PICK1 mediates transient synaptic expression of GluA2-lacking AMPA receptors during glycine-induced AMPA receptor trafficking

Abstract

The number and subunit composition of postsynaptic AMPA receptors (AMPARs) is a key determinant of synaptic transmission. The vast majority of AMPARs contain GluA2 subunit, which renders the channel impermeable to calcium. However, a small proportion are GluA2 lacking and therefore calcium permeable (CP-AMPARs). It has been proposed recently that long-term potentiation (LTP) involves not only an increase in the total number of AMPARs at the synapse but also a transient switch to CP-AMPARs in the first few minutes after LTP induction. The molecular mechanisms that underlie this switch to CP-AMPARs and the subsequent switch back to calcium-impermeable AMPARs are unknown. Here, we show that endogenous GluA1 is rapidly inserted at the synaptic plasma membrane of rat hippocampal neurons immediately after stimulation with elevated glycine, a treatment known to induce LTP. In contrast, GluA2 is restricted from trafficking to the cell surface by a glycine-induced increase in PICK1-GluA2 binding on endosomal compartments. Between 5 and 20 min after stimulus, activation of CP-AMPARs triggers a release of GluA2 from PICK1, allowing GluA2-containing AMPARs to traffic to the synaptic plasma membrane. These results define a PICK1-dependent mechanism that underlies transient alterations in the subunit composition and calcium permeability of synaptic AMPARs that is important during the early phase after stimulation with glycine and therefore is likely to be important during the expression of LTP.

Figure 1.

Transient insertion of endogenous GluA2-lacking AMPARs at the plasma membrane after glycine stimulation (immunocytochemistry). Dissociated hippocampal neurons expressing EGFP were exposed to elevated glycine for 3 min, returned to normal medium for the specified time, and fixed at 0, 5, 10, or 20 min after stimulus. Controls did not receive the stimulus. Surface and internal GluA1 (A), GluA2 (B), and GluA3 (C) were sequentially labeled under nonpermeabilized conditions and permeabilized conditions with different colored secondary antibodies. Representative images are shown for all conditions, with zoomed sections of dendrites taken from the boxes marked. Graphs show surface/internal ratio relative to unstimulated controls. *p < 0.0125, **p < 0.005, ***p < 0.001. n = 4 independent experiments, with 12–15 cells in each. Scale bars, 20 μm.

Figure 2.

Transient insertion of endogenous GluA2-lacking AMPARs at the plasma membrane after glycine stimulation (surface biotinylation). Dissociated hippocampal neurons were exposed to elevated glycine for 3 min and then returned to normal medium. Controls did not receive the stimulus. After 5 min (A) or 20 min (B), biotinylation was used to quantify surface levels of AMPAR subunits. Representative Western blots are shown of GluA1–GluA3 present in total lysates (T) and surface (S, biotinylated) GluA1–GluA3 after glycine stimulus or under control conditions. Graphs show surface/total ratio relative to control. *p < 0.05, ***p < 0.005. n = 6 independent experiments.

Figure 3.

Transient incorporation of GluA2-lacking AMPARs at synaptic sites after glycine stimulation. Dissociated hippocampal neurons were exposed to elevated glycine for 3 min, returned to normal medium for the specified time, and fixed at 5 or 20 min after stimulus. Controls did not receive the stimulus. Neurons were sequentially stained for surface AMPAR subunits GluA1 (A), GluA2 (B), GluA3 (C), (green channel), and PSD95 (red channel) under nonpermeabilized and permeabilized conditions, respectively. Representative images are shown for all conditions. Graphs show Manders' colocalization coefficients for the fraction of AMPAR subunit colocalized with PSD95 normalized to control. *p < 0.025, **p < 0.005. n = 3 independent experiments, with 10–15 cells in each. Scale bars, 5 μm.

Figure 4.

PICK1 restricts GluA2 trafficking to the cell surface after glycine stimulation. A, PICK1 knockdown allows the traffic of GluA2 to the plasma membrane within 5 min after glycine stimulation. Dissociated hippocampal neurons transfected with plasmids expressing EGFP alone or EGFP plus PICK1 shRNA were exposed to elevated glycine for 3 min, returned to normal medium for the specified time, and fixed 0, 5, 10, or 20 min after stimulation. Controls did not receive the stimulus. Cells were processed for immunocytochemistry as described for Figure 1. Graph shows surface/internal ratio relative to unstimulated controls. **p < 0.005, ***p < 0.001. n = 4 independent experiments, with 12–15 cells in each. Scale bar, 20 μm. B, PICK1 overexpression further restricts GluA2 trafficking to the plasma membrane in response to glycine stimulation. Dissociated hippocampal neurons transfected with plasmids expressing WT–PICK1–IRES–EGFP were treated as in A. Graph shows surface/internal ratio relative to unstimulated controls. ***p < 0.001. n = 4 independent experiments, with 12–15 cells in each. Scale bar, 20 μm. C, Coexpression of sh-resistant WT–PICK1 rescues the shRNA phenotype. Dissociated hippocampal neurons transfected with plasmids expressing shRNA against PICK1 plus sh-resistant WT–PICK1–IRES–EGFP were treated as in A. Graph shows surface/internal ratio relative to unstimulated controls. *p < 0.0125. n = 4 independent experiments, with 12–15 cells in each. Scale bar, 20 μm.

Figure 5.

Transient increase in PICK1 localization at endosomal compartments after glycine stimulation. A, Colocalization between GluA2 and PICK1 is transiently increased at endosomal compartments after stimulation with glycine. Dissociated hippocampal neurons were preloaded with Alexa Fluor–transferrin to label endosomes (red channel). Neurons were exposed to elevated glycine for 3 min, returned to normal medium for the specified time, fixed at 0, 5, 10, or 20 min after stimulus, and stained using anti-GluA2 (green channel) and anti-PICK1 (blue channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of GluA2 colocalized with PICK1 within the transferrin compartment normalized to control. *p < 0.0125, ***p < 0.001. n = 3 independent experiments, with 10–15 cells in each. Scale bar, 5 μm. B, Colocalization between PICK1 and EEA1 is transiently enhanced in dendrites at 5 min after glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in A and stained using anti-EEA1 (green channel) and anti-PICK1 (red channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of PICK1 colocalized with EEA1 normalized to control. **p < 0.01. n = 4 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. C, Colocalization between PICK1 and the recycling endosomal marker Rab11 is transiently enhanced in dendrites at 5 min after glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in A and stained using anti-Rab11 (green channel) and anti-PICK1 (red channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of PICK1 colocalized with Rab11 normalized to control condition. **p < 0.01, ***p < 0.001. n = 4 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. D, Colocalization between PICK1 and the synaptic marker PSD95 is unchanged at 5 min after glycine stimulation but increases at 20 min. Dissociated hippocampal neurons were glycine stimulated as in A and returned to normal medium for the specified time, fixed at 5 or 20 min after stimulus, and stained using anti-PSD95 (green channel) and anti-PICK1 (red channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graph shows Manders' colocalization coefficients for the fraction of PICK1 colocalized with PSD95 normalized to control condition. *p < 0.025. n = 3 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. E, Association between PICK1 and Rab11 analyzed by live imaging. Dissociated hippocampal neurons expressing GFP–Rab11 and mCherry–PICK1 were exposed to elevated glycine for 3 min, followed by washout with normal medium. Images were collected every minute, and fluorescence intensity for mCherry–PICK1 was measured within a mask generated from the GFP–Rab11 signal. Images show representative images from selected time points, and graph shows fluorescence normalized to baseline (before glycine application). n = 7. Scale bar, 2 μm.

Figure 6.

Interaction between GluA2 and PICK1 is transiently enhanced by glycine stimulation and is subsequently disrupted as a result of CP-AMPAR activation. A, GluA2–PICK1 interaction is transiently increased at 5 min after stimulus. Dissociated hippocampal neurons were stimulated with glycine for 3 min and returned to normal medium for the specified time. Lysates were prepared and IPs performed using anti-PICK1 antibody. Bound proteins were detected by Western blotting using anti-GluA2 and anti-PICK1 antibodies. Controls did not receive the stimulus. Left shows representative blots, and the graph shows PICK1–GluA2 binding relative to control. **p < 0.01. n = 7. B, GluA2–GRIP1 interaction is increased at 5 and 20 min after stimulus. Dissociated hippocampal neurons were glycine stimulated as in A. Lysates were prepared and IPs performed using anti-GRIP1 antibody. Bound proteins were detected by Western blotting using anti-GluA2 and anti-GRIP1 antibodies. Controls did not receive the stimulus. Left shows representative blots, and the graph shows GRIP1–GluA2 binding relative to control. *p < 0.05; n = 7. C, PICK1–GRIP1 interaction is increased at 5 min after stimulus. Dissociated hippocampal neurons were glycine stimulated as in A. Lysates were prepared and IPs performed using anti-PICK1 antibody. Bound proteins were detected by Western blotting using anti-PICK1 and anti-GRIP1 antibodies. Controls did not receive the stimulus. Left shows representative blots, and the graph shows GRIP1–PICK1 binding relative to control. *p < 0.05; n = 9. D, Blockade of GluA2-lacking AMPARs with NASPM completely blocks the release of GluA2 from PICK1 between 5 and 20 min after glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in A, but 30 μm NASPM was present throughout. Lysates were prepared and IPs performed using anti-PICK1 antibody. Bound proteins were detected by Western blotting using anti-GluA2 and anti-PICK1 antibodies. Controls did not receive the stimulus. Left shows representative blots, and the graph shows PICK1–GluA2 binding relative to control. *p < 0.05; n = 6. E, Blockade of GluA2-lacking AMPARs with NASPM has no effect on GluA1 trafficking in response to glycine stimulation and blocks the increase in GluA2 surface expression at 20 min after stimulus. Dissociated hippocampal neurons were stimulated with glycine for 3 min and returned to normal medium for the specified time. NASPM (30 μm) was present throughout. Controls did not receive the stimulus. Cells were fixed at 5 or 20 min after stimulus and processed for immunocytochemistry as described for Figure 1. **p < 0.01; n = 3 independent experiments, with 10–12 cells in each. Scale bar, 5 μm.

Figure 7.

Calcium-sensing function of PICK1 is required for the regulation of GluA2 trafficking to the plasma membrane after glycine stimulation. A, Schematic diagram showing PICK1 mutants used. B, Calcium-sensing regions of PICK1 are involved in GluA2 trafficking to the plasma membrane after glycine stimulation. Dissociated hippocampal neurons expressing DD4,6AA–PICK1^flag–IRES–EGFP or Δ380–390–PICK1^flag–IRES–EGFP were stimulated with glycine for 3 min, returned to normal medium for the specified time, and fixed 5 or 20 min after stimulus. Controls did not receive the stimulus. Cells were processed for immunocytochemistry as described for Figure 1. *p < 0.025; n = 3 independent experiments, with 12–15 cells in each. Scale bar, 20 μm. C, Colocalization between WT–PICK1^flag and the recycling endosomal marker Rab11 is transiently enhanced in dendrites at 5 min after glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in B and stained using anti-Rab11 (red channel) and anti-flag (green channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graphs show Manders' colocalization coefficients for the fraction of WT–PICK1^flag colocalized with Rab11 normalized to control condition. *p < 0.05, n = 3 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. D, Colocalization between DD4,6AA–PICK1^flag and the recycling endosomal marker Rab11 is unaffected by glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in B and stained using anti-Rab11 (red channel) and anti-flag (green channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graphs show Manders' colocalization coefficients for the fraction of DD4,6AA–PICK1^flag colocalized with Rab11 normalized to control condition. n = 3 independent experiments, with 10–12 cells in each. Scale bar, 5 μm. E, Colocalization between Δ380–390–PICK1^flag and the recycling endosomal marker Rab11 is unaffected by glycine stimulation. Dissociated hippocampal neurons were glycine stimulated as in B and stained using anti-Rab11 (red channel) and anti-flag (green channel). Controls did not receive the stimulus. Representative images are shown for all conditions. Graphs show Manders' colocalization coefficients for the fraction of Δ380–390–PICK1^flag colocalized with Rab11 normalized to control condition. n = 3 independent experiments, with 10–12 cells in each. Scale bar, 5 μm.

Figure 8.

Model for subunit-specific AMPAR trafficking during LTP. A, Under basal conditions, the vast majority of synaptic AMPARs are GluA1/2 and GluA2/3 heteromers. These receptors continually exchange with endosomal compartments in rounds of recycling. It is assumed that a small proportion of intracellular AMPARs are GluA2 lacking (GluA1/3 heteromers or GluA1 homomers). A proportion of PICK1 associates with the recycling endosome system. B, Immediately after LTP induction, PICK1–GluA2 interactions are increased, and a larger proportion of PICK1 associates with endosomes, leading to retention of GluA2-containing AMPARs in endosomes. GluA2-lacking AMPARs are inserted at the plasma membrane and move laterally to the synapse. C, At 5 min after LTP, a proportion of synaptic AMPARs are GluA2 lacking, which are calcium permeable. D, Between 5 and 20 min after LTP induction, activation of GluA2-lacking CP-AMPARs leads to disruption of the PICK1–GluA2 interaction, allowing GluA2-containing AMPARs to be inserted into the plasma membrane. During this time, GluA2-lacking AMPARs are removed from the synaptic plasma membrane. E, This process leads to a net increase in synaptic AMPARs, the vast majority of which contain GluA2.