Introductory Review Cellscience Reviews Vol 2 No.1 ISSN 1742-8130

Modulation of AMPA receptor activity by associated proteins

Karen E. Smith^†, Jessica A. Gorski^†, and Mark L. Dell’Acqua^*

Excitatory synaptic plasticity in the brain is believed to underlie many aspects of learning and memory. This plasticity is in part the result of changes in both AMPA receptor number and activity at the synapse. AMPA receptors are ionotropic glutamate receptors present throughout the mammalian brain, which carry the bulk of normal fast synaptic transmission. The tetrameric functional receptor is comprised of four subunits whose stoichiometry varies with cell type, brain region and developmental stage. The individual subunits all have an extracellular agonist-binding domain, 3 transmembrane loops and one inter-membrane loop. The cytoplasmic C-terminal tails are crucial for the regulation of AMPA receptor function and exhibit the largest variability between subunits. GluR1 and GluR4 have long tails, while GluR2 and GluR3 have short tails (Song and Huganir, 2002). Specific regions within the C-terminal tails determine trafficking properties of the receptor complexes. In the hippocampus, subunit complexes containing GluR1 are inserted in response to activity, while complexes containing GluR2 are constitutively cycled into and out of the synaptic membrane (Shi et al., 2001).

AMPA receptor activity is dictated in part by its subunit composition and the phosphorylation state of these subunits. GluR2-containing channels have lower open channel conductance and are calcium-impermeable, whereas receptors lacking GluR2 have higher open channel conductance and are calcium permeable. Both GluR1 and GluR2 have phosphorylation sites within their C-terminal tail. Phosphorylation of GluR1-Ser831 by CaMKII increases its single channel conductance during hippocampal long-term potentiation (LTP), whereas dephosphorylation of the Ser845 PKA phosphorylation site has been shown to play a role in hippocampal long-term depression (LTD, Song and Huganir, 2002). Phosphorylation of AMPA receptors and binding to scaffold proteins associated with the receptor both play roles in regulation receptor trafficking in LTP and LTD (Malenka and Bear, 2004). GluR1 binds to the PDZ domains of the membrane-associated guanylate kinase (MAGUK) scaffold protein SAP97 via its 4 amino acid PDZ-binding motif at the carboxy terminus (Leonard et al., 1998). The SAP97 interaction appears to be important for trafficking of AMPA receptor through the secretory pathway (Sans et al., 2001), but no role in plasticity has been directly confirmed. However, the C-terminal PDZ ligand site in GluR1 is necessary for the activity dependent delivery to synapses that is associated with LTP (Hayashi et al., 2000; Passafaro et al., 2001; Shi et al., 2001). AMPA receptors can also be linked to the MAGUK scaffold protein PSD-95 through association with a family of transmembrane proteins known as stargazin/transmembrane AMPA receptor regulatory proteins (TARPs). Importantly, overexpresssion of PSD-95 can recruit AMPA receptors to synapses in a manner that involves stargazin/TARPs and induces synaptic potentiation mimicking and occluding LTP (El-Husseini et al., 2002; Schnell et al., 2002; Ehrlich and Malinow, 2004). Somewhat surprisingly, PSD-95 expression promotes delivery of GluR1-containing AMPA receptors despite the fact that all four AMPA receptor subunits can associate with stargazin/TARPs via their intracellular and extracellular domains (Tomita et al., 2004).

Two prominent cytoplasmic domains of GluR2 involved in membrane trafficking during synaptic plasticity include the 4 amino acid PDZ-binding motif at the carboxy terminus and the juxtamembrane region (reviewed in Collingridge and Isaac, 2003 and references therein). The carboxy terminus binds to PDZ domains in two associated proteins, GRIP and PICK1. GRIP1 is thought to be required for AMPA receptor stabilization at synapses. Phosphorylation of GluR2 S880 causes GRIP to dissociate from GluR2, whereas the GluR2-PICK1 interaction is maintained after S880 phosphorylation. PICK1 is required for AMPA receptor trafficking during LTD in the cerebellar Purkinje neurons (Xia et al., 2000), and plays important roles, though not completely defined, in receptor endocytosis and surface expression in the hippocampus (Daw et al., 2000; Osten et al., 2000; Kim et al., 2001; Seidenman et al., 2003). The juxtamembrane of GluR2 binds to NSF and AP2. NSF, a protein involved in membrane fusion events, stabilizes AMPA receptors at postsynaptic membranes. Indeed, NSF competing peptides cause rundown of synaptic AMPA receptors, without altering total surface expression (Lee et al., 2002). The intrinsic ATPase activity of NSF is stimulated by binding to GluR2, which disrupts the GluR2-PICK1 interaction (Hanley et al., 2002).

Cerebellar stellate cell plasticity alters AMPA receptor subunit composition

A novel form of synaptic plasticity, Calcium-permeable AMPA Receptor Plasticity (CARP), has recently been identified in cerebellar stellate cells (Liu and Cull-Candy, 2000; Liu and Cull-Candy, 2002). These cells lack significant GluR1, thus plasticity is expressed by GluR2-, 3- and 4-containing AMPA receptors. During this form of plasticity the current-voltage relationship changes from inward rectifying to linear, due to replacement of Ca²⁺-permeable AMPA receptors, which lack GluR2, by Ca²⁺-impermeable GluR2-containing AMPA receptors. This plasticity is observed by a change in the extracellular postsynaptic currents of depolarized (+40mV clamp) or hyperpolarized (-60mV clamp) stellate neurons after high frequency stimulation. Namely, the I+40 currents, which are mediated by Ca²⁺-impermeable GluR2-containing AMPA receptor, are increased and the I-60 currents, mediated by both GluR2-lacking and GluR2-containing AMPA receptors, are reduced. These changes are due to an activity-dependent insertion of GluR2-containing AMPA receptors into the synapse, and concomitant loss of Ca²⁺-permeable AMPA receptors, while maintaining the approximate number of receptors constant. This contrasts with hippocampal synaptic plasticity, where AMPA receptors are either added or removed in response to potentiation or depression, altering the total number of receptors at the synapse. Several recent papers have helped elucidate the mechanism by which this receptor subtype switch occurs, and have highlighted the involvement of several familiar AMPA receptor binding proteins, NSF, GRIP and PICK1 (Gardner et al., 2005; Liu and Cull-Candy, 2005).

The role of GRIP1 in stellate cerebellar cells appears similar to its role in Purkinje neurons and hippocampal neurons, namely to maintain basal synaptic GluR2. Indeed, Liu and Cull-Candy found that using a peptide that interferes with both GRIP and PICK1 resulted in a 30% reduction in mEPSC over the course of 2 hours, whereas a peptide that interfered only with PICK1 did not show this effect. GRIP was not shown to be required for plasticity-induced increases in synaptic GluR2, as the peptide that interferes with both GRIP and PICK1 did not show a greater effect than the peptide that interfered only with PICK1.

Both Liu and Cull-Candy and Gardner et al. observed that peptides that specifically disrupt GluR2-PICK interactions prevented HFS-induced changes in EPSC amplitude. Thus, HFS failed to recruit GluR2 AMPA receptors when the interaction with PICK1 was acutely disrupted. These results were confirmed by analysis of mice lacking the last 7 amino acids at the GluR2 carboxy terminus and PICK1 null mutant mice (Gardner et al., 2005). Additionally, Gardner et al. found that extrasynaptic membranes had a lower rectification after addition of the PICK1-inhibitory peptide, demonstrating lower GluR2 levels. Analysis of mice lacking the last 7 amino acids at the GluR2 carboxy terminus and PICK1 null mutant mice supported this finding.

Interestingly, these results differ from results obtained when studying hippocampal plasticity and highlight how similar protein interactions can result in alternate signaling outcomes in different cell types. Indeed, in the hippocampus, viral overexpression of PICK1 resulted in increased AMPA receptor-mediated EPSC amplitude, due to a decrease in surface GluR2. Changes in surface GluR2 were detected as increased rectification at positive potentials and higher sensitivity to polyamine toxins (Terashima et al., 2004). These findings were confirmed by use of PICK1-blocking peptides. The difference in these activity-driven changes might stem from the fact that the two cell types begin with different GluR subunit composition. GluR1 is abundant in hippocampal cells, whereas cerebellar cells lack significant GluR1 subunits.

Using peptides that disrupt GluR2-NSF interaction, both Liu and Cull-Candy and Gardner et al. found that the rectification ratio (R+40/-60) did not increase after HFS. Thus the GluR2-NSF interaction is required for the replacement of GluR2-lacking by GluR2-containing AMPA receptors. In stellate cells, a model emerges in which at rest, the synapse is comprised of GluR2-containing and GluR2-lacking receptors, that are anchored in part by binding to GRIP. An extrasynaptic pool of PICK1-anchored GluR2 is at the ready. After HFS, GRIP is released from AMPA receptors due to PKC phosphorylation of the cytoplasmic tail of GluR3, potentially allowing for removal of GluR2-lacking receptors. NSF displaces PICK1 from extrasynaptic GluR2-containing receptors, thus allowing their lateral mobilization into the synapse.

Transmembrane stargazin controls AMPA receptor activity

In addition to cytoplasmic accessory proteins, GluR trafficking is also directed by binding to transmembrane proteins stargazin/TARPs. Stargazin was discovered through a spontaneous mutation in the stargazer mouse and was originally believed to be a calcium channel subunit (Letts et al., 1998; Klugbauer et al., 2000). Further research indicated that stargazin regulates AMPA receptor targeting in the cerebellar granule cells from which it was lacking (Chen et al., 2000). Stargazin is one of a large family of proteins that are structurally related to the γ subunit of calcium channels. There are three additional related proteins in the stargazin subfamily, γ-3, γ-4 and γ-8 (Klugbauer et al., 2000), which together make up the TARP family (Tomita et al., 2003). TARPs are present throughout the brain and have distinct but overlapping distribution. TARPs cluster with AMPA receptors at the synapse and are required for surface expression of the AMPA receptor (Tomita et al., 2003). Recently, it was demonstrated that rather than acting solely as a regulatory trafficking protein, stargazin is also an AMPA receptor auxiliary subunit that is required to form the surface expressed, functional channel in the cerebellum (Vandenberghe et al., 2005).

Now, recent work has for the first time described a role for stargazin as an auxiliary subunit controlling AMPA receptor channel biophysical properties. Tomita et al (Tomita et al., 2005) showed that injection of stargazin mRNA with AMPA receptor subunit mRNA into oocytes causes changes in receptor activity and desensitization. Expression of stargazin with AMPA receptors caused a large increase in the current recorded from the channel, which was much greater than the increase in the surface expression of the receptor. Additionally, stargazin increased the large conductance openings and burst length in response to glutamate. These results suggest that stargazin increased the efficacy of glutamate at the receptor. Experiments using a series of chimeras generated from stargazin and an inactive homologue, γ-5, showed that these effects on channel properties were mediated by the first extracellular region and the first transmembrane domain of stargazin. Viral mediated expression of a stargazin construct containing the extracellular region and first transmembrane domain from γ-5 in organotypic hippocampal slices showed that stargazin slows AMPA receptor desensitization and deactivation in addition to its effects on glutamate efficacy. Expression of this construct altered both the shape and size of the EPSC in the slice. These results indicate a functional effect of stargazin at the synapse. Additional analysis of the mutant constructs also allowed the determination of the regions of stargazin required for trafficking of AMPA receptors to the surface of the cell. Exchanging the cytoplasmic domain of the C-terminal tail of stargazin for the same region of γ-5 showed that it is the sole region responsible for the trafficking of AMPA receptors.

In results published shortly before the work from the Bredt laboratory, Priel et al also found that expression of stargazin with AMPA receptor GluR1 or GluR2 subunits significantly increased the response to glutamate and reduced receptor desensitization in both oocytes and HEK293 cells (Priel et al., 2005). Notably, these changes were not due to large increases in surface expression of the receptor determined by biotinlyation. Additionally, Priel et al examined stargazin mutants containing deletions of the PDZ binding domain and portions of the C-terminus and found no effect of these mutations on AMPA receptor desensitization. This work provides some confirmation of the results from Tomita et al.

In summary, the work presented here from several laboratories has given new insight into the roles accessory proteins play in both the trafficking and function of AMPA receptors. Associated proteins GRIP, PICK1 and NSF are involved in trafficking of GluR2 receptors, thereby altering the subunit composition of surface AMPA receptors and influencing channel properties related to subunit composition. The directionality of these changes appears to be specific to individual cell types; in hippocampal pyramidal cells, PICK1 can promote removal of GluR2 from synapses thereby increasing AMPA-mediated currents through GluR1-containing Ca²⁺ permeable receptors, whereas in cerebellar stellate cells activity drives GluR2 into the synapse through a pathway that requires PICK1, decreasing overall AMPA-mediated currents through replacement of Ca²⁺ permeable GluR3 and GluR4 receptors. Interestingly, in both cell types NSF and GRIP1 promote synaptic insertion and retention of GluR2. Through additional mechanisms that are not subunit-specific, interactions of AMPA receptors with stargazin/TARPs not only promotes cell surface and synaptic localization of the receptors but also increases AMPA-mediated currents by modifying the size and shape of the response to glutamate.

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