NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1 (original) (raw)

Proc Natl Acad Sci U S A. 2010 Aug 3; 107(31): 13924–13929.

Nicole Guetg,a,1 Said Abdel Aziz,a,1 Niklaus Holbro,b Rostislav Turecek,a,c Tobias Rose,b Riad Seddik,a Martin Gassmann,a Suzette Moes,d Paul Jenoe,d Thomas G. Oertner,b Emilio Casanova,a,2 and Bernhard Bettlera,3

Nicole Guetg

aDepartment of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, 4056 Basel, Switzerland;

Said Abdel Aziz

aDepartment of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, 4056 Basel, Switzerland;

Niklaus Holbro

bFriedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland;

Rostislav Turecek

aDepartment of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, 4056 Basel, Switzerland;

cInstitute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic; and

Tobias Rose

bFriedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland;

Riad Seddik

aDepartment of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, 4056 Basel, Switzerland;

Martin Gassmann

aDepartment of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, 4056 Basel, Switzerland;

Suzette Moes

dBiozentrum, University of Basel, 4056 Basel, Switzerland

Paul Jenoe

dBiozentrum, University of Basel, 4056 Basel, Switzerland

Thomas G. Oertner

bFriedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland;

Emilio Casanova

aDepartment of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, 4056 Basel, Switzerland;

Bernhard Bettler

aDepartment of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, 4056 Basel, Switzerland;

aDepartment of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, 4056 Basel, Switzerland;

bFriedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland;

cInstitute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic; and

dBiozentrum, University of Basel, 4056 Basel, Switzerland

Edited* by Richard L. Huganir, Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 24, 2010 (received for review January 22, 2010)

Author contributions: N.G., S.A.A., N.H., R.T., T.R., R.S., M.G., P.J., E.C., and B.B. designed research; N.G., S.A.A., N.H., R.T., T.R., R.S., S.M., and E.C. performed research; T.G.O. contributed new reagents/analytic tools; N.G., S.A.A., N.H., R.T., T.R., R.S., M.G., S.M., P.J., and E.C. analyzed data; and N.G., M.G., and B.B. wrote the paper.

1N.G. and S.A.A. contributed equally to this work.

2Present address: Ludwig Boltzmann Institute for Cancer Research, A-1090 Vienna, Austria.

Supplementary Materials

Supporting Information

GUID: C04587BD-3EA4-4146-AF56-52682D7B2AF8

GUID: 8B65D449-DA49-4DDD-826F-797BB8991D0B

Abstract

GABAB receptors are the G-protein–coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GABAB receptors are abundant on dendritic spines, where they dampen postsynaptic excitability and inhibit Ca2+ influx through NMDA receptors when activated by spillover of GABA from neighboring GABAergic terminals. Here, we show that an excitatory signaling cascade enables spines to counteract this GABAB-mediated inhibition. We found that NMDA application to cultured hippocampal neurons promotes dynamin-dependent endocytosis of GABAB receptors. NMDA-dependent internalization of GABAB receptors requires activation of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), which associates with GABAB receptors in vivo and phosphorylates serine 867 (S867) in the intracellular C terminus of the GABAB1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABAB receptors refractory to NMDA-mediated internalization. Time-lapse two-photon imaging of organotypic hippocampal slices reveals that activation of NMDA receptors removes GABAB receptors within minutes from the surface of dendritic spines and shafts. NMDA-dependent S867 phosphorylation and internalization is predominantly detectable with the GABAB1b subunit isoform, which is the isoform that clusters with inhibitory effector K+ channels in the spines. Consistent with this, NMDA receptor activation in neurons impairs the ability of GABAB receptors to activate K+ channels. Thus, our data support that NMDA receptor activity endocytoses postsynaptic GABAB receptors through CaMKII-mediated phosphorylation of S867. This provides a means to spare NMDA receptors at individual glutamatergic synapses from reciprocal inhibition through GABAB receptors.

Keywords: γ-aminobutyric acid, spines, trafficking, synaptic plasticity, GABAB

GABAB receptors modulate the excitability of neurons throughout the brain. They are therapeutic targets for a variety of disorders, including cognitive impairments, addiction, anxiety, depression, and epilepsy (1). Depending on their subcellular localization GABAB receptors exert distinct regulatory effects on synaptic transmission (2–4). Presynaptic GABAB receptors inhibit neurotransmitter release (5, 6). Postsynaptic GABAB receptors dampen neuronal excitability by gating Kir3-type K+ channels, which generates slow inhibitory postsynaptic potentials and local shunting (7). Molecular diversity in the GABAB system arises from the GABAB1a and GABAB1b subunit isoforms, both of which combine with GABAB2 subunits to form heteromeric GABAB(1a,2) and GABAB(1b,2) receptors (8). Genetically modified mice revealed that the two receptors convey nonredundant synaptic functions at glutamatergic synapses, owing to their differing distribution to axonal and dendritic compartments (3, 9). Selectively GABAB(1a,2) receptors control the release of glutamate, whereas predominantly GABAB(1b,2) receptors activate postsynaptic Kir3 channels in dendritic spines (10–12). Activation of GABAB receptors on spines inhibits NMDA receptors through hyperpolarization and the PKA pathway, which enhances Mg2+ block (13, 14) and reduces Ca2+ permeability (15) of NMDA receptors. Reciprocally, there is evidence that glutamate receptors decrease surface expression of GABAB receptors (16–18). This supports that glutamate receptors and GABAB receptors cross-talk in dendrites and spines.

Neither the glutamate receptors nor the signaling pathways controlling surface availability of GABAB receptors have yet been identified. Here we show that NMDA receptor-dependent phosphorylation via CaMKII targets GABAB receptors for internalization. This postsynaptic regulation of GABAB receptors has implications for the control of local excitability and Ca2+-dependent neuronal functions.

Results

NMDA Receptors Mediate GABAB Receptor Internalization.

We used transfected cultured hippocampal neurons to identify the glutamate receptors regulating cell surface expression of GABAB receptors. Robust cell surface expression of tagged GABAB1b subunits (HA-GB1b-eGFP) was observed upon cotransfection with GABAB2 subunits (Fig. 1_A_), which are mandatory for GABAB1 surface expression (8, 19, 20). GABAB1b surface expression was monitored by immunolabeling of the extracellular HA-tag before permeabilization of cells (red fluorescence). Total GABAB1b expression was monitored by immunolabeling of the intracellular eGFP-tag after permeabilization of cells (green fluorescence). To quantify the level of surface GABAB1b protein, we calculated the ratio of red to green fluorescence intensity (Fig. 1 B and C). Upon glutamate treatment (50 μM glutamate/5 μM glycine for 30 min), surface GABAB1b protein was significantly reduced (41.4 ± 5.3% of control, n = 10, P < 0.001), consistent with published data (18). The NMDA receptor antagonist APV (100 μM for 2 h) prevented the glutamate-induced decrease in surface GABAB1b protein (98.4 ± 12.6% of control, _n_ = 9, _P_ > 0.05). We tested whether a selective activation of NMDA receptors is sufficient to decrease surface GABAB1b protein. Following NMDA treatment (75 μM NMDA/5 μM glycine for 3 min) and recovery in conditioned medium for 27 min, surface GABAB1b protein was significantly reduced (54.8 ± 3.2% of control, n = 10, P < 0.01). Heteromerization with GABAB2 is mandatory for exit of GABAB1 from the endoplasmic reticulum and for receptor function (19, 20). As expected from the assembly with GABAB1, surface GABAB2 protein was also significantly decreased following glutamate or NMDA application, and this decrease was prevented by APV (Fig. S1_A_). We also observed a trend toward decreased surface GABAB1a protein in response to glutamate or NMDA but this did not reach statistical significance (Fig. S1_B_). Although surface biotinylation experiments in cultured cortical neurons revealed significant internalization of endogenous GABAB1b as well as GABAB1a protein in response to NMDA (GB1b NMDA: 52.9 ± 9.9% of control, n = 3, P < 0.01; GB1a NMDA: 74.1 ± 3.4% of control, n = 3, P < 0.05; Fig. 2_B_), significantly more GABAB1b than GABAB1a protein was internalized (P < 0.05; ANOVA with Bonferroni test). Endogenous surface GABAB2 protein was also significantly down-regulated following NMDA treatment (57.0 ± 6.0% of control, n = 3, P < 0.001; Fig. 2_B_). This supports that preferentially GABAB(1b,2) receptors are removed from the cell surface in response to NMDA application, possibly as a consequence of their selective localization in the somatodendritic compartment (10).

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NMDA-dependent removal of surface GABAB receptors. (A) Rat hippocampal neurons coexpressing exogenous HA-GB1b-eGFP and GABAB2 were treated at DIV14 as indicated. Surface GABAB1b (GB1b) protein was fluorescence-labeled with anti-HA antibodies before permeabilization. Total GB1b protein was fluorescence-labeled with anti-eGFP antibodies after permeabilization. Single optical planes captured with a confocal microscope are shown. (Scale bar, 15 μm.) (Insets) Representative spines at higher magnification. (B) Surface GABAB1b protein was quantified by the ratio of surface to total fluorescence intensity. Values were normalized to control values in the absence of any pharmacological treatment. Surface GABAB1b protein was significantly decreased following glutamate or NMDA treatment. No significant reduction was observed with glutamate treatment after preincubation with APV. n = 9–10, **P < 0.01, ***P < 0.001. (C) Dynasore but not CGP54626A prevented the NMDA-induced reduction of surface GABAB1b protein. n = 8–10, *P < 0.05. Quantification was from nonsaturated images. Data are presented as mean ± SEM.

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NMDA-induced removal of surface GABAB receptors requires CaMKII. (A) Rat hippocampal neurons coexpressing exogenous HA-GB1b-eGFP and GABAB2 were analyzed at DIV14. Surface GABAB1b protein was quantified by the ratio of surface to total fluorescence intensity. Preincubation of neurons with the Ca2+-chelator EGTA-AM or the CaMKII inhibitor KN-93 prevented the NMDA-induced reduction in surface GABAB1b protein. KN-92 was ineffective. Data are means ± SEM, n = 9–10, **P < 0.01. (B) NMDA-mediated removal of endogenous surface GABAB receptors. Live cortical neurons were treated at DIV14 as indicated and then biotinylated. Cell homogenates (total) and avidin-purified cell surface proteins (surf) were probed on Western blots with anti-GABAB1 (anti-GB1) and anti-GABAB2 (anti-GB2) antibodies. While all GABAB subunits were removed from the cell surface in response to NMDA, GABAB1b was more efficiently removed than GABAB1a (P < 0.05). NMDA-mediated removal of surface protein was inhibited by KN-93. Anti-tubulin antibodies were used as a control. Of note, we consistently observed that significantly more GABAB1b protein was detected at the cell surface under control conditions, albeit GABAB1a is more abundant in cortical neurons (GB1a-to-GB1b ratio: surface, 0.71 ± 0.08; total, 1.32 ± 0.05; n = 3, P < 0.01). (C) CaMKII interacts with GABAB receptors in the brain. Anti-GB1 and anti-GB2 antibodies coimmunoprecipitated CaMKII from purified mouse brain membranes, whereas control rabbit (serum rb) or guinea-pig serum (serum gp) did not. (D) Pull-down assays with GST-fusion proteins containing the entire C-terminal domain of GABAB1 (GST-GB1) or GABAB2 (GST-GB2) and whole-brain lysates. CaMKII bound to a larger extent to GST-GB1 than to GST-GB2. Control assays were with glutathione beads alone or with beads together with GST protein. (E) In vitro phosphorylation of GST-fusion proteins with [γ-32P]-ATP in the presence or absence of recombinant CaMKII. Phosphorylated proteins were separated by SDS/PAGE and exposed to autoradiography. CaMKII specifically phosphorylated GST-GB1 but not GST-GB2 or GST alone. Coomassie blue staining controlled for loading. The GST-GB2 fusion protein tended to degrade (50).

We examined whether the decrease in surface GABAB1b protein following glutamate or NMDA treatment is due to endocytosis. We observed basal endocytosis of surface GABAB1b protein under control conditions (Fig. S2_A_), as previously reported (21). Glutamate or NMDA treatment visibly increased endocytosis of GABAB1b protein, which was inhibited in the presence of APV (Fig. S2_A_). Constitutively internalized GABAB1b protein colocalized with Rab11-eGFP (22, 23), a marker for recycling endosomes (Fig. S2_B_). Following glutamate or NMDA treatment, a fraction of internalized GABAB1b protein segregated into structures devoid of Rab11-eGFP, possibly indicating GABAB1b protein targeted for degradation (24, 25). Preincubation of neurons with dynasore (80 μM for 15 min), a cell-permeable inhibitor of dynamin-dependent endocytosis (26), interfered with the NMDA-mediated removal of surface GABAB1b protein (NMDA: 58.8 ± 5.4% of control, n = 8, P < 0.05; NMDA + dynasore: 105 ± 10% of control, _n_ = 10, _P_ > 0.05; dynasore: 98.4 ± 9.0% of control, n = 9, P > 0.05; Fig. 1_C_). Agonists accelerate basal endocytosis of GABAB receptors (25). Antagonizing GABAB receptor activity with CGP54626A (2 μM for 10 min) did not attenuate NMDA-mediated removal of surface GABAB1b protein (NMDA + CGP54626A: 60.2 ± 9.6% of control, n = 10, P < 0.05; Fig. 1_C_). Thus NMDA receptor activation triggers dynamin-dependent endocytosis of GABAB receptors, irrespective of whether GABAB receptors are active or not.

Removal of Surface GABAB Receptors Requires CaMKII Activity.

NMDA failed to reduce surface GABAB1b protein in transfected hippocampal neurons in the presence of the membrane-permeable Ca2+-chelator EGTA-AM (100 μM for 10 min; NMDA: 51.7 ± 6.7% of control, n = 10, P < 0.01; NMDA + EGTA-AM: 83.4 ± 9.0% of control, _n_ = 9, _P_ > 0.05; Fig. 2_A_). NMDA also failed to reduce surface GABAB1b protein in the presence of the CaMKII inhibitor KN-93 (10 μM for 10 min; NMDA + KN-93: 85.6 ± 11.1% of control, n = 10, P > 0.05), implicating activation of CaMKII by NMDA receptors (27) in the removal of surface GABAB1b. Likewise, KN-93 also prevented the NMDA-induced decrease of exogenous GABAB2 protein (Fig. S1_A_). In contrast, the NMDA-mediated decrease in surface GABAB1b protein was not inhibited in the presence of KN-92 (28), an inactive structural analog of KN-93 (10 μM for 10 min; NMDA + KN-92: 48.9 ± 7.9% of control, n = 9, P < 0.01; Fig. 2_A_). Biotinylation experiments with cultured cortical neurons demonstrated that surface levels of endogenous GABAB subunits were significantly less reduced when NMDA was applied in the presence of KN-93 (GB1b: 80.3 ± 6.2% of control, _P_ > 0.05 versus control, P < 0.05 versus NMDA alone; GB1a: 91.7 ± 4.1% of control, _P_ > 0.05 versus control, P < 0.05 versus NMDA; GB2: 89.2 ± 3.0% of control, _P_ > 0.05 versus control, P < 0.01 versus NMDA; n = 3; ANOVA with Tukey's multiple comparison test; Fig. 2_B_). Thus KN-93 also interferes with NMDA-mediated internalization of endogenous GABAB subunits.

S867 in GABAB1 Is Phosphorylated by CaMKII.

Both anti-GABAB1 and anti-GABAB2 antibodies efficiently coimmunoprecipitated CaMKII from purified mouse brain membranes, whereas control sera did not (Fig. 2_C_). This indicates that CaMKII associates with the GABAB1 and/or GABAB2 subunits of heteromeric GABAB receptors (29). We corroborated this finding by performing pull-down assays with GST fusion proteins encoding the GABAB1 and GABAB2 C-termini (GST-GB1, GST-GB2). CaMKII in whole-brain lysate associated to a larger extent with GST-GB1 than with GST-GB2, supporting that CaMKII preferentially associates with GABAB1 (Fig. 2_D_). For in vitro phosphorylation, the GST-fusion proteins were incubated for 30 min at 30 °C with [γ-32P]-ATP and recombinant CaMKII. CaMKII-dependent phosphorylation was detectable on GST-GB1 but not on GST-GB2 or GST alone (Fig. 2_E_). Thus CaMKII associates with native GABAB receptors and phosphorylates site(s) in the C terminus of GABAB1.

To identify the CaMKII phosphorylation site(s) in GABAB1, we digested phosphorylated GST-GB1 protein with LysC and trypsin, separated the resulting peptides by reverse-phase HPLC (RP-HPLC) and collected fractions at 1-min intervals (Fig. 3_A_). The majority of radiolabel eluted in a single peak in fraction 54, which we further analyzed using electrospray ionization mass spectrometry (ESI-MS/MS). Database searches of the ESI-MS/MS scans revealed the presence of the phosphopeptide GEWQSETQDTMK (the methionine of which was oxidized). The fragmentation spectrum indicated phosphorylation of the serine residue corresponding to S867 in the full-length GABAB1a protein (Fig. 3_B_). S867 is localized in the juxtamembrane domain, a regulatory region for many transmembrane proteins, including G-protein–coupled receptors (30). S867 does not conform to the consensus sequence for phosphorylation by CaMKII (31) or other kinases (Table S1). Nonetheless, alanine substitution of putative phosphorylation sites within the GEWQSETQDTMK motif (GST-GB1S867A, GST-GB1T869A, GST-GB1T872A, GST-GB1T869A/T872A) confirmed that recombinant CaMKII only phosphorylates S867 in this sequence (Fig. 3_C_). In addition, we found that CaMKII in brain extracts also specifically phosphorylates S867 in GST-GB1 (Fig. S3).

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CaMKII phosphorylates S867 in the GABAB1 subunit. (A) RP-HPLC analysis of proteolytically digested GST-GB1 after phosphorylation with recombinant CaMKII and [γ-32P]-ATP. Peptide elution was monitored at 214 nm and radioactivity (red) determined by liquid scintillation counting. Asterisk marks elution of the 32P-labeled peptide in fraction 54. (B) Fragmentation spectrum of the doubly charged 768.29 Da precursor from the phosphorylated peptide of fraction 54. Fragmentation pattern agrees with predicted ESI-MS/MS spectrum for the phosphopeptide GEWQpS867ETQDTMK. The y- and b-ions matching the GEWQpS867ETQDTMK sequence are labeled. Asterisks mark phosphorylated ions. (C) In vitro phosphorylation of GST fusion proteins with recombinant CaMKII and [γ-32P]-ATP. Phosphorylated proteins were separated by SDS/PAGE and exposed to autoradiography. Substitution of S867 with alanine in GST-GB1S867A prevented phosphorylation by CaMKII, whereas alanine substitutions of other putative phosphorylation sites in proximity of S867 (GST-GB1T869A, GST-GB1T872A and GST-GB1T869A/T872A) did not. Coomassie blue staining controlled for loading.

NMDA Increases Phosphorylation at S867 in Native GABAB Receptors.

To analyze S867 phosphorylation in native tissue, we generated a S867 phosphorylation-state specific antibody, anti-GB1pS867. After phosphorylation of GST fusion proteins with recombinant CaMKII this antibody labeled GST-GB1 but not GST-GB1S867A (Fig. S4_A_). No S867 phosphorylation was seen when using recombinant PKC instead of CaMKII for phosphorylation (Fig. S4_B_). Importantly, the anti-GB1pS867 antibody revealed weak basal S867 phosphorylation (i) in mouse brain membranes after enrichment of GABAB receptors by immunoprecipitation and (ii) in synaptic plasma membranes after subcellular fractionation (Fig. 4 A and B). Application of NMDA to cultured cortical neurons significantly increased phosphorylation of S867 (Fig. 4_C_). Phosphorylation of S867 in brain membranes or cortical neurons was detectable only in the GABAB1b subunit isoform. However, we cannot exclude that NMDA treatment also weakly phosphorylates the GABAB1a subunit and that this phosphorylation is below our detection limit. Of note, GABAB(1b,2) but not GABAB(1a,2) receptors reside in spines (10) where NMDA and GABAB receptors are particularly abundant (15). This may explain why NMDA receptor activation preferentially targets GABAB1b for phosphorylation.

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S867 phosphorylation in brain tissue and cultured neurons. (A) S867 phosphorylation was detectable after immunoprecipitation of GABAB receptors with anti-GABAB1 antibodies (IP:GB1) from WT but not GB1−/− brain membranes. S867 phosphorylation was detected on Western blots with a phosphorylation-state specific antibody (anti-GB1pS867). The same blot was reprobed with anti-GB1 antibodies. Immunoprecipitation with rabbit IgG (IP:IgG) was used as a control. Note the specific phosphorylation of the GABAB1b subunit. (B) S867 phosphorylation of GABAB1b was clearly detectable in synaptic plasma membranes (SPM) and barely detectable in the P2 membrane fraction purified from total mouse brain homogenates. (C) NMDA application to cultured cortical neurons increased S867 phosphorylation in the GABAB1b subunit. Neurons were treated with NMDA for 3 min and harvested at the times indicated. Whole-cell lysates (input) were subjected to immunoprecipitation with anti-GB1 antibodies (IP:GB1). S867 phosphorylation was detected on Western blot with anti-GB1pS867; anti-tubulin antibodies were used as control. (D) Alanine mutation of S867 in GABAB1b prevents NMDA-induced internalization. Cultured hippocampal neurons expressing exogenous HA-GB1b-eGFP (GB1b) or HA-GB1bS867A-eGFP (GB1bS867A) together with GABAB2 were analyzed at DIV14. Surface GABAB1b protein was quantified by the ratio of surface to total fluorescence intensity. Values were normalized to GB1b control in the absence of NMDA. Data are means ± SEM, n = 8–10. **P < 0.01.

Removal of Surface GABAB Receptors Requires S867 Phosphorylation.

Cultured hippocampal neurons expressing HA-GB1b-eGFP or HA-GB1bS867A-eGFP in combination with exogenous GABAB2 were analyzed for surface expression of transfected GABAB1b protein (Fig. 4_D_ and Fig. S5). HA-GB1bS867A-eGFP exhibited a similar surface expression level as HA-GB1b-eGFP, showing that lack of S867 phosphorylation does not prevent surface expression. However, HA-GB1bS867A-eGFP was refractory to removal from the surface upon NMDA treatment, as determined by the ratio of surface to total fluorescence intensity (GB1b NMDA: 52.6 ± 4.8%, n = 10, P < 0.01; GB1bS867A control: 82.4 ± 6.3%, _n_ = 9, _P_ > 0.05; GB1bS867A NMDA: 98.1 ± 16.0, n = 8, P > 0.05; data normalized to GB1b control; Fig. 4_D_). This implicates S867 phosphorylation in GABAB receptor removal from the cell surface.

NMDA-Mediated CaMKII Activation Reduces GABAB-Induced K+ Currents.

Well-known effectors of dendritic GABAB receptors are the Kir3-type K+ channels, which cluster with GABAB receptors in spines (12). We used whole-cell patch-clamp recording to address whether NMDA-treatment reduces baclofen-induced K+ currents due to GABAB receptor internalization. Baclofen-evoked K+ currents were recorded from cultured hippocampal neurons clamped at −50 mV after pharmacological blockade of Na+ channels, GABAA, glycine, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptors (Fig. 5_A_). Baclofen-induced K+ currents were recorded before and 30 min after NMDA treatment (30 μM NMDA/5 μM glycine in Mg2+-free solution for 1 min). During NMDA applications, neurons were held at −70 mV to minimize Ca2+ entry through voltage-gated Ca2+ channels. Following NMDA treatment, the maximal amplitudes of the baclofen-induced K+ currents were reduced (19.1 ± 6.5%, n = 5; Fig. 5_C_). Likewise, baclofen-induced K+ currents was decreased following glutamate treatment (5 μM glutamate/5 μM glycine in Mg2+-free solution for 1 min; 35.2 ± 5.1%, n = 3), and this decrease was prevented by the NMDA-receptor antagonist dCPP (20 μM; 93.5 ± 0.5%, n = 3, P < 0.001 compared with NMDA alone; Fig. 5 A and C). Intracellular dialysis with the CaMKII inhibitor KN-93 (5 μM) significantly attenuated the NMDA-mediated reduction of K+ currents (65.5 ± 9.0%, _n_ = 5, _P_ < 0.001 compared with NMDA alone). Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) promotes phosphorylation of the GABAB2 subunit by 5′AMP-dependent protein kinase (32). Intracellular dialysis with the CaMKK inhibitor STO-609 (5 μM) resulted in a modest attenuation of NMDA-mediated reduction of K+-currents that, however, did not reach significance (41.3 ± 2.9%, _n_ = 5, _P_ > 0.05 compared with NMDA alone; Fig. 5 A and C). We next addressed whether phosphorylation at S867 is critical for the NMDA-mediated decrease in K+-current amplitude. We transfected cultured hippocampal neurons from GABAB1−/− (GB1−/−) mice (33) with expression constructs for GABAB1b (GB1b), GABAB1a (GB1a) or GB1bS867A. All exogenous GABAB1 subunits, including GB1bS867A, fully rescued GABAB receptor function, demonstrating that they heteromerize with endogenous GABAB2 subunits (Fig. 5 B and D). In GB1−/− neurons reconstituted with GB1b, NMDA application decreased the baclofen-induced K+ currents to a similar extent as in wild-type neurons. In contrast, in GB1−/− neurons reconstituted with GB1a or GB1bS867A NMDA application decreased the K+ currents significantly less (GB1b: 28.3 ± 3.0%, n = 5; GB1a: 54.2 ± 7.2%, n = 6, P < 0.05; GB1bS867A: 64.1 ± 7.0%, n = 5, P < 0.01). Thus predominantly phosphorylation of GABAB1b at S867 is implicated in the NMDA-mediated decrease of the K+-current amplitude.

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CaMKII reduces GABAB-mediated K+ currents in cultured hippocampal neurons. (A) Representative baclofen-induced K+ currents recorded at −50 mV before and after application of NMDA or glutamate. Baclofen-induced K+ currents were strongly reduced 30 min after NMDA or glutamate application. KN-93 and dCPP but not STO-609 attenuated the NMDA-mediated K+ current reduction. (B) Representative baclofen-induced K+ currents recorded from neurons of GABAB1−/− (GB1−/−) mice transfected with GABAB1a (GB1a) GABAB1b, (GB1b) or GB1bS867A expression vectors. NMDA was less effective in decreasing the K+ current in neurons transfected with GB1bS867A or GABAB1a. (C) Bar graph illustrating that dCPP and KN-93 attenuated the NMDA-mediated reduction of baclofen-induced K+ currents. (D) NMDA was significantly less effective in decreasing the K+ current in GB1−/− neurons transfected with GB1a or GB1bS867A than with GB1b. Maximal K+-current amplitudes after NMDA application were normalized to the maximal K+-current amplitudes before NMDA application. Data are means ± SEM, n = 3–6. *P < 0.05; **P < 0.01; ***P < 0.001.

NMDA-Mediated Endocytosis of GABAB Receptors in Dendritic Shafts and Spines.

GABAB(1b,2) receptors, which appear to be the main substrate for S867 phosphorylation, resides in dendritic spines and shafts (10). We therefore addressed whether GABAB receptors at these locations internalize in response to NMDA. We transfected GABAB1b fused to a pH-sensitive eGFP [Super Ecliptic pHluorin (SEP-GB1b)] together with GABAB2 into organotypic hippocampal slice cultures. SEP-GB1b selectively visualizes GABAB1b protein at the cell surface (34). In addition, we expressed the freely diffusible red fluorescent protein (RFP) t-dimer2 to visualize the morphology of transfected cells (10). Time-lapse two-photon images of transfected CA1 pyramidal cells were collected at days in vitro (DIV) 14–21 (Fig. 6_A_). Dendrites were imaged at 5-min intervals before and after bath application of NMDA (30 μM for 1 min). NMDA application resulted in a long-lasting decrease in green fluorescence in dendritic spines and shafts, indicating GABAB receptor internalization (Fig. 6 B and C, green traces; SEP-GB1b fluorescence ratio after/before NMDA: spine, 0.82 ± 0.05, n = 22, P < 0.001; shaft, 0.73 ± 0.04, _n_ = 8, _P_ < 0.001; nonparametric Mann–Whitney test; five cells analyzed). NMDA application did not significantly affect RFP fluorescence in dendritic spines and shafts (Fig. 6 B and C, red traces; RFP fluorescence ratio after/before NMDA: spine, 1.04 ± 0.05, _n_ = 22, _P_ > 0.05; shaft, 0.88 ± 0.03, n = 8, P > 0.05). The decrease in green fluorescence was inhibited in the presence of the NMDA receptor antagonist dCPP (20 μM; Fig. 6 B and C, green traces; SEP-GB1b fluorescence ratio after/before NMDA: spine, 1.00 ± 0.05, n = 15, P > 0.05; shaft, 0.99 ± 0.04, n = 3, P > 0.05; 2 cells analyzed). No significant change in the red fluorescence under NMDA receptor blockade was observed (Fig. 6 B and C, red traces; RFP fluorescence ratio after/before NMDA: spine, 1.06 ± 0.07, n = 15, P > 0.05; shaft, 1.02 ± 0.03, n = 3, P > 0.05). Thus NMDA receptor stimulation leads to GABAB receptor internalization in dendritic spines and shafts. In agreement with experiments described above (Figs. 4_D_ and ​5_D_ and Fig. S5_B_) the SEP-GB1bS867A protein is refractory to NMDA-induced internalization in dendritic spines and shafts (Fig. S6).

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NMDA-mediated endocytosis of GABAB receptors in dendritic spines and shafts. (A) Red fluorescence (R), green fluorescence (G), and G/R ratio images of dendrites expressing freely diffusible RFP and SEP-GB1b before and after NMDA application. NMDA application leads to a decrease in green fluorescence in dendritic spines and shafts. G/R ratio is coded in rainbow colors and is scaled to encompass 2 SDs (2σ) of the average dendritic ratio before NMDA application. (Scale bar, 5 μm.) (B and C) Time course of red and green fluorescence in dendritic spines (B) and shafts (C) before and after NMDA application. NMDA leads to a long-lasting decrease in SEP-fluorescence within minutes, which is prevented by prior application of dCPP. Data are mean ± SEM.

Discussion

Activity-Dependent Phosphorylation and Internalization of Dendritic GABAB Receptors.

A previous report showed that glutamate application to cortical neurons decreases the number of GABAB receptors at the cell surface (18). Another report showed that glutamate application increases the steady-state level of GABAB receptor endocytosis while at the same time reducing the rate of endocytosis (35). Here, we show that glutamate acts via NMDA receptors to activate CaMKII (36), which directly phosphorylates S867 in the C terminus of GABAB1 to trigger endocytosis. NMDA-dependent phosphorylation of S867 is detectable only in the GABAB1b subunit, which mostly resides in the dendrites and, in contrast to the GABAB1a subunit, efficiently penetrates spines (10). Consistent with this, we found that GABAB receptors undergo endocytosis in dendritic spines and shafts within minutes of NMDA receptor activation. Notably, endocytosis prevents GABAB receptors from activating effector K+ channels that cluster with GABAB receptors in spines (12).

Physiological Implications.

GABA from interneurons firing in synchrony can spill over to pre- and postsynaptic GABAB receptors on excitatory synapses (11, 37). This will reduce glutamate release and produce hyperpolarizing inhibitory postsynaptic potentials that enhance Mg2+ block of NMDA receptors and thus reduce their Ca2+ signals (13, 14). In addition to modulating the electrical properties of neurons, GABAB receptors can also reduce the Ca2+ permeability of NMDA receptors in dendritic spines via activation of the PKA signaling pathway (15). Here, we demonstrate that NMDA receptors can counter this suppression of Ca2+ signals and rapidly endocytose GABAB receptors from the surface of dendritic shafts and spines. Hence, there appears to be a reciprocal regulation where both NMDA and GABAB receptors can cancel each other out. The temporal interplay of NMDA and GABAB receptors may be particularly relevant to phenomena controlling synaptic strength, where NMDA receptor activity is of importance. Of note, the same NMDA receptor/CaMKII signaling cascade regulating synaptic strength also internalizes GABAB receptors. This provides a means to keep individual glutamatergic synapses modifiable (38, 39) and to spare them from inhibition through spillover of GABA. A previous study reported that NMDA receptor activation promotes surface expression of Kir3 channels in hippocampal neurons (40), which was paralleled with an increase in basal Kir3 currents and adenosine A1-mediated Kir3 currents (41). However, GABAB mediated Kir3 currents were not altered, in apparent conflict with an earlier report (42) and our own findings. The reasons for these discrepancies are unclear but may relate to differences in the signaling pathways activated under the different experimental conditions used. Reciprocal regulation of NMDA and GABAB receptors is reminiscent of the recently reported interplay of NMDA and muscarinic receptors (43). In this cross-talk, NMDA receptors phosphorylate and inactivate muscarinic receptors in a CaMKII-dependent manner, much in the same way as now observed with GABAB receptors.

Materials and Methods

Neuronal Cultures.

Dissociated hippocampal and cortical neurons were prepared from embryonic day 18.5 Wistar rats or from embryonic day 16.5 WT and GABAB1−/− mice (33, 44). Neurons were transfected at DIV7 using Lipofectamin 2000 (Invitrogen). Pharmacological treatments were performed in conditioned medium at 37 °C/5% CO2.

Biochemistry.

Surface biotinylation, immunoprecipitation, in vitro phosphorylation, HPLC analysis, and MS were essentially performed as described (24, 45, 46).

Electrophysiology.

Recordings in cultured hippocampal neurons were performed with an Axopatch 200B patch-clamp amplifier. GABAB responses were evoked by fast application of 100 μM baclofen (47).

Two-Photon Imaging.

Organotypic hippocampal slice cultures for two-photon time-lapse imaging (48) were prepared from Wistar rats at postnatal day 5 (49). For time-lapse imaging, we used a custom-built, two-photon laser scanning microscope based on a BX51WI microscope (Olympus) and a pulsed Ti:Sapphire laser (Chameleon XR, Coherent) tuned to λ = 930 nm, controlled by the open source software ScanImage. Fluorescence was detected in epi- and transfluorescence mode using four photomultiplier tubes (R2896, Hamamatsu).

Data Analysis.

Data are given as mean ± SEM. Statistical significance was assessed using ANOVA, with the Dunnett's multiple comparison test unless otherwise indicated, using GraphPad Prism 5.0.

Additional experimental procedures are described in SI Materials and Methods.

Supplementary Material

Acknowledgments

We thank N. Hardel (Department of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, Basel, Switzerland) for the Rab11-eGFP plasmid, K. Ivankova and A. Cremonesi for technical help, and F. Schatzmann for comments on the manuscript. B.B. was supported by the Swiss Science Foundation (3100A0-117816) and the European Community's Seventh Framework Programme FP7/2007-2013 under Grant Agreement 201714. R.T. was supported by the Wellcome Trust International Senior Research Fellowship and an EU Synapse grant (LSHM-CT-2005-019055).

Footnotes

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