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AMPA Receptor

SAP

Tardin, Choquet et al. (2003)

Direct imaging of lateral movements of AMPA receptors inside synapses. The EMBO Journal, 22(18)

Statements

Trafficking of AMPAR in and out of synapses is crucial for synaptic plasticity. Protocols that induce plasticity of synaptic transmission in culture result in changes of AMPAR concentration at synapses and are thought to mimic at the molecular level the processes of LTP and LTD.

Membrane trafficking may occur outside of the synapse and accumulate at the PSD after a short delay (Passafaro et al. 2001). Altogether, a unified picture of the postsynaptic density could be one where receptors are immobilized for transient periods of time related to the receptor-scaffold affinity. This could also be true of NMDA receptors (Tovar and Westbrook, 2002).

Findings

Application of glutamate increased the diffusion rate of GluR2-containting AMPAR whereas a protocol designed to induce calcium influx (stimulation of NMDAR with glycine, glutamate) reduced the percentage of diffusible AMPARs at the PSD. Bath application of 100 uM glutamate caused an 85% increase in AMPAR endocytosis within 15 min (corresponding to a 22% drop in total membrane expression). Conversely , the calcium influx protocol (20 uM biccuculine, 1 uM strychnine, 200 uM glycine) caused a 59% increase in AMPAR membrane expression. Glutamate caused a 55% increase in AMPAR diffusion within synapses, but did not change diffusion outside synapses. Furthermore, glutamate decreased the number of completely immobile AMPARs by 30%. Interestingly, Glutamate causes endocytosis of AMPARs, and internal AMPARs are immobile. Therefore it seems like glutamate may be causing a general endocytotic episode at non-synaptic AMPARs, perhaps not even at the synapse that received the glutamate application. In a parallel effect, it was found that blocking calcium with BAPTA increased the % of mobile AMPARs. Newly inserted receptors were found to be initially diffusive and then stabilized at synaptic sites. In summary, they found that bath application of glutamate induces rapid depletion of AMPARs from PSDs increases synaptic diffusion rate, decreases % of completely immobile receptors, increases proportion of receptors in the area surrounding the synapse (juxtasynaptic region). Activation of NMDARs results in increased surface expression of AMPARs -- in the first few minutes there is mainly a decrease in the proportion of immobile synaptic receptors, but after 40 min, both diffusion rates and percentages of immobile synaptic receptors are back to control values and the proportion of juxtasynaptic receptors is decreased. This observation relates to the fate of newly exocytosed AMPARs: using cleavable extracellular tags, it was observed that at early times after exocytosis, new GluR1 containing AMPARs are diffusively distributed along dendrites. This is followed by their lateral translocation and accumulation into synapses (Passafaro et al., 2001). GluR2 subunits were addressed directly at synapses. In our experiments, we followed the movement of native GluR2 containing AMPARs, where the data suggests that at the level of synapses themselves, newly added receptors are initially diffusive and then stabilize over time.

AMPA receptors that lack edited GluA2 subunits have high single channel conductance, are permeable to Ca2+, are blocked by polyamines causing inward rectification at depolarized potentials.

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• 100 uM glutamate - within 15 min
• 85% increase in AMPAR endocytosis
• 22% drop in total membrane expression
• 55% increase in AMPAR diffusion rate within synapses
• 0% increase in AMPAR diffusion rate outside synapses
• 30% decrease in completely immobile AMPAR at PSD

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• Start: 100 AMPARs in PSD
• Usual endocytosis rate: -0.25% / min
• Add: 100 uM glutamate
• New endocytosis rate: -1.5% / min
• Time: 15 min
• Final: 77.5 AMPARs

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• calcium influx protocol (20 uM biccuculine, 1 uM strychnine, 200 uM glycine)
• 59% increase in AMPAR expression

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• Start: 100 AMPARs in PSD
• Usual endocytosis rate: -0.25% / min
• Add: NMDA antagonists above
• New exocytosis rate: +4% / min
• Time: 15 min
• Final: 160 AMPARs

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Rao-Ruiz, Spijker, et al.

Retrieval-specific endocytosis of GluA2-AMPARs underlies adaptive reconsolidation of contextual fear

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A consolidated memory returns to a transient destabilized state shortly after reactivation, necessitating a dynamic time-dependent process of reconsolidation to persist further. During this reconstruction, a memory is labile and subject to change. In general, a memory-recall causes internalization of AMPAR at activate synapses for ~2 hours. Then AMPAR repopulate these synapses and return to baseline levels, and sometimes even higher levels.

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GROUPS

• NS: No Shock
• US: Shock
• NR: No Retrieval (24h later)
• R: Retrieval (24h later)

Tissue collected 1 h after retrieval for western blot and ephys

RESULTS

Down-regulation of all AMPAR subtypes and smaller mEPSC amplitudes 1 h after retrieval. Increase at 7 h.

Start at 100% baseline expression

• Post-Recall: 1 h 4 h 7 h
• GluR1 70% 100% 100%
• GluR2 85% 85% 130%
• GluR3 50% 50% 100%

Blocking GluR2 internalization with 3Y peptide (3A is a control peptide) 1 h before or 1 h after recall prevented a subsequent GluR2 increase at 7 hours. The narrow decay time found at 7 hours in 3A controls suggests that synapses are composed more of GluR2 than GluR1 or GluR3 receptors which are permeable to calcium. Furthermore, 3Y increased fear memory when tested 2 h (RT2) after a recall event (RT1) or 24 h (RT3) after recall.

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Start at 100% baseline expression

• Post-Recall: 1 h 4 h 7 h
• GluR1 70% 100% 100%
• GluR2 85% 85% 130%
• GluR3 50% 50% 100%

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Bassani · Folci · Zapata · Passafaro 2013

Abstract

Glutamate ionotropic AMPA receptors (AMPAR) mediate most fast excitatory synaptic transmission in the central nervous system. The content and composition of AMPARs in postsynaptic membranes (which determine synaptic strength) are dependent on the regulated trafficking of AMPAR subunits in and out of the membranes. AMPAR trafficking is a key mechanism that drives nascent synapse development, and is the main determinant of both Hebbian and homeostatic plasticity in mature synapses. Hebbian plasticity seems to be the biological substrate of at least some forms of learning and memory; while homeostatic plasticity (also known as synaptic scaling) keeps neuronal circuits stable by maintaining changes within a physiological range

Introduction

Glutamatergic synapses mediate excitatory transmission in the central nervous system. AMPARs and NMDARs are the two principal types of ionotropic glutamate receptors in glutamatergic synapses, and changes in the trafficking, subunit composition, and signaling of these receptors are fundamental processes underlying synapse strength. AMPARs are tetrameric (four subunit) cation channels that mediate fast excitatory synaptic transmission. NMDARs are also tetrameric cation channels; their activation requires glutamate (ligand-gating) and also membrane depolarization (voltage dependence), which removes the Mg2+ normally blocking the channel. Activated NMDARs allow Ca2+ to enter the neuron; the magnitude of the Ca2+ signal in the postsynaptic neuron largely determines whether long-term potentiation (LTP) or long-term depression (LTD) of AMPAR currents occurs.

In mature synapses, AMPAR trafficking is a major determinant of both Hebbian and homeostatic plasticity. Hebbian plasticity is a mechanism by which long-lasting modifications in synaptic strength occur [4 ]. Synaptic modifications resulting from Hebbian plasticity give rise to LTP and LTD and are largely determined by the delivery and removal of postsynaptic AMPARs. There is good evidence that LTP and LTD are substrates for at least some aspects of learning, memory, and cognition [5 ]; and some diseases characterized by defective learning and cognitive function are directly related to alterations in AMPAR trafficking [6 ]. Homeostatic synaptic plasticity on the other hand is a mechanism ensuring that changes in synaptic activity occur within a limited range, thereby preserving the stability of neuronal circuits and functional integrity of the brain [7 ].

AMPAR structure and subunit composition

AMPARs are homo or hetero tetramers assembled from GluA1-A4 subunits, encoded by genes GRIA1-4 [8 ]. The four subunits that make up each receptor combine in various stoichiometries to form receptor subtypes with distinct channel properties [9 ]. Most AMPARs in adult hippocampus and cortex appear to consist of the GluA1 plus GluA2, or GluA2 plus GluA3 subunits [10 ]. Since GluA3 is expressed at relatively low levels, over 70 % of GluA2 is associated with GluA1 [11 ]. All AMPAR subunits consist of an extracellular amino terminal domain (ATD or NTD), a ligand-binding domain (LBD) (S1 and S2), three membrane-spanning domains (M1, M2 and M3), one cytoplasmic re-entrant loop (P), and a carboxy-terminal intracellular region (Fig. 1) [9, 12, 13]. The extracellular and transmembrane regions of all GluAs are highly homologous and the four GluAs differ from each other mainly in terms of their intracellular cytoplasmic tails. The function of the NTD in AMPARs is to control the initial dimerization of subunits and to prevent heteromerization between AMPAR and kainate receptor subunits [14]. Subsequent tetramerization of dimers is mediated by associations at the ligand binding and membrane domains (M1–M3 and P) and also depends on Q/R editing of GluA2 (see below) [15]. GluA1, GluA4, and GluA2L (alternative splice form of GluA2) have long cytoplasmic tails; GluA2, GluA3, and GluA4S (alternative splice form of GluA4) have short C-terminal tails [16, 17, 18]. Alternative splicing also generates the flop (short) and flip (long) variants encoded by exons 14 and 15, respectively, that differ by a 38-amino-acid insertion into a region that forms part of the extracellular LBD and is localized before the M3 domain [19, 20]. The flip variants of all subunits are prominently expressed before birth and their expression, as determined by in situ hybridization, remains largely unchanged during postnatal development and in the adult; whereas the expression of flop variants increases throughout development, and reaches adult levels by postnatal day 14 in the rat [21]. Flop versions are less responsive to AMPAR potentiators [21, 22] and generally de-sensitize more rapidly in response to glutamate than receptors containing flip variant [19].

Almost all (99 %) GluA2s in adult brain have a positively charged arginine (R) in the M2 channel-forming segment at position 607 (Q/R site), while the other AMPAR subunits have glutamine (Q) at this position [15 ]. This is not due to a primary coding difference, but to site-selective deamination of adenosine to inosine on the pre-mRNA, which changes the glutamine codon to an arginine codon [23 ]. Deamination is performed by ADAR2, a double-stranded-RNA-specific adenosine deaminase and principal RNA-editing enzyme in mammals; ADAR2 can regulate its own expression and activity by editing its own pre-mRNA [24 ]. Unedited (Q) GluA2 exists during embryogenesis where it seems to have the important role of directing human neural progenitor cell differentiation to neurons [25 ]. Edited R-containing subunits remain largely unassembled and are retained at the endoplasmic reticulum, whereas unedited Q subunits readily tetramerize and traffic to synapses. Furthermore, the presence of a positively charged amino acid (R) in the channel-forming segment effectively blocks calcium entry and results in AMPARs with relatively low conductance and a linear current–voltage relationship [26 ]. By contrast, AMPARs containing unedited subunits are Ca2+ permeable, have higher conductance, and are susceptible to voltage-dependent block by endogenous intracellular polyamines [26 ]. Thus, this single amino acid residue not only affects channel composition but also controls ion conduction and channel rectification as well as subunit retention at the endoplasmic reticulum [15 ]. That Q/R editing is essential for correct brain function and is demonstrated by the finding that mice engineered to synthesize only unedited GluA2 subunits [27 , 28 , 29 ] die early and develop seizures, as do ADAR2 knockout (KO) mice [23 ].

AMPAR Interacting Proteins

AMPAR trafficking and synaptic targeting relies on interactions with several types of proteins, comprising those that interact with the extracellular N-terminal domain; those that interact with the intracellular C-terminal domain (including proteins with PDZ domains), and the so-called AMPAR auxiliary proteins.

Proteins interacting with the extracellular domain

The first two proteins demonstrated to interact with all four AMPA receptor subunits were neuronal pentraxin 1 (NP1) and neuronal immediate early gene neuronal activity-regulated pentraxin (NARP) [30 ]. NP1 induces clustering of GluA4 homomeric receptors—the main AMPARs expressed during synaptogenesis [30 , 31 ] whereas NARP induces clustering of GluA1-, GluA2-, and GluA3-containing AMPARs in both neurons and heterologous cells. Experimental overexpression of the gene encoding NARP in neurons increases the number of synaptic AMPAR clusters [32 ], suggesting that NARP may be important for stabilizing synaptic AMPAR clusters at excitatory synapses. The extracellular domain of GluA2 is also the specific site for interaction with N-cadherin—an interaction important for the formation, growth, and maintenance of dendritic spines. The first evidence of this was the finding that GluA2 overexpression in mature cultured hippocampal neurons increased spine length, spine head width, and spine density, and that this activity required NTD [33 ]. GluA2 may therefore stimulate synaptic development and dendritic spine formation via this novel structural interaction at the synaptic junction [34 ] (Fig. 2 ).

Proteins interacting with the intracellular domain

• PDZ domain proteins interacting with the C-terminal domain

The C-terminus of GluA1 binds with a PDZ domain of SAP97—a member of the membrane-associated guanylate kinase (MAGUK) family [35 ]. SAP97 interacts with the protein kinase A (PKA) anchoring molecule AKAP79 [36 ], which may serve to enhance the GluA1 phosphorylation required for LTP [37 ]. Recently, it has been shown that neuronal activity induces AKAP79/150 palmitoylation, which is required for AKAP79/150 recruitment at spines and for spine enlargement [38 , 39 ]. In AKAP79/150-KO mice, PKA does not reach the postsynaptic membrane and the bidirectional modulation of postsynaptic AMPARs is altered, with concomitant alterations in synaptic transmission and memory [40 ]. However, SAP97 has also been shown to act early in the secretory pathway to facilitate AMPAR maturation [41 ]. Furthermore, while some studies have found that SAP97 overexpression in cultured neurons increased the number of synaptic AMPARs and NMDARs [42 , 43 ], other studies report that SAP97 has no significant effect on excitatory postsynaptic currents (EPSCs) mediated by AMPAR or NMDAR [44 , 45 ]. The situation is further complicated by the recent finding that SAP97 conditional KO mice have normal LTP [46 ]. Clearly, further research is required to elucidate the roles of SAP97 in AMPAR trafficking. GluA2 and GluA3 share a C-terminal sequence (-SVKI) that interacts with glutamate receptor-interacting proteins (GRIP1/2) containing seven PDZ domains [47 ]; with AMPAR binding protein (ABP) [48 , 49 ], which has six PDZ domains; and with PICK1, which contains a single PDZ domain. ABP seems to be a splice variant of GRIP2 (also called GRIPrelated protein), which lacks the GRIP2 N-terminus and PDZ7 [48 ]. ABP and GRIP are found at the PSD and also in intracellular punctate structures resembling endosomes. Several studies have implicated GRIP1/2 in the control of AMPAR trafficking, as well as synaptic plasticity and social behavior. For example, genetic ablation of GRIP1/2 abolishes cerebellar LTD [50 ] and affected mice show increased sociability and impaired prepulse inhibition [51 ]. However, it is unclear how GRIP1/2 achieves this control. Some studies suggest that AMPAR delivery to dendrites and synapses requires GRIP1 interaction with kinesin heavy chain [52 ] and liprin-α , which in turn binds microtubule-based motor KIF1A [53 , 54 ] and also the LAR family of tyrosine phosphatase receptors [55 ]. Liprin-α has been shown to be important for both postsynaptic and presynaptic maturation [53 ]. Other studies indicate that GRIP1 retains AMPARs in the intracellular compartment [56 ]. Others again indicate that GRIP1 regulates the endosomal recycling of AMPARs in that GRIP1 binding to neuronenriched endosomal protein 21 kDa (NEEP21) promotes the recycling of internalized AMPARs back to the plasma membrane [57 , 58 ]. It has also been reported that GRIP1 binds KIF5, another microtubule-based motor protein important for vesicular transport along axons and dendrites: the KIF5–GRIP1 complex interacts with GluA2, and appears to be involved in the transport of GluA2 to dendrites [52 ]. The interaction of GluA2 with PICK1 is important for GluA2 endocytosis [59 ]. Furthermore, the interactions of GRIP/ABP and PICK1 with GluA2 depend on the phosphorylation status of serine 880 (S880) and tyrosine 876 (Y876) on the C-terminal of the GluA2 subunit: In particular, phosphorylation of S880 prevents the interaction of GRIP/ABP with GluA2, but not of PICK1 with GluA2 [60 , 61 ]. On the other hand, ABP binding can itself prevent S880 phosphorylation [62 ]. Phosphorylation of Y876 by Src tyrosine kinase also regulates the GRIP/ABP interaction with GluA2, but not the PICK1 interaction [63 ]. GRIP, ABP/GRIP2, and PICK also play critical roles in LTD and are involved in regulating the recycling of internalized following NMDAR activation [64 ], in association with the memory performance-associated protein KIBRA, which also binds PICK1 and AMPAR [65 ]. Recently, PICK1 has been shown to interact with tetraspannin- 7 (Tspan7), a protein involved in X-linked intellectual disability, and this interaction has been shown to be important for the localization of AMPARs at the postsynaptic membrane, and for synapse maturation [66 ].

• Interactions of with 4.1N, NSF, and AP2 with the C-terminal domain

The GluA1 subunit forms a complex with 4.1N—a neuronal specific form of 4.1R, the red blood cell actin cytoskeletonassociated protein—as a result of which AMPARs appear to be stabilized at the cell surface [67 ]. NSF is an ATPase known to play an important role in membrane fusion [68 , 69 , 70 ]. It has also been found that NSF interacts with the C-terminus of GluA2 and that this interaction is required for the insertion of GluA2-containing AMPARs into the membrane, and their stabilization there [68 , 71 , 72 , 73 , 74 , 75 ]. Disruption of the NSF–GluA2 interaction by specific peptides causes a rundown of EPSCs [68 , 70 , 71 , 74 ], while a mutated GluA2 that does not interact with NSF does not arrive at synapses in hippocampal slice cultures [76 ]. It has been shown that inhibition of NSF activity prevents LTP, while the amount of NSF in the PSD appears to be regulated dynamically [77 ]. AP2 is a clathrin adaptor complex involved in endocytosis. It interacts with GluA2 at a binding site in the C-terminal region overlapping (but not identical to) the NSF binding site [71 , 78 ] and this interaction seems to be important for clathrin-mediated endocytosis during NMDA receptor-mediated LTD [71 ]. One study that used a peptide to block the AP2–GluA2 interaction found increased AMPAR-mediated transmission [78 ]; however, an earlier study that used another peptide to block this interaction had no effect on basal transmission but selectively prevented LTD [71 ]. Further study is required to clarify the role of AP2 in AMPAR trafficking, although it does seem clear that defective AP2–GluA2 interaction interferes with LTD.

• AMPA auxiliary proteins

The AMPA auxiliary proteins are another group of molecules important for AMPAR targeting to the postsynaptic membrane. They include the transmembrane AMPAR regulatory proteins (TARPs), cornichon-like proteins, neuropilin, and tolloid-like proteins [79 , 80 , 81 , 82 , 83 , 84 ]. TARPs regulate AMPAR trafficking and channel kinetics and are classified into six isoforms—type 1a (γ -2/stargazin and γ -3), type 1b (γ -4 and γ -8), and type 2 (γ -5 and γ -7)— according to how they control AMPAR trafficking and channel properties [85 , 86 ]. They stabilize AMPARs at the postsynaptic membrane by direct interaction with scaffolding protein PSD95 and other MAGUKs. TARPs also slow deactivation and desensitization, thereby increasing AMPAR conductance, and influencing AMPAR affinity for pharmacological agents [85 , 87 , 88 , 89 , 90 ]. Synaptic TARP phosphorylation is activity regulated and in turn phosphorylation influences stargazin binding to PSD-95 [91 , 92 , 93 ]. CaMKIIdependent phosphorylation of stargazin retains AMPARs at postsynaptic sites by reducing AMPAR diffusion [94 ]. Interaction of AMPARs with the cornichon-like proteins CNIH2 and CNIH3 was recently reported by Schwenk et al. [95 ], who showed that this interaction is involved in the regulation of AMPAR expression in the postsynaptic membrane, and also influences AMPAR channel properties. CNIH2 and CNIH3 bind AMPARs which in turn complex with TARPγ 4 in the hippocampus [96 ]. In this complex, the cornichon-like proteins control AMPAR trafficking to the postsynaptic membrane, whereas TARPγ 4 stabilizes the CNIH2/3–AMPAR interaction in the membrane [97 ].