Regulation of synaptic strength and AMPA receptor subunit composition by PICK1

Abstract

PICK1 (protein interacting with C kinase-1) regulates the surface expression of the AMPA receptor (AMPAR) GluR2 subunit, however, the functional consequences of this interaction are not well understood. Previous work has suggested that PICK1 promotes the internalization of AMPARs. However, we found that when PICK1 is virally expressed in the CA1 region of hippocampal slices, it causes an increase in AMPAR-mediated EPSC amplitude. This effect is associated with increased AMPAR rectification and sensitivity to polyamine toxin. These effects are blocked by PKC or calcium/calmodulin-dependent protein kinase II inhibitors, indicating that the virally expressed PICK1 signals through an endogenous kinase cascade. In contrast, blockade of interactions with GluR2 at the N-ethylmaleimide-sensitive factor site did not cause a change in subunit composition, suggesting that the effects of PICK1 are not simply a nonspecific consequence of removing AMPARs from the surface. Immunocytochemical and biochemical analyses in dissociated cultured hippocampal neurons show that PICK1 causes a decrease in endogenous GluR2 surface expression but no change in GluR1 surface levels. To address the physiological role of PICK1, we virally expressed C-terminal GluR2 peptides. Blockade of endogenous PICK1 PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain interactions produced opposite effects on synaptic strength and AMPAR rectification to those observed with PICK1 expression. This demonstrates that AMPAR subunit composition is physiologically regulated through a mechanism involving PICK1 PDZ domain interactions. These findings suggest that PICK1 acts to downregulate the GluR2 content of AMPARs at hippocampal CA1 synapses, thereby increasing synaptic strength at resting membrane potentials.

Figure 2.

The PICK1-dependent increase in AMPAR-mediated EPSC amplitude is associated with an increase in AMPAR rectification. A, Averaged EPSCs from example experiments (unpaired experiments) at a holding potential of -70 mV (bottom traces) and +40 mV (top traces) for a control noninfected neuron (left) and for a neuron infected with the PICK1 virus (right). B, Pooled data showing AMPA/NMDA ratio (B1) and rectification (AMPA EPSC_{-70 mV}/AMPA EPSC_{+40 mV}; B2) for neurons from uninfected slices (n = 9), noninfected neurons in slices infected with PICK1 virus (Control; n = 13), and neurons infected with the PICK1 virus (n = 13). ^*p < 0.05. C, Pooled data showing I-V relationships (I_AMPA normalized to data at -70 mV) in the presence of 50 μm d-AP-5 for control neurons (C1; n = 7) and neurons virally expressing PICK1 (C2; n = 7). The dashed lines are linear regression fits to the data at negative holding potentials.

Figure 1.

Viral expression of PICK1 in CA1 pyramidal neurons in acute cultured hippocampal slices increases AMPAR-mediated EPSC amplitude. A, Low-power transmission (A1) and fluorescence (A2) images of a hippocampal slice cultured overnight with Sindbis virus bicistronically expressing EGFP. B, High-power images (transmission and fluorescence images superimposed) of whole-cell patch-clamp recordings from noninfected control (B1) and neighboring infected (B2) neurons in the CA1 pyramidal cell layer (same slice as in A). C, Schematic showing the experimental configuration for recording from control and infected neurons. D, Averaged EPSCs (bottom traces, -70 mV; top traces, +40 mV) from an example pairwise experiment for a control noninfected neuron (left), for a neighboring neuron infected with virus expressing PICK1 (center), and EPSCs from these two neurons superimposed (right). E, Summary analysis of all pairwise comparisons (n = 9) of the effects of viral expression of PICK1 on EPSC amplitude recorded at a holding potential of -70 mV. ^*p < 0.05. F, Summary analysis of all pairwise comparisons (n = 9) of effects of PICK1 overexpression on the NMDAR-mediated EPSC.

Figure 3.

A functional PDZ domain on PICK1 is required for the regulation of AMPARs by PICK1. A, Schematic showing the domain structure of wild-type PICK1 and the position of the amino acid substitutions in the mutant form, PICK1-AA, that lacks a functional PDZ domain. B, Averaged EPSCs from an example control neuron (left) and a neuron expressing PICK-AA (right) at a holding potential of -70 mV (bottom traces) and +40 mV (top traces). C, Pooled data (n = 12) showing the effects of PICK1-AA on the AMPA/NMDA ratio (C1) and rectification (C2).

Figure 4.

PICK1 causes an increase in the sensitivity of AMPAR-mediated EPSCs to PhTx. A, Pooled data (n = 11) for EPSC amplitude (normalized to mean amplitude before PhTx application) versus time for experiments in which PhTX (1 μm) was bath applied to cultured hippocampal slices during recordings from control (uninfected) neurons. B, Pooled data for the effects of PhTx on neurons infected with the PICK1 virus (n = 5). For A and B, the inset shows averaged EPSCs from an example experiment taken at the time points indicated. C, Summary analysis for the effects of PhTx on EPSC amplitude (averaged at 25 min as percentage of baseline) under both conditions. ^*p < 0.05.

Figure 5.

PICK1 causes a reduction in surface-expressed endogenous GluR2 puncta but not GluR1 puncta in cultured hippocampal neurons. A, Examples of EGFP fluorescence (left) and surface GluR2 staining (N-terminal GluR2 antibody, nonpermeabilizing conditions; right) for a control neuron (top) and a neuron infected with the PICK1 virus (bottom). ForA-C, right panels are close-up images of antibody staining of the region of the dendrite indicated by the box (for all images, scale bars are 20 μm for low-power and 2 μm for close-up). B, Examples of EGFP fluorescence (left) and surface GluR1 staining (N-terminal GluR1 antibody, nonpermeabilizing conditions; right) for a control neuron (top) and a neuron infected with the PICK1 virus (bottom). C, Examples of EGFP fluorescence (left) and whole-cell GluR2 staining (N-terminal GluR2 antibody, permeabilizing conditions; right) for a control neuron (top) and a neuron infected with the PICK1 virus (bottom). D, Quantitative analysis of the effects of PICK1 viral expression on GluR2 and GluR1 surface levels (n = 10 neurons from three separate experiments for all experimental groups). ^****p < 0.001. E, Quantitative analysis of the effects of PICK1 viral expression on GluR2 whole-cell levels in absolute intensity [arbitrary units (AU)] values for soma and dendrites (left) and ratio of dendritic/somatic fluorescence (right; n = 10 neurons from three separate experiments for control; n = 9 neurons from three separate experiments for PICK1).

Figure 6.

PICK1 causes a reduction in surface GluR2 but not in surface GluR1. A, Western blot analyses of total GluR2 (left) and total GluR1 (right) levels in cultured hippocampal neurons under control conditions and in sister cultures infected with the PICK1 virus (n = 3 experiments for both; for this and subsequent quantifications, values are expressed as a percentage of levels in sister control cultures). B, Western blot analysis of cell surface biotinylation of GluR2 in control cultures (n = 7) and in sister cultures infected with either the PICK1 virus (n = 7) or the PICK-AA virus (n = 3). ^**p < 0.01. C, Western blot analysis of cell surface biotinylation of GluR1 in control cultures (n = 6) and in sister cultures infected with either the PICK1 virus (n = 6) or the PICK-AA virus (n = 3).

Figure 7.

Blockade of interactions at the NSF site on GluR2 reduces AMPAR-mediated EPSC amplitude but does not affect rectification. A, Averaged EPSCs from example experiments at a holding potential of -70 mV (bottom traces) and +40 mV (top traces) for a control uninfected neuron (left) and for a neuron expressing pep2m (right). B, Pooled data (n = 16) for the effects of pep2m expression on AMPA/NMDA ratio (B1) and rectification (B2). ^*p < 0.05. C, Pooled data of EPSC amplitude versus time for experiments in which PhTx (1 μm) was bath applied during recordings from neurons expressing pep2m (n = 5). D, Summary data showing the effects of PhTx on EPSC amplitude (averaged at 25 min as percentage of baseline) in neurons expressing pep2m compared with interleaved controls (same control data set as shown in Fig. 4).

Figure 8.

The regulation of AMPARs by PICK1 requires endogenous PKC and CaMKII activity. A1, Averaged EPSCs from example experiments at a holding potential of -70 mV (bottom traces) and +40 mV (top traces) for a control uninfected neuron (left) and for a neuron virally expressing PICK1 (right) in slices cultured in the continuous presence of the PKC inhibitor BIS (1 μm). Summary data for the AMPA/NMDA ratio (A2) and rectification (A3) for these experiments (right pair of bars: n = 10 for Control+BIS; n = 10 for PICK1+BIS). Also shown are data from interleaved experiments for control neurons and neurons virally expressing PICK1 cultured under control conditions (left pair of bars: n = 22 for Control; n = 22 for PICK1). B, Similar experiments showing the results of CaMKII inhibition by KN62 (10 μm) on the effects of PICK1 expression (n = 12 for Control+KN62; n = 11 for PICK1+KN62; interleaved control experiments: n = 17 for Control; n = 7 for PICK1). ^*p < 0.05; ^***p < 0.005.

Figure 9.

Endogenous regulation of AMPAR subunit composition by a mechanism involving PDZ domain interactions. A1, Averaged EPSCs from example experiments at a holding potential of -70 mV (bottom traces) and +40 mV (top traces) for a control uninfected neuron (left) and for a neuron expressing pep2-SVKI (right). Pooled data (n = 7) for the effects of pep2-SVKI expression on the AMPA/NMDA ratio (A2) and rectification (A3). B1, Averaged EPSCs from example experiments at a holding potential of 70 mV (bottom traces) and +40 mV (top traces) for a control uninfected neuron (left) and for a neuron expressing pep2-EVKI (right). Pooleddata (n = 8) for the effects of pep2-EVKI expression on the AMPA/NMDA ratio (B2) and rectification (B3). ^*p < 0.05; ^***p < 0.005.