NSF binds calcium to regulate its interaction with AMPA receptor subunit GluR2

N-ethylmaleimide-sensitive fusion protein (NSF) is essential for numerous Ca(2+)-triggered vesicle trafficking events. It functions as a molecular chaperone to regulate trafficking protein complexes such as the soluble NSF attachment protein (SNAP) receptor complex and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-protein interacting with C-kinase (PICK1) complex. AMPAR trafficking is fundamental to processes of synaptic plasticity, which may underlie learning and memory. Changes in synaptic strength brought about by AMPAR trafficking are triggered by a post-synaptic influx of Ca(2+), which may have numerous molecular targets including PICK1. NSF binds AMPAR subunit glutamate receptor subunit 2 (GluR2) and functions to maintain receptors at the synapse. In this study, it was showed that NSF is a Ca(2+)-binding protein and that GluR2-NSF interactions are inhibited by the presence of 15 mumol/L Ca(2+). NSF Ca(2+)-binding is reciprocally inhibited by the presence of GluR2 C-terminus. Mutant of NSF that binds Ca(2+) with reduced affinity and binds GluR2 with reduced sensitivity to Ca(2+) was identied. In addition, the interaction of betaSNAP with PICK1 is sensitive to Ca(2+). This study demonstrates that the GluR2-NSF-betaSNAP-PICK1 complex is regulated directly by Ca(2+), allowing for the transduction of Ca(2+) signals into concerted alterations in protein-protein interactions to bring about changes in AMPAR trafficking during synaptic plasticity.     

NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICK1 complex

AMPA receptor (AMPAR) trafficking is crucial for synaptic plasticity that may be important for learning and memory. NSF and PICK1 bind the AMPAR GluR2 subunit and are involved in trafficking of AMPARs. Here, we show that GluR2, PICK1, NSF, and alpha-/beta-SNAPs form a complex in the presence of ATPgammaS. Similar to SNARE complex disassembly, NSF ATPase activity disrupts PICK1-GluR2 interactions in this complex. Alpha- and beta-SNAP have differential effects on this reaction. SNAP overexpression in hippocampal neurons leads to corresponding changes in AMPAR trafficking by acting on GluR2-PICK1 complexes. This demonstrates that the previously reported synaptic stabilization of AMPARs by NSF involves disruption of GluR2-PICK1 interactions. Furthermore, we are reporting a non-SNARE substrate for NSF disassembly activity.     

Clathrin adaptor AP2 and NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA receptor trafficking and hippocampal LTD

Proteins that bind to the cytoplasmic tails of AMPA receptors control receptor trafficking and thus the strength of postsynaptic responses. Here we show that AP2, a clathrin adaptor complex important for endocytosis, associates with a region of GluR2 that overlaps the NSF binding site. Peptides used previously to interfere with NSF binding also antagonize GluR2-AP2 interaction. Using GluR2 mutants and peptide variants that dissociate NSF and AP2 interaction, we find that AP2 is involved specifically in NMDA receptor-induced (but not ligand-dependent) internalization of AMPA receptors, and is essential for hippocampal long-term depression (LTD). NSF function, on the other hand, is needed to maintain synaptic AMPA receptor responses, but is not directly required for NMDA receptor-mediated internalization and LTD.     

Subunit rules governing the sorting of internalized AMPA receptors in hippocampal neurons

Removal of synaptic AMPA receptors is important for synaptic depression. Here, we characterize the roles of individual subunits in the inducible redistribution of AMPA receptors from the cell surface to intracellular compartments in cultured hippocampal neurons. The intracellular accumulation of GluR2 and GluR3 but not GluR1 is enhanced by AMPA, NMDA, or synaptic activity. After AMPA-induced internalization, homomeric GluR2 enters the recycling pathway, but following NMDA, GluR2 is diverted to late endosomes/lysosomes. In contrast, GluR1 remains in the recycling pathway, and GluR3 is targeted to lysosomes regardless of NMDA receptor activation. Interaction with NSF plays a role in regulated lysosomal targeting of GluR2. GluR1/GluR2 heteromeric receptors behave like GluR2 homomers, and endogenous AMPA receptors show differential activity-dependent sorting similar to homomeric GluR2. Thus, GluR2 is a key subunit that controls recycling and degradation of AMPA receptors after internalization.     

Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting

Both acute and chronic changes in AMPA receptor (AMPAR) localization are critical for synaptic formation, maturation, and plasticity. Here I report that AMPARs are differentially sorted between recycling and degradative pathways following endocytosis. AMPAR sorting occurs in early endosomes and is regulated by synaptic activity and activation of AMPA and NMDA receptors. AMPAR intemalization triggered by NMDAR activation is Ca2+-dependent, requires protein phosphatases, and is followed by rapid membrane reinsertion. Furthermore, NMDAR-mediated AMPAR trafficking is regulated by PKA and accompanied by dephosphorylation and rephosphorylation of GluR1 subunits at a PKA site. In contrast, activation of AMPARs without NMDAR activation targets AMPARs to late endosomes and lysosomes, independent of Ca2+, protein phosphatases, or PKA. These results demonstrate that activity regulates AMPAR endocytic sorting, providing a potential mechanistic link between rapid and chronic changes in synaptic strength.     

Long-Term Potentiation in Isolated Dendritic Spines

Background

In brain, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation can induce long-lasting changes in synaptic α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor (AMPAR) levels. These changes are believed to underlie the expression of several forms of synaptic plasticity, including long-term potentiation (LTP). Such plasticity is generally believed to reflect the regulated trafficking of AMPARs within dendritic spines. However, recent work suggests that the movement of molecules and organelles between the spine and the adjacent dendritic shaft can critically influence synaptic plasticity. To determine whether such movement is strictly required for plasticity, we have developed a novel system to examine AMPAR trafficking in brain synaptosomes, consisting of isolated and apposed pre- and postsynaptic elements.

Methodology/Principal Findings

We report here that synaptosomes can undergo LTP-like plasticity in response to stimuli that mimic synaptic NMDAR activation. Indeed, KCl-evoked release of endogenous glutamate from presynaptic terminals, in the presence of the NMDAR co-agonist glycine, leads to a long-lasting increase in surface AMPAR levels, as measured by [³H]-AMPA binding; the increase is prevented by an NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). Importantly, we observe an increase in the levels of GluR1 and GluR2 AMPAR subunits in the postsynaptic density (PSD) fraction, without changes in total AMPAR levels, consistent with the trafficking of AMPARs from internal synaptosomal compartments into synaptic sites. This plasticity is reversible, as the application of AMPA after LTP depotentiates synaptosomes. Moreover, depotentiation requires proteasome-dependent protein degradation.

Conclusions/Significance

Together, the results indicate that the minimal machinery required for LTP is present and functions locally within isolated dendritic spines.     

Quaternary structure, protein dynamics, and synaptic function of SAP97 controlled by L27 domain interactions

Single-particle electron microscopy (EM) combined with biochemical measurements revealed the molecular shape of SAP97 and a monomer-dimer transition that depended on the N-terminal L27 domain. Overexpression of SAP97 drove GluR1 to synapses, potentiated AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs), and occluded LTP. Synaptic potentiation and GluR1 delivery were dissociable by L27 domain mutants that inhibit multimerization of SAP97. Loss of potentiation was correlated with faster turnover of monomeric SAP97 mutants in dendritic spines. We propose that L27-mediated interactions of SAP97 with itself or other proteins regulate the synaptic delivery of AMPARs. RNAi knockdown of endogenous PSD-95 depleted surface GluR1 and impaired AMPA EPSCs. In contrast, RNAi knockdown of endogenous SAP97 reduced surface expression of both GluR1 and GluR2 and inhibited both AMPA and NMDA EPSCs. Thus SAP97 has a broader role than its close relative, PSD-95, in the maintenance of synaptic function.     

Differential roles of NR2A- and NR2B-containing NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking

NMDA receptors (NMDARs) control bidirectional synaptic plasticity by regulating postsynaptic AMPA receptors (AMPARs). Here we show that NMDAR activation can have differential effects on AMPAR trafficking, depending on the subunit composition of NMDARs. In mature cultured neurons, NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1, primarily by regulating its surface insertion. In mature neurons, NR2B is coupled to inhibition rather than activation of the Ras-ERK pathway, which drives surface delivery of GluR1. Moreover, the synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDARs in brain and is required for inhibition of NMDAR-dependent ERK activation. Preferential coupling of NR2B to SynGAP could explain the subtype-specific function of NR2B-NMDARs in inhibition of Ras-ERK, removal of synaptic AMPARs, and weakening of synaptic transmission.     

An essential role for PICK1 in NMDA receptor-dependent bidirectional synaptic plasticity

PICK1 is a calcium-sensing, PDZ domain-containing protein that interacts with GluR2 and GluR3 AMPA receptor (AMPAR) subunits and regulates their trafficking. Although PICK1 has been principally implicated in long-term depression (LTD), PICK1 overexpression in CA1 pyramidal neurons causes a CaMK- and PKC-dependent potentiation of AMPAR-mediated transmission and an increase in synaptic GluR2-lacking AMPARs, mechanisms associated with NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here, we directly tested whether PICK1 participates in both hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1 potentiation of AMPAR-mediated transmission is NMDAR dependent and fully occludes LTP. Conversely, blockade of PICK1 PDZ interactions or lack of PICK1 prevents LTP. These observations demonstrate an important role for PICK1 in LTP. In addition, deletion of PICK1 or blockade of PICK1 PDZ binding prevented NMDAR-dependent LTD. Thus, PICK1 plays a critical role in bidirectional NMDAR-dependent long-term synaptic plasticity in the hippocampus.     

Molecular constituents of neuronal AMPA receptors

Dynamic regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) underlies aspects of synaptic plasticity. Although numerous AMPAR-interacting proteins have been identified, their quantitative and relative contributions to native AMPAR complexes remain unclear. Here, we quantitated protein interactions with neuronal AMPARs by immunoprecipitation from brain extracts. We found that stargazin-like transmembrane AMPAR regulatory proteins (TARPs) copurified with neuronal AMPARs, but we found negligible binding to GRIP, PICK1, NSF, or SAP-97. To facilitate purification of neuronal AMPAR complexes, we generated a transgenic mouse expressing an epitope-tagged GluR2 subunit of AMPARs. Taking advantage of this powerful new tool, we isolated two populations of GluR2 containing AMPARs: an immature complex with the endoplasmic reticulum chaperone immunoglobulin-binding protein and a mature complex containing GluR1, TARPs, and PSD-95. These studies establish TARPs as the auxiliary components of neuronal AMPARs.     

A role for cGMP-dependent protein kinase II in AMPA receptor trafficking and synaptic plasticity

Regulated trafficking of AMPA receptors (AMPARs) is an important mechanism that underlies the activity-dependent modification of synaptic strength. Trafficking of AMPARs is regulated by specific interactions of their subunits with other proteins. Recently, we have reported that the AMPAR subunit GluR1 binds the cGMP-dependent kinase type II (cGKII) adjacent to the kinase catalytic site, and that this interaction is increased by cGMP. In this complex, cGKII phosphorylates GluR1 at serine 845 (S845), a site known to be phosphorylated also by PKA. S845 phosphorylation leads to an increase of GluR1 on the plasma membrane. In neurons, cGMP is produced by soluble guanylate cyclase (sGC), which is activated by nitric oxide (NO). Calcium flux through the NMDA receptor (NMDAR) activates neuronal nitric oxide synthase (nNOS), which produces NO. Using a combination of biochemical and electrophysiological experiments, we have shown that trafficking of GluR1 is under the regulation of NO, cGMP and cGKII. Moreover, our study indicates that the interaction of cGKII with GluR1, which is under the regulation of the NMDAR and NO, plays an important role in hippocampal plasticity.     

A role for cGMP-dependent protein kinase II in AMPA receptor trafficking and synaptic plasticity

Regulated trafficking of AMPA receptors (AMPARs) is an important mechanism that underlies the activity-dependent modification of synaptic strength. Trafficking of AMPARs is regulated by specific interactions of their subunits with other proteins. Recently, we have reported that the AMPAR subunit GluR1 binds the cGMP-dependent kinase type II (cGKII) adjacent to the kinase catalytic site, and that this interaction is increased by cGMP. In this complex, cGKII phosphorylates GluR1 at serine 845 (S845), a site known to be phosphorylated also by PKA. S845 phosphorylation leads to an increase of GluR1 on the plasma membrane. In neurons, cGMP is produced by soluble guanylate cyclase (sGC), which is activated by nitric oxide (NO). Calcium flux through the NMDA receptor (NMDAR) activates neuronal nitric oxide synthase (nNOS), which produces NO. Using a combination of biochemical and electrophysiological experiments, we have shown that trafficking of GluR1 is under the regulation of NO, cGMP and cGKII. Moreover, our study indicates that the interaction of cGKII with GluR1, which is under the regulation of the NMDAR and NO, plays an important role in hippocampal plasticity.     

PICK1 is a calcium-sensor for NMDA-induced AMPA receptor trafficking

Regulation of AMPA receptor (AMPAR) trafficking results in changes in receptor number at the postsynaptic membrane, and hence modifications in synaptic strength, which are proposed to underlie learning and memory. NMDA receptor-mediated postsynaptic Ca2+ influx enhances AMPAR internalisation, but the molecular mechanisms that trigger such trafficking are not well understood. We investigated whether AMPAR-associated protein-protein interactions known to regulate receptor surface expression may be directly regulated by Ca2+. PICK1 binds the AMPAR GluR2 subunit and is involved in AMPAR internalisation and LTD. We show that PICK1 is a Ca2+-binding protein, and that PICK1-GluR2 interactions are enhanced by the presence of 15 muM Ca2+. Deletion of an N-terminal acidic domain in PICK1 reduces its ability to bind Ca2+, and renders the GluR2-PICK1 interaction insensitive to Ca2+. Overexpression of this Ca2+-insensitive mutant occludes NMDA-induced AMPAR internalisation in hippocampal neurons. This work reveals a novel postsynaptic Ca2+-binding protein that provides a direct mechanistic link between NMDAR-mediated Ca2+ influx and AMPAR endocytosis.     

The phosphorylation state of GluR1 subunits determines the susceptibility of AMPA receptors to calpain cleavage

The alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) is an ionotropic glutamate receptor that governs most of excitatory synaptic transmission in neurons. In vitro biochemical assay has shown that calpain, a Ca2+-activated protease, can cleave AMPAR GluR1 subunits. Our physiological study found that calpain, which was activated by prolonged stimulation of the N-methyl-D-aspartate receptor (100 microM, 10 min), caused a substantial suppression of AMPAR currents in cortical neurons. Since the phosphorylation sites of GluR1 by several protein kinases are located in close proximity to the calpain cleavage sites, we investigated the effect of phosphorylation on the susceptibility of GluR1 to calpain cleavage. Interestingly, we found that the calpain regulation of AMPAR currents was diminished by inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) but was augmented by inhibition of protein phosphatase 1/2A (PP1/2A). In agreement with this, in vitro assay showed that the calpain-induced proteolytic cleavage of GluR1 C-terminal fusion protein was strongly potentiated by adding the purified active CaMKII, and GluR1 phosphorylated at Ser831 by CaMKII is much more sensitive to calpain cleavage. Taken together, our data suggest that calpain activation suppresses AMPA receptor currents via proteolytic cleavage of GluR1 subunits, and the susceptibility of AMPARs to calpain cleavage is determined by the phosphorylation state of GluR1 subunits, which is mediated by CaMKII-PP1/2A activity.     

Postsynaptic complexin controls AMPA receptor exocytosis during LTP

Long-term potentiation (LTP) is a compelling synaptic correlate of learning and memory. LTP induction requires NMDA receptor (NMDAR) activation, which triggers SNARE-dependent exocytosis of AMPA receptors (AMPARs). However, the molecular mechanisms mediating AMPAR exocytosis induced by NMDAR activation remain largely unknown. Here, we show that complexin, a protein that regulates neurotransmitter release via binding to SNARE complexes, is essential for AMPAR exocytosis during LTP but not for the constitutive AMPAR exocytosis that maintains basal synaptic strength. The regulated postsynaptic AMPAR exocytosis during LTP requires binding of complexin to SNARE complexes. In hippocampal neurons, presynaptic complexin acts together with synaptotagmin-1 to mediate neurotransmitter release. However, postsynaptic synaptotagmin-1 is not required for complexin-dependent AMPAR exocytosis during LTP. These results suggest?a complexin-dependent molecular mechanism for regulating AMPAR delivery to synapses, a mechanism that is surprisingly similar to presynaptic exocytosis but controlled by regulators other than synaptotagmin-1.     

S-nitrosylation of c-Src via NMDAR-nNOS module promotes c-Src activation and NR2A phosphorylation in cerebral ischemia/reperfusion

Previous studies suggested that activated c-Src promote the tyrosine phosphorylation of NMDA receptor subunit NR2A, and thus aggravate the injury induced by transient cerebral ischemia/reperfusion (I/R) in rat hippocampus CA1 region. In this study, we examined the effect of nitric oxide (NO) on the activation of c-Src and the tyrosine phosphorylation of NMDA receptor NR2A subunit. The results show that S-nitrosylation and the phosphorylation of c-Src were induced after cerebral I/R in rats, and administration of nNOS inhibitor 7-NI, nNOS antisense oligonucleotides and exogenous NO donor sodium nitroprusside diminished the increased S-nitrosylation and phosphorylation of c-Src during cerebral I/R. The cysteine residues of c-Src modified by S-nitrosylation are Cys489, Cys498, and Cys500. On the other hand, NMDAR antagonist MK-801 could attenuate the S-nitrosylation and activation of c-Src. Taken together, the S-nitrosylation of c-Src is provoked by NO derived from endogenous nNOS, which is activated by Ca²⁺ influx from NMDA receptors, and promotes the auto-phosphorylation at tyrosines and further phosphorylates NR2A. The molecular mechanism we outlined here is a novel postsynaptic NMDAR-nNOS/c-Src-mediated signaling amplification, the ‘NMDAR-nNOS → NO → SNO-c-Src → p-c-Src → NMDAR-nNOS’ cycle, which presents the possibility as a potential therapeutic target for stroke treatment.     

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2

Trafficking of AMPA-type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1-binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR-mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21-GRIP1 binding is crucial for GluR2-AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.     

Hippocampal LTD expression involves a pool of AMPARs regulated by the NSF-GluR2 interaction

We investigated whether the interaction between the N-ethyl-maleimide-sensitive fusion protein (NSF) and the AMPA receptor (AMPAR) subunit GluR2 is involved in synaptic plasticity in the CA1 region of the hippocampus. Blockade of the NSF-GluR2 interaction by a specific peptide (pep2m) introduced into neurons prevented homosynaptic, de novo long-term depression (LTD). Moreover, saturation of LTD prevented the pep2m-induced reduction in AMPAR-mediated excitatory postsynaptic currents (EPSCs). Minimal stimulation experiments indicated that both pep2m action and LTD were due to changes in quantal size and quantal content but were not associated with changes in AMPAR single-channel conductance or EPSC kinetics. These results suggest that there is a pool of AMPARs dependent on the NSF-GluR2 interaction and that LTD expression involves the removal of these receptors from synapses.     

Redox modulation by S-nitrosylation contributes to protein misfolding, mitochondrial dynamics, and neuronal synaptic damage in neurodegenerative diseases

The pathological processes of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases engender synaptic and neuronal cell damage. While mild oxidative and nitrosative (nitric oxide (NO)-related) stress mediates normal neuronal signaling, excessive accumulation of these free radicals is linked to neuronal cell injury or death. In neurons, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation and subsequent Ca(2+) influx can induce the generation of NO via neuronal NO synthase. Emerging evidence has demonstrated that S-nitrosylation, representing covalent reaction of an NO group with a critical protein thiol, mediates the vast majority of NO signaling. Analogous to phosphorylation and other posttranslational modifications, S-nitrosylation can regulate the biological activity of many proteins. Here, we discuss recent studies that implicate neuropathogenic roles of S-nitrosylation in protein misfolding, mitochondrial dysfunction, synaptic injury, and eventual neuronal loss. Among a growing number of S-nitrosylated proteins that contribute to disease pathogenesis, in this review we focus on S-nitrosylated protein-disulfide isomerase (forming SNO-PDI) and dynamin-related protein 1 (forming SNO-Drp1). Furthermore, we describe drugs, such as memantine and newer derivatives of this compound that can prevent both hyperactivation of extrasynaptic NMDARs as well as downstream pathways that lead to nitrosative stress, synaptic damage, and neuronal loss.     

N-methyl-D-aspartate-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor down-regulation involves interaction of the carboxyl terminus of GluR2/3 with Pick1. Ligand-binding studies using Sindbis vectors carrying AMPA receptor decoys

The dynamics of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors, as represented by their exocytosis, endocytosis and cytoskeletal linkage, has often been implicated in N-methyl-d-aspartate (NMDA)-dependent synaptic plasticity. To explore the molecular mechanisms underlying the AMPA receptor dynamics, cultured hippocampal neurons were stimulated with 100 microm NMDA, and the biochemical and pharmacological changes in the ligand binding activity of AMPA receptor complexes and its subunits, GluR1 and GluR2/3, were investigated. The NMDA treatment reduced the total amount of bound [(3)H]AMPA on the surface of the neurons but not in their total membrane fraction. This process was mimicked by a protein kinase C activator, phorbol ester, but blocked by an inhibitor of the same kinase, calphostin C. The NMDA-induced down-regulation of the ligand binding activity was also reflected by the decreased AMPA-triggered channel activity as well as by the cells' reduced immunoreactivity for GluR1. In parallel, the NMDA treatment markedly altered the interaction between the AMPA receptor subunits and their associating molecule(s); the association of PDZ molecules, including Pick1, with GluR2/3 was enhanced in a protein-kinase-C-dependent manner. Viral expression vectors carrying GluR1 and GluR2 C-terminal decoys, both fused to enhanced green fluorescent protein, were transfected into hippocampal neurons to disrupt their interactions. The overexpression of the C-terminal decoy for GluR2 specifically and significantly blocked the NMDA-triggered reduction in [(3)H]AMPA binding, whereas that for GluR1 had no effects. Co-immunoprecipitation using anti-Pick1 antibodies revealed that the overexpressed GluR2 C-terminal decoy indeed prevented Pick1 from interacting with the endogenous GluR2/3. Therefore, these observations suggest that the NMDA-induced down-regulation of the functional AMPA receptors involves the interaction between GluR2/3 subunits and Pick1.