The role of the PDZ protein GIPC in regulating NMDA receptor trafficking.

The NMDA receptor is an important component of excitatory synapses in the CNS. In addition to its synaptic localization, the NMDA receptor is also present at extrasynaptic sites where it may have functions distinct from those at the synapse. Little is known about how the number, composition, and localization of extrasynaptic receptors are regulated. We identified a novel NMDA receptor-interacting protein, GIPC (GAIP-interacting protein, C terminus), that associates with surface as well as internalized NMDA receptors when expressed in heterologous cells. In neurons, GIPC colocalizes with a population of NMDA receptors on the cell surface, and changes in GIPC expression alter the number of surface receptors. GIPC is mainly excluded from the synapse, and changes in GIPC expression do not change the total number of synaptic receptors. Our results suggest that GIPC may be preferentially associated with extrasynaptic NMDA receptors and may play a role in the organization and trafficking of this population of receptors.

The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2.

The NMDA receptor (NMDAR) is a component of excitatory synapses and a key participant in synaptic plasticity. We investigated the role of two domains in the C terminus of the NR2B subunit--the PDZ binding domain and the clathrin adaptor protein (AP-2) binding motif--in the synaptic localization of NMDA receptors. NR2B subunits lacking functional PDZ binding are excluded from the synapse. Mutations in the AP-2 binding motif, YEKL, significantly increase the number of synaptic receptors and allow the synaptic localization of NR2B subunits lacking PDZ binding. Peptides corresponding to YEKL increase the synaptic response within minutes. In contrast, the NR2A subunit localizes to the synapse in the absence of PDZ binding and is not altered by mutations in its motif corresponding to YEKL of NR2B. This study identifies a dynamic regulation of synaptic NR2B-containing NMDARs through PDZ protein-mediated stabilization and AP-2-mediated internalization that is modulated by phosphorylation by Fyn kinase.

Export from the endoplasmic reticulum of assembled N-methyl-d-aspartic acid receptors is controlled by a motif in the c terminus of the NR2 subunit.

Functional N-methyl-d-aspartic acid (NMDA) receptors are formed from the assembly of NR1 and NR2 subunits. When expressed alone, the major NR1 splice variant and the NR2 subunits are retained in the endoplasmic reticulum (ER), reflecting a quality control mechanism found in many complex multisubunit proteins to ensure that only fully assembled and properly folded complexes reach the cell surface. Recent studies have identified an RRR motif in the C terminus of the NR1 subunit, which controls the ER retention of the unassembled subunit. Here we investigated the mechanisms controlling the ER retention of the NR2 subunit and the export of the assembled complex from the ER. We found that Tac chimeras of the C terminus of the NR2B subunit show that an ER retention signal is also present in the NR2B subunit. In assembled complexes, ER retention signals on the individual subunits must be overcome to allow the complex to leave the ER. One common mechanism involves mutual masking of the signals on the individual subunits. Our data do not support such a mechanism for regulating the release of assembled NMDA receptors from the ER. We found that the motif, HLFY, immediately following transmembrane domain 4 of the NR2 subunit, is required for the assembled complex to exit from the ER. Mutation of this motif allowed the assembly of NR1 and NR2 subunits into a complex that was functional, based on MK-801 binding, but it is retained in the ER. These results are consistent with HLFY functioning as a signal that is necessary for the release of the assembled functional NMDA receptor complex from the ER.

NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex.

NMDA (N-methyl-D-aspartate) receptors (NMDARs) are targeted to dendrites and anchored at the post-synaptic density (PSD) through interactions with PDZ proteins. However, little is known about how these receptors are sorted from the endoplasmic reticulum and Golgi apparatus to the synapse. Here, we find that synapse-associated protein 102 (SAP102) interacts with the PDZ-binding domain of Sec8, a member of the exocyst complex. Our results show that interactions between SAP102 and Sec8 are involved in the delivery of NMDARs to the cell surface in heterologous cells and neurons. Furthermore, they suggest that an exocyst-SAP102-NMDAR complex is an important component of NMDAR trafficking.

PSD-95 regulates NMDA receptors in developing cerebellar granule neurons of the rat.

We transfected a green fluorescent protein-tagged PSD-95 (PSD-95gfp) into cultured rat cerebellar granule cells (CGCs) to investigate the role of PSD-95 in excitatory synapse maturation. Cells were grown in low potassium to favour functional synapse formation in vitro. Transfected cells displayed clear clusters of PSD-95gfp, often at the extremities of the short dendritic trees. We recorded NMDA and AMPA miniature excitatory postsynaptic currents (NMDA- and AMPA-mESPCs) in the presence of TTX and bicuculline. At days in vitro (DIV) 7-8 PSD-95gfp-transfected cells had NMDA-mEPSCs with faster decay and smaller amplitudes than matching controls. In contrast, AMPA-mEPSC frequencies and amplitudes were increased. Whole-cell current density and ifenprodil sensitivity were reduced in PSD-95gfp cells, indicating a reduction of NR2B subunits containing NMDA receptors. No changes were observed compared to control when cells were transfected with cDNA for PSD-95gfp with palmitoylation site mutations that prevent targeting to the synapse. Overexpression of the NMDA receptor NR2A subunit, but not the NR2B subunit, prevented NMDA-mEPSC amplitude reduction when cotransfected with PSD-95gfp. PSD-95gfp overexpression produced faster NMDA-mEPSC decay when transfected alone or with either NR2 subunit. Surface staining of the epitope-tagged NR2 subunits revealed that colocalization with PSD-95gfp was higher for flag-tagged NR2A subunit clusters than for flag-tagged NR2B subunit clusters. These data suggest that PSD-95 overexpression in CGCs favours synaptic maturation by allowing synaptic insertion of NR2A and depressing expression of NR2B subunits.

Trafficking of NMDA receptors.

The NMDA receptor (NMDAR) plays a central role in the function of excitatory synapses. Recent studies have provided interesting insights into several aspects of the trafficking of this receptor in neurons. The NMDAR is not a static resident of the synapse. Rather, the number and composition of synaptic NMDARs can be modulated by several factors. The interaction of PDZ proteins, generally thought to occur at the synapse, appears to occur early in the secretory pathway; this interaction may play a role in the assembly of the receptor complex and its exit from the endoplasmic reticulum. This review addresses recent advances in our understanding of NMDAR trafficking and its synaptic delivery and maintenance.

Relationship between availability of NMDA receptor subunits and their expression at the synapse.

The effect of increasing the expression of NMDA subunits in cerebellar granule cells (CGCs) by transfection was studied to determine how the availability of various NMDA subunits controls both the total pool of functional receptors and the synaptic pool. Overexpression of either NR2A or NR2B, but not splice variants of NR1, by transfection caused a significant increase in the total number of functional NMDA receptors and in surface NR1 subunit cluster density in CGCs in primary culture. These data solidify the central role of NR2 subunit availability in determining the number of cell surface receptors. Overexpression of either NR2A or NR2B significantly altered the deactivation kinetics of NMDA-mediated miniature EPSCs (NMDA-mEPSCs). However, there was no significant effect of NR2 subunit overexpression on the mEPSC amplitude or single-channel conductance. NR2 subunit overexpression did not change the rate of block by MK-801 of NMDA-mediated currents in excised patches from CGCs, indicating that subunit composition does not regulate peak open probability of the channel in CGCs. With the overexpression of a mutant of NR2B lacking the PDZ binding domain, there was an increase in the total number of NMDA receptors without a change in mEPSC kinetics. Therefore, the entry of NMDA receptors into the synapse requires a PDZ binding domain and is limited by means other than receptor subunit availability.

Silent synapses in developing cerebellar granule neurons.

Silent synapses are excitatory synapses endowed exclusively with N-methyl-D-aspartate (NMDA) responses that have been proposed to acquire alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) responses during development and after long-term potentiation (LTP). These synapses are functionally silent because of the Mg(2+) block of NMDA receptors at resting potentials. Here we provide evidence for the presence of silent synapses in developing cerebellar granule cells. Using the patch-clamp technique in the whole-cell configuration, we recorded the spontaneous excitatory postsynaptic currents (sEPSCs) from rat cerebellar granule cells in culture and in slices at physiological concentration of Mg(2+) (1 mM). A holding potential of +60 mV removes Mg(2+) block of NMDA channels, allowing us to record NMDA-sEPSCs. We thus compared the frequency of AMPA-sEPSCs, recorded at -60 mV, with that of NMDA-sEPSCs, recorded at +60 mV. NMDA-sEPSCs occurred at higher frequency than the AMPA-sEPSCs in most cells recorded in slices from rats at postnatal day (P) <13 and in culture at 6-8 days after plating (DIV6-8). In a few cells from young rats (P6-9) and in most neurons in culture at DIV6 we recorded exclusively NMDA-sEPSCs, supporting the hypothesis of existence of functional synapses with NMDA and without AMPA receptors. Increasing glutamate release in the slice with cyclothiazide and temperature increased AMPA and NMDA-sEPSCs frequencies but failed to alter the relative ratio of frequency of occurrence. Frequency ratio of NMDA versus AMPA-sEPSCs in slices was correlated with the weighted time constant of decay (tau(w)) of NMDA-sEPSCs and decreased with development along the reported decrease of tau(w). We suggest that the prevalence of synaptic NR2A subunits that confer faster kinetics is paralleled by the disappearance of silent synapses early in cerebellar development.