECM with hyaluronidase does not affect lateral receptor mobility on the timescale of seconds. Similarly, cross-linking with specific antibodies against the extracellular domain of the GluA1 receptor subunit, which affects lateral receptor mobility on spiny neurons, does not influence receptor mobility on aspiny neurons. AMPARs on aspiny interneurons are characterized by strong inward rectification indicating a significant fraction of Ca2+-permeable receptors. Therefore, we tested whether Ca2+ controls AMPAR mobility in these neurons. Application of the membrane-permeable Ca2+ chelator BAPTA-AM significantly increased the lateral mobility of GluA1-containing synaptic and extrasynaptic receptors. These data indicate that the perisynaptic ECM affects the lateral mobility differently on spiny and aspiny neurons. Although ECM structures on interneurons appear much more prominent, their influence on AMPAR mobility seems to be negligible at short timescales. 相似文献
The C terminus of AMPA-type glutamate receptor (AMPAR) GluA1 subunits contains several phosphorylation sites that regulate AMPAR activity and trafficking at excitatory synapses. Although many of these sites have been extensively studied, little is known about the signaling mechanisms regulating GluA1 phosphorylation at Thr-840. Here, we report that neuronal depolarization in hippocampal slices induces a calcium and protein phosphatase 1/2A-dependent dephosphorylation of GluA1 at Thr-840 and a nearby site at Ser-845. Despite these similarities, inhibitors of NMDA-type glutamate receptors and protein phosphatase 2B prevented depolarization-induced Ser-845 dephosphorylation but had no effect on Thr-840 dephosphorylation. Instead, depolarization-induced Thr-840 dephosphorylation was prevented by blocking voltage-gated calcium channels, indicating that distinct Ca2+ sources converge to regulate GluA1 dephosphorylation at Thr-840 and Ser-845 in separable ways. Results from immunoprecipitation/depletion assays indicate that Thr-840 phosphorylation inhibits protein kinase A (PKA)-mediated increases in Ser-845 phosphorylation. Consistent with this, PKA-mediated increases in AMPAR currents, which are dependent on Ser-845 phosphorylation, were inhibited in HEK-293 cells expressing a Thr-840 phosphomimetic version of GluA1. Conversely, mimicking Ser-845 phosphorylation inhibited protein kinase C phosphorylation of Thr-840 in vitro, and PKA activation inhibited Thr-840 phosphorylation in hippocampal slices. Together, the regulation of Thr-840 and Ser-845 phosphorylation by distinct sources of Ca2+ influx and the presence of inhibitory interactions between these sites highlight a novel mechanism for conditional regulation of AMPAR phosphorylation and function. 相似文献
The prevailing view at present is that postsynaptic expression of the classical NMDA receptor-dependent long-term potentiation relies on an increase in the numbers of local AMPA receptors (AMPARs). This is thought to parallel an expansion of postsynaptic cell specializations, for instance dendritic spine heads, which accommodate synaptic receptor proteins. However, glutamate released into the synaptic cleft can normally activate only a hotspot of low-affinity AMPARs that occur in the vicinity of the release site. How the enlargement of the AMPAR pool is causally related to the potentiated AMPAR current remains therefore poorly understood. To understand possible scenarios of postsynaptic potentiation, here we explore a detailed Monte Carlo model of the typical small excitatory synapse. Simulations suggest that approximately 50% increase in the synaptic AMPAR current could be provided by expanding the existing AMPAR pool at the expense of 100–200% new AMPARs added at the same packing density. Alternatively, reducing the inter-receptor distances by only 30–35% could achieve a similar level of current potentiation without any changes in the receptor numbers. The NMDA receptor current also appears sensitive to the NMDA receptor crowding. Our observations provide a quantitative framework for understanding the ‘resource-efficient’ ways to enact use-dependent changes in the architecture of central synapses. 相似文献
Aging causes multiple changes in the mammalian brain, including changes in synaptic signaling. Previous reports have shown increased extracellular adenosine in the aging brain, and we recently reported that activation of adenosine A1 receptors (A1Rs) induces AMPA receptor (AMPAR) internalization in rat hippocampus. This study investigated whether aging-related changes in the rat hippocampus include altered surface expression of adenosine A1 and A2A receptors, and whether these changes correspond to changes in AMPAR surface expression and altered synaptic plasticity. We found reduced A1R surface expression in middle-aged rat hippocampus, and also reduced GluA1 and GluA2 AMPAR subunit surface expression. Using a chemically-induced LTP (cLTP) experimental protocol, we recorded fEPSPs in young (1 month old) and middle-aged (7–12 month old) rat hippocampal slices. There were significant impairments in cLTP in middle-aged slices, suggesting impaired synaptic plasticity. Since we previously showed that the A1R agonist N6-cyclopentyladenosine (CPA) reduced both A1Rs and GluA2/GluA1 AMPARs, we hypothesized that the observed impaired synaptic plasticity in middle-aged brains is regulated by A1R-mediated AMPAR internalization by clathrin-mediated endocytosis. Following cLTP, we found a significant increase in GluA1 and GluA2 surface expression in young rats, which was blunted in middle-aged brains or in young brains pretreated with CPA. Blocking A1Rs with 8-cyclopentyl-1,3-dipropylxanthine or AMPAR endocytosis with either Tat-GluA2-3Y peptide or dynasore (dynamin inhibitor) similarly enhanced AMPAR surface expression following cLTP. These data suggest that age-dependent alteration in adenosine receptor expression contributes to increased AMPAR endocytosis and impaired synaptic plasticity in aged brains. 相似文献
Regulation of striatal medium spiny neuron synapses underlies forms of motivated behavior and pathological drug seeking. A primary mechanism for increasing synaptic strength is the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynapse, a process mediated by GluA1 AMPAR subunit phosphorylation. We have examined the role of converging glutamate and dopamine inputs in regulating biochemical cascades upstream of GluA1 phosphorylation. We focused on the role of Ca2+-permeable AMPARs (CPARs), which lack the GluA2 AMPAR subunit. Under conditions that prevented depolarization, stimulation of CPARs activated neuronal nitric oxide synthase and production of cGMP. CPAR-dependent cGMP production was sufficient to induce synaptic insertion of GluA1, detected by confocal microscopy, through a mechanism dependent on GluA1 Ser-845 phosphorylation. Dopamine D1 receptors, in contrast, stimulate GluA1 extra synaptic insertion. Simultaneous activation of dopamine D1 receptors and CPARs induced additive increases in GluA1 membrane insertion, but only CPAR stimulation augmented CPAR-dependent GluA1 synaptic insertion. This incorporation into the synapse proceeded through a sequential two-step mechanism; that is, cGMP-dependent protein kinase II facilitated membrane insertion and/or retention, and protein kinase C activity was necessary for synaptic insertion. These data suggest a feed-forward mechanism for synaptic priming whereby an initial stimulus acting independently of voltage-gated conductance increases striatal neuron excitability, facilitating greater neuronal excitation by a subsequent stimulus. 相似文献
An increase in the number of AMPA-type glutamate receptors (AMPARs) is critical for long-term potentiation (LTP), synaptic plasticity regarded as a basal mechanism of learning and memory. However, when and how each type of AMPAR reaches the postsynaptic membrane remain unclear. We have developed experimental methods to form postsynaptic-like membrane (PSLM) on a glass surface to precisely visualize the location and movement of receptors. We observed fluorescence-labeled AMPAR subunits (GluA1-3) around PSLM with total internal reflection fluorescence microscopy. The increases of GluA1, 2, and 3 in PSLM showed different time courses after LTP induction. GluA1 increased first, and was exocytosed to the periphery of PSLM soon after LTP induction. GluA2 and GluA3 initially decreased, and then increased. Exocytosis of GluA2 and GluA3 occurred primarily in non-PSLM, and later than exocytosis of GluA1. Thus, GluA1-3 appear to increase in the postsynaptic membrane through distinct pathways during LTP. 相似文献