SUMOylation and phosphorylation of GluK2 regulate kainate receptor trafficking and synaptic plasticity

Summary Phosphorylation or SUMOylation of the kainate receptor (KAR) subunit GluK2 have both individually been shown to regulate KAR surface expression. However, it is unknown if phosphorylation and SUMOylation of GluK2 are important for activity-dependent KAR synaptic plasticity. Here, we show that PKC-mediated phosphorylation of GluK2 at serine 868 promotes GluK2 SUMOylation at lysine 886 and that both these events are necessary for the internalization of GluK2 containing KARs that occurs during long-term depression of KAR-mediated synaptic transmission at rat hippocampal mossy fiber synapses. Conversely, phosphorylation of GluK2 at serine 868 in the absence of SUMOylation leads to an increase in KAR surface expression by facilitating receptor recycling between endosomal compartments and the plasma membrane. Thus, we describe a role for the dynamic control of synaptic SUMOylation in the regulation of KAR synaptic transmission and plasticity.


Introduction
Synaptic plasticity is a cellular correlate of learning and memory and occurs through the processes of long-term potentiation (LTP) or long-term depression (LTD) 1 . Changes in synaptic strength can be achieved by increases or decreases in the surface expression of neurotransmitter receptor proteins which has been shown to depend on interactions with specific intracellular binding partners. These interactions are often regulated by posttranslational receptor modifications such as phosphorylation [2][3][4] .
KARs are ionotropic glutamate receptors made up of tetrameric arrangements of five subunits (GluK15, previously known as GluR57 and KA12) and are found pre-and post-synaptically at many synapses throughout the brain [5][6] . KAR surface expression at the plasma membrane depends on protein interactions at the intracellular c-termini of the receptor subunits [7][8][9] , for example, the PDZ domain-containing proteins PICK1 and GRIP [10][11] . In GluK2, amino acids 868-880 in the c-terminus are important for surface expression in heterologous systems or cultured neurons 9, 12 . Phosphorylation of KARs by PKC can enhance or depress KAR-mediated synaptic transmission 10,[13][14][15][16] and is involved in LTD in DRG neurons 16 , the perirhinal cortex 13,15 , and the hippocampus 14 . More recently, it has been shown that GluK2 can be SUMOylated in response to agonist binding 17 , which leads to internalisation of GluK2 containing KARs. SUMOylation is a post-translational modification of lysine residues similar to ubiquitination, whereby a member of the Small Ubiquitin-like Modifier (SUMO) family is conjugated to target proteins, altering substrate function. SUMOylation is emerging as a major regulator of nuclear and extranuclear protein function in neurons [18][19][20] . While the mechanisms that modulate SUMOylation remain largely unknown, protein phosphorylation has been reported to either enhance or inhibit SUMOylation, depending on the substrate protein [21][22][23] .
The SUMOylation site at lysine 886 (K886) in GluK2 is located near to the PKC phosphorylation sites at serine 846 (S846) and 868 (S868). It has been demonstrated that PKC phosphorylation of S868 but not S846 promotes SUMOylation of K886 24 . We therefore hypothesised that PKC phosphorylation of GluK2 may control SUMOylation and subsequent removal of GluK2 containing KARs during activity dependent LTD. We show that phosphorylation and SUMOylation are necessary for the PKC-dependent LTD of KARs at mossy fiber-CA3 synapses in the hippocampus. Furthermore, phosphorylation of S868 in the absence of SUMOylation results in an increase in KAR recycling between endosomal compartments and the plasma membrane.

Phosphorylation promotes SUMOylation and loss of KARs
We have previously demonstrated SUMOylation of the KAR subunit GluK2 at hippocampal mossy fiber synapses 17 . The functional consequences of SUMOylation were a removal of KARs from the postsynaptic membrane leading to a decrease in KAR-mediated synaptic transmission. Here we reproduce this finding by recording pharmacologically isolated KARmediated, synaptically evoked excitatory postsynaptic currents (KAR EPSCs) from CA3 neurons in hippocampal slices and included either SUMO-1 (active SUMO, 4.2 μM) or the conjugation-deficient mutant SUMO-1-ΔGG (inactive SUMO, 4.2 μM) in the recording pipette. We confirmed that inclusion of SUMO-1 caused a rapid decrease in the amplitude of the KAR EPSC to 46.6 ± 5.0% ( Fig. 1a; n = 13; P < 0.001) of the initial amplitude obtained within 1 minute of rupturing the membrane inside the patch electrode whereas inclusion of SUMO-1-ΔGG had no effect on KAR EPSC amplitude ( Fig. 1a; 103.4 ± 11.2%; n = 9; P > 0.05).
Phosphorylation of proteins can either facilitate or inhibit SUMOylation 21-23 and PKCmediated phosphorylation of KARs regulates their subcellular localisation [13][14]25 . Since PKC-mediated phosphorylation of GluK2 promotes GluK2 SUMOylation 24 , we reasoned that activation of PKC should facilitate, and inhibition reduce, the effects of SUMO on KAR EPSCs. To test this, we recorded KAR EPSCs from CA3 neurons following pre-incubation of the slices in either PMA (1 μM) or chelerythrine (5 μM) for a minimum of 15 minutes. In the presence of PMA (1 μM), inclusion of active SUMO in the recording pipette decreased the amplitude of KAR EPSCs to 22.9 ± 4.7%, a greater effect than seen in control conditions ( Fig. 1b; n = 8; P < 0.05). In addition, in the presence of chelerythrine (5 μM), active SUMO no longer had any effect ( Fig. 1b; 98.2 ± 6.0%, n=8; P>0.05). These results support the hypothesis that phosphorylation of GluK2 promotes SUMOylation and further suggests that phosphorylation is required for SUMO-dependent internalization of GluK2.
To determine the sites of phosphorylation and SUMOylation of GluK2 that regulate the surface expression of KARs, we expressed recombinant wild-type or mutated GluK2 where the putative PKC phosphorylation site S868 [24][25] was altered to either a phosphomimetic aspartate or a non-phosphorylatable (phosphonull) alanine or the SUMOylation site K886 was altered to a non-SUMOylatable (SUMOnull) arginine 24 . We first characterised kainate responses in HEK cells by expressing wild-type (WT) GluK2. This produced stable KARmediated response amplitudes to 300 ms applications of 100 μM kainate. Inclusion of inactive SUMO (SUMO-1-ΔGG) in the patch pipette had no effect on response amplitude ( Fig. 2a; 106.3 ± 5.1%; n = 7; P > 0.05) but inclusion of active SUMO in the recording pipette induced a rapid depression of response amplitude ( Fig. 2a; 52.5 ± 3.6%; n = 6; P < 0.0001). The speed of depression was faster than that seen in neurons but the magnitude was similar. The depression of KAR-mediated responses was directly due to SUMOylation of GluK2 as neither active nor inactive SUMO had any effect on KAR-mediated responses in HEK cells expressing the non-SUMOylatable (SUMOnull) GluK2 mutant K886R 17 ( Fig.  2b; 106.6 ± 8.9% and 100.5 ± 12.6% inactive and active SUMO respectively; n = 6 for each; P > 0.05).
We next used the phosphomimetic and non-phosphorylatable mutations of serine 868 to test the role of phosphorylation in SUMO-mediated removal of surface KARs. In HEK cells expressing the S868A (phosphonull) GluK2 mutant, infusion of active SUMO via the recording pipette had no significant effect on the KAR mediated responses when compared to the inactive control ( Fig. 2c; 98.2 ± 9.4% vs. 105.0 ± 8.3% inactive and active SUMO respectively; n = 6 for each; P > 0.05). However, in HEK cells expressing the S868D (phosphomimetic) GluK2 mutant, infusion of active SUMO caused a depression in KARmediated responses to 27.8 ± 3.5% (n = 6)compared to inactive SUMO ( Fig. 2d; vs. 142.5 ± 11.2%; n = 6; P < 0.001), but not different from infusion of active SUMO with wild-type GluK2 (Fig. 2a). These data suggest that phosphorylation of GluK2 at S868 is required for SUMO-mediated removal of KARs from the plasma membrane.
A previous study from our labs reported that phosphorylation of S868 can enhance SUMOylation of GluK2 in Cos-7 cells 24 . To confirm this finding we quantified the amount of SUMOylated GluK2 in HEK cells expressing wild-type GluK2 or the S868A, S868D or K886R mutants. Similar to the situation in neurons, some SUMOylation of wild-type GluK2 was detectable under basal conditions. However, SUMOylation of the S868D phosphomimetic mutant was increased compared to the wild-type ( Supplementary Fig. 1), suggesting that phosphorylation of S868 enhances SUMOylation of GluK2.

Phosphorylation of GluK2 increases KAR EPSC amplitude
Surprisingly, infusion of inactive SUMO into HEK cells expressing the phosphomimetic S868D mutant of GluK2 led to an increase in the amplitude of the KAR-mediated current when compared to wild-type ( Fig. 2d; 142.5 ± 11.2% vs. 106.3 ± 5.1%; P < 0.05). These data suggest that phosphorylation of S868 combined with receptor activation may increase surface expression of GluK2, which would directly oppose the increased removal of GluK2 by SUMOylation. Consistent with this interpretation, PMA (1 μM) caused an increase in the amplitude of the KAR EPSC recorded from CA3 neurons to 139.3 ± 12.2% ( Fig. 3a; n = 7; P < 0.05). Furthermore, the PKC inhibitor chelerythrine (5 μM) caused a decrease in KAR EPSC to 68.5 ± 8.0% ( Fig. 3b; n = 8; P < 0.01). PKC inhibition by infusion of the PKC inhibitory peptide PKC 19-36 also caused a decrease in KAR EPSC confirming the role of PKC inhibition (Supplementary Fig. 2a; 57.4 ± 12.4%; n = 5; P < 0.05).
To investigate this further we recorded from HEK cells expressing GluK2 and applied PMA. Perfusion of PMA (1 μM) onto HEK cells expressing wild-type GluK2 led to a significant increase in the amplitude of the KAR current to 170.3 ± 23.6% ( Fig. 3c; n = 6; P < 0.05).
Perfusion of PMA (1 μM) onto HEK cells expressing the non-SUMOylatable K886R GluK2 mutant also led to an increase in KAR-mediated response amplitude to 178.3 ± 38.3% ( Fig.  3d; n = 6; P < 0.05). Interestingly, whereas the increase was not significant after 10 minutes of PMA application in HEKs expressing wild-type GluK2 ( Fig. 3e; 146.0 ± 20.3%; n = 6; P > 0.05), it was significant after 10 minutes of PMA application in HEKs expressing K886R ( Fig. 3e; 184.0 ± 40.6%; n = 6; P < 0.05) suggesting that the increase is faster in HEKs expressing the non-SUMOylatable K886R GluK2 mutant. We also tested the effects of PMA (1 μM) perfusion on HEK cells expressing the S868A non-phosphorylatable mutant. In this case, PMA no longer led to an increase in amplitude of the KAR current but to a small decrease ( Fig. 3f; 77.5 ± 8.4%; n = 5; P > 0.05). These data indicate that, in the absence of SUMOylation, PKC-mediated phosphorylation of GluK2 at S868 leads to an increase in GluK2 surface expression.

GluK2 phosphorylation regulates kainate receptor recycling
Our results implicate PKC in both increased and decreased GluK2 surface expression. We therefore reasoned that PKC phosphorylation might be involved in both exo-and endocytosis components of GluK2 trafficking. We first addressed whether the PKCdependent increase in the amplitude of the KAR EPSC is due to changes in the rate of delivery and surface expression of de-novo synthesised GluK2 in neurons. We used Sindbis virus to express SEP-GluK2 26 in neurons and monitored the dynamics of SEP-GluK2 membrane insertion using anti-GFP antibody labelling. In these experiments live neurons were first incubated with unlabelled anti-GFP antibody for 5 minutes occupy and block the GFP epitope on all existing surface expressed SEP-GluK2. Following washing, to remove all unbound unlabelled anti-GFP antibody, the amount of newly inserted SEP-GluK2 was assessed at 0, 10 or 20 min using anti-GFP antibody directly coupled to Alexa594. We found that the amount of SEP-GluK2 inserted into the plasma membrane was unaffected after 10 or 20 min treatment with PMA or chelerythrine compared to control (Fig. 4a). As expected, inhibition of protein synthesis with cycloheximide prevented any increase in GluK2 inserted into the plasma membrane. These results suggest that PKC activation does not affect the rate of insertion of de-novo GluK2 in neurons.
Since inserting newly synthesised receptors does not account for the increased surface expression of GluK2 we investigated whether there was increased recycling to the plasma membrane from early endosomes 27 by functional colocalization analysis with the transferrin receptor (TfR). In the presence of transferrin, the TfR is internalized into early endosomes and then constitutively sorted to recycling endosomes 28 . As expected, after 5 min of pulse and 15 min of chase in the presence of either chelerythrine or vehicle, Alexa594-conjugated transferrin (Alexa-Trf) was localised predominantly in intracellular endosomes with a moderate colocalization with GluK2 (Fig. 4b). In the presence of the PKC inhibitor chelerythrine, GluK2 showed limited colocalization with TfR (Pearson's coefficient 0.38 ± 0.01, n = 28) and this level of colocalization was increased in cells treated with PMA (Pearson's coefficient 0.64 ± 0.01, n = 31). This suggests PKC activation increases the proportion of GluK2 sorted into recycling pathways. Intermediate levels of colocalization between GluK2 and TfR were observed under control conditions (Pearson's coefficient 0.55 ± 0.04, n = 25), however this value was highly variable, likely due to differences in basal PKC activity.
Receptor recycling consists of both activity-dependent and independent components 29 . In the presence of tetrodotoxin (TTX), to block the activity-dependent component of GluK2 recycling, the colocalization of GluK2 with TfR was significantly decreased (Pearson's coefficient 0.44 ± 0.03, n = 19). Interestingly, this level of colocalization in the presence of TTX was unaffected by the addition of chelerythrine (Pearson's coefficient 0.38 ± 0.09, n = 31, P > 0.05), suggesting that chelerythrine acts on the activity-dependent component of GluK2 recycling, leaving basal recycling unaffected. Thus, these results suggest that PKC phosphorylation regulates activity-dependent recycling of GluK2.
An increase in receptor recycling leads to greater numbers of receptors in early endosomal compartments. Therefore, PKC phosphorylation of GluK2 is expected to increase the amount of GluK2 associated with early endosomes. Consistent with our hypothesis, addition of PMA to cultured neurons transduced with wild-type GluK2 led to an increase in GluK2 colocalization with the early endosomal marker EEA1 ( Fig. 4c; Pearson's coefficient 0.42 ± 0.02 in control to 0.53 ± 0.04 (n = 12-15, P = 0.004). In contrast, PMA had no effect on the colocalization of EEA1 with S868A, S868D or K886R GluK2 mutants. Interestingly, in the absence of PMA the K886R GluK2 mutant showed an increased colocalization in comparison to wild-type (p < 0.001) whereas the phosphomimetic S868D GluK2 mutant showed a small decrease (p<0.05) and the non-phosphorylatable S868A GluK2 mutant showed no change (p>0.05). Importantly, the pattern of GluK2 expression was not dramatically altered by GluK2 mutations when colocalized with PSD95 staining (WT -0.57 ± 0.02, n = 10; S868A -0.59 ± 0.03 n = 10; S868D -0.55 ± 0.08, n = 10; K886RR -0.56 ± 0.02, n = 15) and the pattern of EEA1 expression was not affected by application of PMA (Fig. 4c), although chronic chelerythrine treatment did change EEA1 staining to a less punctate distribution ( Supplementary Fig 3) preventing analysis of mutant GluK2 colocalization with EEA1 under these conditions. These data support the conclusion that PKC phosphorylation of S868 increases recycling of GluK2 between the plasma membrane and early endosomal compartments.
To address the influence of PKC activation on GluK2 recycling we compared recycling in neurons incubated in PMA or chelerythrine. Using live-cell antibody feeding we quantified the recycling of SEP-GluK2, again using SEP as an extracellular epitope for anti-GFP antibody. Neurons were incubated with Alexa594 coupled to anti-GFP antibody together with PMA or chelerythrine for 10 mins to allow internalisation of surface labelled receptors. The Alexa594-coupled anti-GFP antibody was then stripped from any remaining surface receptors and the reappearance (recycling) of Alexa594-tagged SEP-GluK2 at the plasma membrane after 10 or 20 min (Fig. 5a). The intracellular signal from internalized Alexa594tagged SEP-GluK2 was diffuse and largely below detection threshold and was subtracted for analysis (see methods). The amount of Alexa594-tagged SEP-GluK2 that was previously surface expressed then internalised and returned back to the surface was increased at 10 and 20 min in cells treated with PMA. In contrast, GluK2 recycling was undetectable in cells treated with chelerythrine (Fig. 5a). These data indicate that PKC activation increases the rate of GluK2 recycling in neurons.
We further confirmed the effect of PKC phosphorylation of GluK2 on recycling using primaquine, an inhibitor of exocytosis from early endosomes 30 . Application of primaquine to slices produced a dose-dependent decrease in KAR EPSC amplitude. 100 μM primaquine decreased KAR EPSC amplitude to 6.0 ± 1.1% after 10 minutes whereas 1μM primaquine decreased KAR EPSC amplitude to 57.0 ± 8.5% that stabilised after 60 minutes ( Supplementary Fig. 4). Application of PMA following 1 hour pre-incubation with 1 μM primaquine failed to elicit an increase in KAR EPSC ( Fig. 5b; 106.8 ± 6.5; n = 7; P > 0.05) in contrast to control conditions (data from Figure 3a is reproduced for comparison) To establish that PMA exerts its effects directly via phosphorylation of GluK2, we used livecell antibody feeding experiments to quantify the PKC-dependent recycling in HEK cells expressing WT or the non-phosphorylatable S868A GluK2 mutant. We initially confirmed that GluK2 is recycled in HEK cells by comparing the colocalization of GluK2 and TfR ( Supplementary Fig. 5) 17 . In HEK cells expressing WT GluK2, application of PMAincreased the amount of recycled (reinserted) GluK2 at 10 and 20 min. In contrast, GluK2 recycling was undetectable in control cells or cells treated with chelerythrine (Fig.  6a). However, in HEK cells expressing the non-phosphorylatable S868A GluK2 mutant, there was no detectable reinsertion in the presence of either PMA or chelerythrine (Fig. 6b) demonstrating that phosphorylation of this specific residue on GluK2 is required for the enhancement of GluK2 recycling by PKC.

Phosphorylation and SUMOylation are required for KAR LTD
Phosphorylation of KARs at mossy fiber-CA3 synapses is involved in KAR LTD 14 but the possible contribution of SUMOylation has not been investigated. Indeed, it is unknown if SUMOylation is involved in any form of activity-dependent synaptic plasticity. We induced KAR LTD by stimulating mossy fiber synapses for 5 minutes at a frequency of 1 Hz whilst maintaining the membrane potential at −70 mV. This produced a reduction in KAR EPSC amplitude to 56.1 ± 1.0% 30 minutes after induction of LTD ( Fig. 7a; n = 9; P < 0.001). In this, and all other LTD experiments, there were no changes in paired pulse ratio after induction of LTD ( Fig. 7a; 1.26 ± 0.15 vs 1.34 ± 0.19, baseline vs LTD respectively; n = 9; P > 0.05). Similar to previous findings 14 , KAR LTD was not dependent on changes in postsynaptic [Ca 2+ ] since it was insensitive to inclusion of BAPTA (10 mM) in the patch solution ( Fig. 7b; 63.9 ± 3.4%, n = 7; P < 0.05), but was sensitive to blockade of mGluR5 receptors by bath perfusion with 30 μM MPEP ( Fig. 7c; 94.3 ± 12.5%, n = 8; P > 0.05). We also confirmed that PKC phosphorylation is required for KAR LTD since inclusion of the PKC inhibitor peptide PKC 19-36 (4.8 μM)in the recording pipette, but not the inactive (Glu27)PKC 19-36 peptide 31 (4.8 μM), blocked KAR LTD ( Fig. 7d; 100.1 ± 7.1%; n = 7; vs. 44.9 ± 4.9%; n = 5; P < 0.05). This was confirmed using chelerythrine (5 μM), which also completely blocked KAR LTD ( Supplementary Fig. 2b).

Discussion
Our results demonstrate that PKC phosphorylation of GluK2 is necessary for SUMOylationdependent internalisation of KARs from the synaptic membrane. Thus, we provide evidence for the dynamic interaction of two forms of post-translational protein modification controlling the subcellular localisation of KARs. Further, we show that this is the mechanism for the removal of KARs from the postsynaptic membrane during activitydependent LTD.
The observations that the phophomimetic GluK2 mutant shows an increase in KAR EPSC under baseline stimulation conditions (Fig. 2d) and that PMA causes an increase in KAR responses in HEK cells expressing wild-type or the non-SUMOylatable GluK2 mutant (Fig.  3c,d), but not the non-phosphorylatable GluK2 mutant (Fig. 3f), suggest that phosphorylation of S868 is also involved in non-SUMOylation dependent trafficking of KARs. This is further supported by the increase in KAR recycling between the plasma membrane and intracellular compartments after phosphorylation of GluK2 at S868 (Figs. 4, 5 and 6). We propose that increased recycling leads to an increase in surface expressed KARs. Interestingly, either preincubation in PMA (Fig. 1c) or expression of the phosphomimetic S868D GluK2 mutant (Fig. 2d) produced a greater decrease in KAR response amplitude when SUMO was applied compared to control conditions. This suggests that PKC phosphorylation in the absence of SUMOylation promotes recycling and increased surface insertion of KARs but can also promote enhanced endocytosis through SUMOylation of GluK2 17 . Thus, we propose that SUMOylation is the molecular switch that determines either enhanced or decreased KAR surface expression.
We show that phosphorylation of GluK2 at S868 by PKC has dual, competing effects on KAR localisation. Phosphorylation of GluK2 increases recycling and the proportion of KARs on the plasma membrane, but also promotes SUMOylation that leads to a loss of surface KARs. This is highlighted by the observation that PMA increases KAR EPSC amplitude faster in the SUMOnull K886R GluK2 mutant than in wild-type GluK2 (Fig.  3c,d). In addition, PMA causes an increase in KAR EPSC amplitude in CA3 neurons while chelerythrine leads to a decrease (Fig. 3a,b) strongly suggesting that the increase in receptor insertion outweighs the increase in SUMOylation-dependent internalisation under these conditions. Intriguingly, the equilibrium between these two events may help to explain why, in different recording conditions, different studies have shown opposing effects of PKC inhibitors at mossy fibre CA3 synapses 10,14 . Phosphorylation of S846 by PKC has been shown to increase KAR endocytosis 25 even though it does not promote SUMOylation of K886 24 . Our data suggest that the phosphorylation of S868 and subsequent SUMOylation of K886 override the role of S846 phosphorylation. Furthermore, phosphorylation of the S868 residue on GluK2 has also been shown to increase KAR retention in the ER 25 providing evidence for an additional role of S868 phosphorylation in controlling KAR localization.
Crucially, we show that SUMOylation is critical for KAR LTD at mossy fiber-CA3 synapses. The degree of depression seen after LTD (Figs. 7 and 8) is comparable to the proportion of KARs internalised after infusion of SUMO into the cell (Fig. 1a). This suggests that the inclusion of SUMO in the patch solution occludes LTD via prior removal of SUMO sensitive KARs (Fig. 8a). Conversely, infusion of SENP blocks LTD by removing SUMO from GluK2 before KARs can be internalised (Fig. 8b). The dominant negative effect of transduced GluK2 (Fig. 8) implies that expressed mutant GluK2 supplants endogenous GluK2 at mossy fiber synapses. Interestingly, KAR LTD at mossy Fiber-CA3 synapses has also been shown to require the destabilisation of surface KARs containing the GluK5 subunit by SNAP-25 14 . Our results do not directly address the role of GluK5 but taken together these results suggest that KARs removed from the synaptic membrane during LTD contain both GluK2 and GluK5 and that endocytosis during LTD requires dual synergistic processes initiated by PKC phosphorylation that leads to both the binding of PICK1 and SNAP-25 to GluK5 and the SUMOylation of GluK2.
In conclusion, our results demonstrate a role for SUMOylation in synaptic plasticity of KARs at mossy fiber synapses in the hippocampus opening the possibility that this form of post-translational protein modification may play a role in other forms of synaptic plasticity. SUMO proteins and the enzymes required for SUMO conjugation are found throughout dendrites and are concentrated at spines 17 and accumulating evidence suggests that many synaptic proteins other than KARs are also SUMOylated 32 . Thus, post-translational protein modifications such as SUMOylation, phosphorylation and ubiquitination may dynamically interact to coordinate neurotransmitter receptor localisation and function.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Electrophysiological recordings in hippocampal slices
Transverse hippocampal slices (400-500 μm thickness) were prepared from p14 Wistar rats. Slices were cut in ice cold artificial cerebrospinal fluid (aCSF) containing (in mM): 119 NaCl, 10 Glucose, 26 NaHCO 3 , 2. All data were sampled at 10 kHz, filtered at 5 kHz and recorded onto computer using Signal software (CED). Analyses were performed with scripts written in this software environment. Data are presented as mean ± s.e.m. Values used for statistical analyses were taken from the same timepoints as example traces. Statistical significance was determined using paired or unpaired two-tailed Student's t-tests as appropriate. Experiments were performed at room temperature.

Receptor Exocytosis Experiments
Dissociated hippocampal neuronal cultures were pretreated with PMA, chelerythrine or vehicle for 15 min at 37°C prior to live-cell imaging experiments in the continued presence of the drugs. At the beginning of the experiment, GFP reactive sites on the cell surface of neurons expressing SEP-GluK2 were blocked by pretreatment with an excess of unlabeled anti-GFP rabbit polyclonal antibody (rAb; 1:200, Invitrogen) at room temperature for 5 min (rather than at 37°C, in order to limit receptor endocytosis). Neurons were immediately placed on a microscope stage heated to 37 °C and incubated with diluted anti-GFP rAb directly coupled to Alexa594 (anti-GFP-Alexa594, 1:500) for 2 min to visualise the plasma membrane. Neurons were quickly washed twice and the fluorescence at time zero acquired. Cells were incubated either a further 10 or 20 min in the presence of PMA, chelerythrine or vehicle and then reprobed with anti-GFP-Alexa594 (1:200), rapidly washed twice and imaged to determine the SEP-GluK2 inserted during the 10 or 20 min incubation interval.

Receptor Recycling Experiments
SEP-GluK2 recycling was quantified by live-cell antibody feeding in the presence of PMA or chelerythrine. Cells were incubated in PMA or chelerythrine for 10 min with anti-GFP-Alexa594 (1:200) at 37 °C to allow the endocytosis of the labelled surface receptors. Cells were washed twice in aCSF and the total surface SEP-GluK2 determined by confocal microscopy. Cells were then washed with stripping buffer (recording buffer pH 3.2 plus MesNa 50 mM) for 30 seconds. Because MesNa is membrane impermeant it removes anti-GFP antibody from surface expressed SEP-GluK2 but not from SEP-GluK2 that has been internalised. Immediately after stripping, the cells were imaged (t = 0) and then incubated at 37 °C for 10 min and 20 min (t = 10 and t = 20) to allow recycling to occur in the presence of either PMA or chelerythrine.
The red fluorescence corresponding to anti-GFP labelled SEP-GluK2 returning to the plasma membrane was recorded at time 0, 10 and 20 min from the same cell as a series of Z stacks (0.25 μm spacing between single confocal slices) using a Zeiss LSM 510 confocal laser-scanning station with an oil-immersion 63× 1.4 NA objective (Zeiss). Alexa594 was visualized using a 543 nm HeNe laser line and a 585 nm long-pass filter, whereas SEP was excited with the 488 nm laser line from an argon-krypton laser and the emitted light was detected using a 505-550 nm band pass filter.
The efficiency of the stripping was normalized in each cell as the ratio of fluorescence prestripping/post-stripping. If the efficiency of stripping was not greater than 40%, then the analysis did not continue. Differences in expression were normalized to the mean of the fluorescence at time 0 and the rate of receptor reappearance at the cell surface within individual cells was then determined. To ensure that the red fluorescence from the area of interest came only from surface-expressed KARs all SEP-GluK2 experiments included a brief (10 s) pH 6 wash at the end of the experiment to quench surface SEP-GluK2 fluorescence. Any areas of interest that retained SEP fluorescence during the pH 6 wash were excluded from the analysis of the red Alexa594 fluorescence ( Supplementary Fig. 5).
In HEK cells, the same control was performed by incubating the cells with anti-rabbit Cy5 at the end of the experiment for 2 mins at room temperature ( Supplementary Fig. 6). Statistical analysis of differences between experimental groups was performed using one way ANOVA followed by post-hoc Tukey test calculated using SigmaStat software. Data are presented as mean ± SD.

GluK2 colocalization analysis
48 hrs after transfection, HEK cells grown on coverslips were washed and pre-incubated with serum free DMEM for 2 hrs. For transferrin assays in neurons, the neurons were preincubated with B27 free Neurobasal during 1h. HEK293 or neurons were then incubated with 10 μg ml −1 of Alexa594 conjugated transferrin (Molecular Probes) in serum/B27-free medium for 5 min at room temperature. Cells were then washed with PBS twice and shifted to 37°C for 20 min to label recycling endosomes in the presence of PMA, chelerythrine or vehicle. Following incubation the cells were washed with PBS twice and fixed in 4.0% paraformaldehyde. For the EEA1 GluK2 colocalization experiment, 24 h after infection, the neurons were incubated with PMA, chelerythrine or vehicle for 30 min. For EEA1 or PSD95 GluK2 colocalization neurons were then washed with PBS, fixed in 4.0% paraformaldehyde and stained with specific primary antibodies against EEA1(BD Biosciences) or PSD95 (Millipore).
Three-dimensional volumes of z stacks (0.25 μm spacing between single confocal slices) were analysed using Image processing and analysis in Java (ImageJ). The degree of colocalization was assessed in whole cell volumes and sub-volumes by calculating the Pearson's correlation coefficient in the region of interest using a semiautomated algorithm embedded in the JaCoP plugin 33 of the ImageJ software. The colocalization plugin also performed a two-step analysis to calculate the Pearson's correlation coefficient for the original data and for a large set (approximately 500) of images randomized with a grain size determined by the point spread function of the microscope objective. If the Pearson's correlation coefficient of the original image was not greater than 95% of the randomized images, then the samples were not used. In addition, cells displaying saturated or low, nearthreshold signals were discarded, and user bias in setting analysis parameters was avoided by using an automated thresholding procedure 33 . Histograms presenting the mean correlation coefficient (derived from 19 to 31 cells assessed per treatment condition) are shown with standard deviation bars in all figures. Statistical significance for differences between paired combinations of images was calculated using the two-tailed Student's t-test.

GluK2 SUMOylation in HEK cells
HEK cells in 35mm dishes were transfected with 1μg plasmid DNA encoding YFP-myc-GluK2 WT, S868D, S868A or K886R along with FLAG-SUMO-1 and FLAG-Ubc9. 36 hours post-transfection, cells were washed twice with PBS and lysed in 500 μl lysis buffer (10 mM tris-HCl, 150 mM NaCl, 1% triton X-100, 0.1% SDS, 20 mM NEM and protease inhibitors (Roche)). Lysates were then sonicated briefly and GluK2 solubilised by rotation for 1 hour at 4°C. Lysates were then cleared by centrifugation at 16000g for 15 minutes. 200 μl cleared lysate was then taken and 600 μl lysis buffer lacking NEM added. GluK2 was immunoprecipitated by addition of 1 μg sheep anti-myc (made in-house) and left rotating at 4°C overnight. To each IP, 20μl (bed volume) of protein G sepharose (Sigma) pre-washed in lysis buffer (lacking NEM) was added, and IPs incubated for a further 2 hours at 4°C. After extensive washing in lysis buffer lacking NEM, beads were boiled in 50 μl 2× Laemmli buffer. IPs and corresponding inputs were then subjected to SDS-PAGE and Western blotting for FLAG (M2 monoclonal antibody, Sigma) or myc (9E10, Santa Cruz), respectively. Blots were analysed using ImageJ by normalizing the band representing SUMOylated GluK2 to total GluK2. Statistical significance was determined using two-tailed Student's t-test.