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Originally published In Press as doi:10.1074/jbc.M611533200 on April 18, 2007

J. Biol. Chem., Vol. 282, Issue 24, 17855-17865, June 15, 2007
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Identification of the Sites for CaMK-II-dependent Phosphorylation of GABAA Receptors*

Catriona M. Houston{ddagger}, Henry H. C. Lee{ddagger}§, Alastair M. Hosie{ddagger}, Stephen J. Moss{ddagger}§, and Trevor G. Smart{ddagger}1

From the {ddagger}Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom and the §Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication, December 18, 2006 , and in revised form, April 17, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphorylation can affect both the function and trafficking of GABAA receptors with significant consequences for neuronal excitability. Serine/threonine kinases can phosphorylate the intracellular loops between M3-4 of GABAA receptor beta and {gamma} subunits thereby modulating receptor function in heterologous expression systems and in neurons (1, 2). Specifically, CaMK-II has been demonstrated to phosphorylate the M3-4 loop of GABAA receptor subunits expressed as GST fusion proteins (3, 4). It also increases the amplitude of GABAA receptor-mediated currents in a number of neuronal cell types (5-7). To identify which substrate sites CaMK-II might phosphorylate and the consequent functional effects, we expressed recombinant GABAA receptors in NG108-15 cells, which have previously been shown to support CaMK-II modulation of GABAA receptors containing the beta3 subunit (8). We now demonstrate that CaMK-II mediates its effects on {alpha}1beta3 receptors via phosphorylation of Ser383 within the M3-4 domain of the beta subunit. Ablation of beta3 subunit phosphorylation sites for CaMK-II revealed that for {alpha}beta{gamma} receptors, CaMK-II has a residual effect on GABA currents that is not mediated by previously identified sites of CaMK-II phosphorylation. This residual effect is abolished by mutation of tyrosine phosphorylation sites, Tyr365 and Tyr367, on the {gamma}2S subunit, and by the tyrosine kinase inhibitor genistein. These results suggested that CaMK-II is capable of directly phosphorylating GABAA receptors and activating endogenous tyrosine kinases to phosphorylate the {gamma}2 subunit in NG108-15 cells. These findings were confirmed in a neuronal environment by expressing recombinant GABAA receptors in cerebellar granule neurons.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The {gamma}-aminobutyric acid type A (GABAA)2 receptor is a pentameric ligand-gated ion channel responsible for fast synaptic and tonic inhibition in the brain. The function of GABAA receptors can be modulated by phosphorylation, which affects inhibitory synaptic plasticity and thus has significant consequences for the control of neuronal network excitability (9, 10). Phosphorylation of the intracellular domains between M3-4 of the beta and {gamma} subunits by serine (Ser)/threonine (Thr) and tyrosine (Tyr) kinases has been shown to modulate receptor function either through a direct effect on receptor properties, such as the probability of channel opening or desensitization, or by regulating trafficking of the receptor to and from the cell surface (1, 2, 9).

The use of glutathione-based fusion proteins and site-directed mutagenesis has enabled the sites of phosphorylation within beta and {gamma} subunits to be identified. The beta2 subunit has one main site for phosphorylation at Ser410 (the equivalent of Ser409 in beta1), which can be phosphorylated by PKG, PKA, PKC, and CaMK-II (3). Similarly, Ser409 has also been identified as the major site of phosphorylation in the beta3 subunit for PKC, PKA, PKG, and CaMK-II with neighboring Ser408 additionally phosphorylated by PKC. Furthermore, Ser383 can also be phosphorylated, but only by CaMK-II. In comparison, the {gamma}2S subunit can be phosphorylated by PKC at Ser327, and at Ser348 and Thr350 by CaMK-II (4, 11).

In addition to phosphorylation by Ser/Thr kinases both the beta and {gamma} subunits are substrates for tyrosine kinases (12, 13). Mutation of the tyrosine residues at 365 and 367 on the {gamma}2S subunit (corresponding to Tyr373 and Tyr375 on {gamma}2L) removed the functional modulation of GABAA receptors expressed in HEK293 cells by the tyrosine kinase, Src (12).

CaMK-II has been demonstrated to potentiate GABAA receptor function by increasing the fraction of receptors (measured as a Bmax) in synaptosomal membranes (14, 15). CaMK-II can also increase the amplitudes of GABA whole cell currents and IPSCs measured in hippocampal, spinal cord dorsal horn, and cortical neurons, in addition to cerebellar Purkinje and granule neurons (5-8, 16). Furthermore, we have recently reported that NG108-15 cells, which lack endogenous GABAA receptors, form a suitable environment for CaMK-II modulation of transiently expressed {alpha}1beta3 and {alpha}1beta3{gamma}2S GABAA receptors (10). Using this heterologous expression system, and by expressing recombinant GABAA receptors in cerebellar granule neurons, we were able to identify which CaMK-II phosphorylation sites were important for functional modulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—In brief, NG108-15 cells were maintained in Dulbecco's modified Eagle's medium (4 g/liter glucose) supplemented with 10% v/v fetal calf serum (Invitrogen), 2 mM glutamine (Sigma), 100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine (Sigma), and 20 units/ml penicillin G and 20 µg/ml streptomycin (Sigma) and incubated at 37 °C, in 90% air, 10% CO2. Subculturing was performed 2-3 times/week. Cells used for transfection were plated onto poly-L-ornithine-coated coverslips (500 µg/ml) and used for electrophysiology 2-3 days later (8).

Cerebellar granule cell cultures were prepared from postnatal day (P) 0-1 Sprague-Dawley rats as follows: cerebella were enzymatically dissociated in trypsin (0.1%, 10 min at 37 °C) followed by trituration in DNase I with glass pipettes of decreasing diameter (x3), and plated onto poly-L-ornithine (500 µg/ml) coated coverslips and maintained in BME (Invitrogen) supplemented with 0.5% w/v glucose, 5 mg/liter insulin, 5 mg/liter transferrin, 5 mg/liter selenium (Sigma), 20 units/ml penicillin G, and 20 µg/ml streptomycin, 0.14 mM glutamine, 1.4 mM NaCl, 0.01 mg/ml bovine serum albumin (Sigma), and 20 mM KCl for 7-10 days before use (8). All animals were used in accordance with UK home office guidelines.

cDNA Constructs and Transfection—Murine GABAA receptor subunit {alpha}1, beta3, and {gamma}2S cDNAs were cloned into the plasmid vector pRK5 (17). The 9E10 (Myc) epitope was inserted between the fourth and fifth residue of the mature protein by site-directed mutagenesis (18, 19). Wild-type rat {alpha}-CaMK-II and mutant {alpha}-CaMK-II T286A cDNAs were cloned into the plasmid vector pcDNA3. NG108-15 cells and cerebellar granule cells were transfected using the reagent, Effectene (Qiagen Ltd, West Sussex, UK) in the presence of 0.3-0.5 µg of total cDNA/dish, with cDNAs encoding for GABAA receptor subunits and enhanced GFP (for cell identification) present in equal ratio.

Patch Clamp Electrophysiology—Whole cell membrane currents were recorded from single cells with an Axopatch 1-C amplifier (Molecular Devices, Union City, CA). Patch pipettes (resistance 5-6 M{Omega} for NG108-15 cells; 8-9 M{Omega} for granule neurons) were filled with the following solution containing (mM): 150 CsCl, 1 MgCl2, 10 HEPES, 4 Na2ATP, 0.1 CaCl2, and 1.1 mM EGTA, pH adjusted to 7.2 with CsOH (290-310 mOsm). The cells were perfused with the following Krebs solution containing (mM): 140 NaCl, 4.7 KCl, 1.2 MgCl2, 2.52 CaCl2, 11 D-glucose, 5 HEPES adjusted to pH 7.4 with 1 M NaOH (290-310 mOsm). Currents were filtered at 3 kHz (8-Pole Bessel filter) and analyzed using Clampex 8.2. Cells were not used for analysis if their access or series resistances changed by more than 15%. GABA (dissolved in Kreb's solution) was applied using a modified Y-tube rapid perfusion system as described previously (20). All experiments were carried out at room temperature (25 °C).

CaMK-II—A purified truncated recombinant form of {alpha}-CaMK-II (New England Biolabs, Beverly, MA) with the same substrate specificity as the full-length form (21) was pre-activated (40-50 ng/µl; specific activity ~200-250 units/µl) by incubation in a reaction buffer containing: 50 mM Tris-HCl, 10 mM MgCl2, 2 mM dithiothreitol, 0.1 mM Na2EDTA, supplemented with 1.2 µM calmodulin (CaM), 1.5 mM CaCl2, and 0.4 mM ATP{gamma}S for 15 min at 25 °C (8). Pre-activated {alpha}-CaMK-II was diluted within the patch pipette solution to give a final {alpha}-CaMK-II concentration of 60 nM and then maintained on ice throughout the recording period (5, 22, 23). Control whole cell recordings were made with a patch pipette solution containing an appropriate dilution of the pre-activation buffer, which omitted the {alpha}-CaMK-II.

Analysis—The peak amplitudes of membrane currents activated by GABA (10 µM) were determined at -50 mV (NG108-15) or -60 mV holding potential (granule cells). Statistical analyses were performed using either one-way analyses of variance (ANOVA) with a Bonferroni post-test to compare selected groups, or the nonparametric Kruskal-Wallis test with Dunn's post-test if the group variances were significantly different. The ANOVA post-test compared GABA-activated currents in the presence and absence of CaMK-II ({dagger}), as well as the effect of receptor mutations on the modulation by CaMK-II (*). In all cases, p < 0.05 was considered significant. All time points were tested for statistical significance, but for clarity only a selected few are shown for illustrative purposes.

Biochemistry—NG108-15 cells were transfected using electroporation and cultured for 48 h (17). Expressing cells were then labeled with 0.5-1.0 mCi/ml [32P]orthophosphoric acid (12,000 Ci/mmol) for 4 h at 37 °C, before treatment with specific kinase/phosphatase inhibitors and/or 50 mM KCl (24-27). Cells were then lysed in 2% boiling SDS, immunoprecipitated in a buffer supplemented with 2% Triton X-100 using antibodies against GABAA receptor subunits, followed by SDS-PAGE. The levels of incorporated radioactivity were measured using a phosphorimager (26, 27). Immunoprecipitated material was subject to SDS-PAGE and 32P-labeled. Isolated GABAA receptor subunits were also subject to phosphopeptide mapping and phosphoamino acid analysis (24). To specifically measure tyrosine phosphorylation of the {gamma}2 subunit, SDS-soluble lysates were prepared from transfected NG108-15 cells. These were subject to SDS-PAGE prior to immunoblotting with antibodies specific for phosphorylated Tyr365 and Tyr367 (anti-pY365/7) in the {gamma}2 subunit and with antibodies against total {gamma}2 subunits (anti-{gamma}2) coupled to 125I-protein A (12, 25, 26). The ratios of pY365/7 to {gamma}2 signals were then determined with levels measured under control conditions assigned a value of 100%.

Immunocytochemistry—Cerebellar granule cell cultures were fixed in 4% paraformaldehyde for 10 min and quenched with 50 mM NH4Cl (in phosphate-buffered saline (PBS); two washes) followed by three washes in PBS and then one wash with 10% v/v FCS and 0.5% w/v bovine serum albumin in PBS. The cells were then incubated with the primary antibody (45 min, rabbit anti-Myc, 1:200, Santa Cruz Biotechnology) followed by three washes in 10% v/v fetal calf serum and 0.5% w/v bovine serum albumin in PBS, prior to incubation with the secondary antibody (45 min, anti-rabbit TRITC conjugate 1:250, Sigma). Immunofluorescent images were acquired using a Zeiss confocal microscope (LSM 510 Meta), equipped with Argon (488 nm) and Helium Neon lasers (543 nm), with a x63 objective.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Modulation of {alpha}beta GABAA Receptors by CaMK-II: Role of M3-4 Serine Residues—Previously, we demonstrated that the internal application of pre-activated (8, 22, 23) {alpha}-CaMK-II to {alpha}1beta3/{gamma}2S GABAA receptors, expressed in NG108-15 cells, caused a significant increase in the amplitude of GABA whole cell currents (8). This effect was solely due to CaMK-II activity because heat-inactivated {alpha}-CaMK-II had no effect on the amplitude of {alpha}1beta3{gamma}2S GABAA receptor-mediated currents in NG108-15 cells (8). To determine if this potentiation resulted from GABAA receptor phosphorylation by CaMK-II, we mutated consensus sites for phosphorylation by CaMK-II. We presumed that one or more of the three phosphorylation sites identified in previous studies, Ser383,408,409 in beta3 subunits (3, 28) were responsible. Although the {alpha}1 subunit has been reported to be phosphorylated by CaMK-II (15), no specific residues were identified by fusion protein analysis (4, 29). Moreover, in our previous study, {alpha}1beta2 receptors were not modulated by CaMK-II (8). Therefore, we initially assumed that the beta3 subunit was the primary site of CaMK-II phosphorylation. Accordingly, all three serines in the beta3 subunit were converted to non-phosphorylated alanines. Using NG108-15 cells expressing {alpha}1beta3S383,408,409A GABAA receptors, the repeated application of 10 µM GABA elicited currents that were quite stable over 20 min when recording with the control patch pipette solution, which always contained the pre-activation buffer without {alpha}-CaMK-II (8). In parallel experiments, by internally applying {alpha}-CaMK-II (60 nM), the GABA current amplitudes retained their stability (93.2 ± 7.6%) compared with the controls (101.3 ± 4.6% at t = 6 min, Fig. 1A). This strongly indicated that phosphorylation of at least one or more of these three serine residues mediated the {alpha}-CaMK-II modulation of the {alpha}1beta3 receptor.


Figure 1
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  		FIGURE 1.
Serine 383 on the intracellular M3-M4 loop of the beta3 subunit mediates CaMK-II-dependent effects on {alpha}1beta3 GABAA receptors expressed in NG108-15 cells. Whole cell GABA-activated currents were recorded for 20 min from NG108-15 cells expressing; {alpha}1beta3S383,408,409A (A); {alpha}1beta3S408;409A (B); and {alpha}1beta3S383A (C) GABAA receptors in the presence of pre-activated {alpha}-CaMK-II (60 nM), which was applied via the patch pipette solution. Peak amplitude whole cell currents were normalized to the peak currents recorded 3-4 min after achieving the whole-cell configuration (defined as t = 0 min and 100%). Control recordings, displayed in all figures, were obtained with a patch pipette solution supplemented with the pre-activation buffer without {alpha}-CaMK-II. Representative 10 µM GABA-activated currents are shown at different time points in control conditions or in the presence of {alpha}-CaMK-II. All data points in this and proceeding figures represent the mean ± S.E. (n = 4-8 cells). Data from wild-type receptors, which were obtained in parallel experiments, have been previously published and are shown for comparison (8). The symbol (*) indicates significant differences between wild-type and mutant receptors in the presence of {alpha}-CaMK-II (A and C) and ({dagger}) indicates significance between mutant receptors in the presence of CaMK-II and in control conditions (B).

 
PKA phosphorylation of beta3 subunits has been reported to increase the amplitude of GABA-activated currents by phosphorylating Ser408,409 (28). However, the application of {alpha}-CaMK-II to {alpha}1beta3S408,409A receptors still potentiated current amplitudes to 120.6 ± 6.2% compared with the controls (93.9 ± 3.8% at t = 6 min). This was not significantly different from the effect of {alpha}-CaMK-II on {alpha}1beta3 wild-type receptors (118 ± 4.8%, Fig. 1B (8)). The time courses for the potentiating effects of {alpha}-CaMK-II were also similar for both the SerS408,409A mutant and wild-type receptors, attaining a steady-state within 6 min. By contrast to SerS408,409A, application of {alpha}-CaMK-II to {alpha}1beta3S383A receptors prevented the GABA current potentiation (94 ± 6.8%) compared with control (101.7 ± 1.5% at t = 6 min, Fig. 1C). This indicated that the {alpha}-CaMK-II modulation of {alpha}1beta3 receptor function is mediated solely through phosphorylation of Ser383.

Serine 383 Is Phosphorylated by CaMK-II—To confirm whether Ser383 is the main site of CaMK-II phosphorylation, NG108-15 cells expressing {alpha}1beta3{gamma}2 receptors were exposed to [32P]orthophosphoric acid prior to activating CaMK-II by depolarizing the cells with 50 mM KCl-based external solution. Receptor beta3 subunits were isolated by immunoprecipitation using subunit-selective antisera linked to protein A-Sepharose. Precipitated subunits were resolved using SDS-PAGE and visualized with autoradiography. The beta3 subunits exhibited basal phosphorylation, which was enhanced by the KCl external solution and inhibited back to control levels by exposure to the CaMK-II inhibitor, KN-62 (Fig. 2, A and B). To identify those residues that are phosphorylated we subjected gel slices to phosphoamino acid analysis. All the phosphorylation was located to serine residues (Fig. 2C). By mutating Ser383 to alanine, basal phosphorylation remained associated with the serine residues, but the increase caused by KCl was ablated (Fig. 2C). Quantitative analysis revealed that KCl increased beta3 phosphorylation in wild-type {alpha}1beta3{gamma}2 receptors by 50 ± 15% (Fig. 3, A and B), which was reduced to basal levels by 1 µM KN-62. In addition, phosphorylation remained unaltered by KCl if only a mutant form of CaMK-IIT286A was present which lacked the ability to autophosphorylate and become Ca2+ independent. Although beta3S383A prevented phosphorylation of beta3 subunits by CaMK-II, the mutation beta3S408,409A did not, with phosphorylation levels in the presence of CaMK-II increasing by over 50% (Fig. 3B). Together these experiments strongly suggest that Ser383 is the principal site of CaMK-II phosphorylation in the receptor beta3 subunit.


Figure 2
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  		FIGURE 2.
GABAA receptor beta3 subunit is phosphorylated by CaMK-II. The GABAA beta3 subunit was co-expressed with the {alpha}1 and {gamma}2 subunits and wild-type {alpha}-CaMK-II in NG108-15 cells. A,[35S]methionine metabolic labeling in live cells, either transfected with GABAA receptor cDNAs or mock-transfected, and immunoprecipitation of beta3 subunits with beta3 subunit antisera. B, mock-transfected and {alpha}1beta3{gamma}2 subunit-expressing cells were labeled with [32P]orthophosphoric acid and were then either left untreated or exposed to high K+ (50 mM for 2 min) and/or KN-62 (1 µM for 5 min). Lysates were immunoprecipitated with beta3 subunit antisera. C, phosphoamino acid analysis of wild-type (wt) beta3 and mutant beta3S383A subunits taken from immunoprecipitated receptors exposed to normal external solution or high K+ solution. Subunits were isolated by acid hydolysis and electrophoresis. The positions of phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) are indicated.

 
CaMK-II Regulation of {alpha}beta{gamma} Receptors—For {alpha}1beta3{gamma}2S receptors expressed in NG108-15 cells, CaMK-II induced a significantly larger potentiation of GABA currents when compared with its modulation at {alpha}1beta3 receptors (8). This suggested that {alpha}-CaMK-II modulation may be partly mediated through the {gamma}2S subunit. Indeed, in parallel experiments, applying {alpha}-CaMK-II (60 nM) to {alpha}1beta3S383,408,409A{gamma}2S receptors still resulted in a significant potentiation of 10 µM GABA current amplitudes to 117.8 ± 5% as compared with the controls (93.4 ± 4%; at t = 8 min; Fig. 4A). The potentiation required longer to peak compared with the time course for wild-type receptors. The effect was also transient with current amplitudes returning to baseline levels within 20 min (Fig. 4A). This residual potentiation was significantly smaller than that observed with {alpha}-CaMK-II on {alpha}1beta3{gamma}2S wild-type receptors ({alpha}1beta3S383,408,409A{gamma}2S, 107.5 ± 3.5; {alpha}1beta3{gamma}2S, 145.8 ± 10.8%; at t = 4 min (8)). These results confirmed that {alpha}-CaMK-II is mediating part of its potentiating effect via the {gamma}2S subunit.


Figure 3
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  		FIGURE 3.
Serine 383 is the major CaMK-II phosphorylation site in beta3 subunits. NG108-15 cells expressing {alpha}1beta3{gamma}2, {alpha}1beta3S383A{gamma}2, or {alpha}1beta3S408,409A{gamma}2 subunits with either wild-type or a mutant form of {alpha}-CaMK-IIT286A incapable of autophosphorylation were labeled with [35S]methionine. A, cells were exposed to normal external or high K+ solutions. Expressed GABAA receptors were immunoprecipitated with beta3 subunit-selective antisera. Immune complexes were resolved by SDS-PAGE and analyzed using a phosphorimager. B, quantitative analysis of beta3 subunit phosphorylation for {alpha}1beta3{gamma}2, {alpha}1beta3S383A{gamma}2, and {alpha}1beta3S408,409A{gamma}2 receptors treated with normal external solution or high K+ solution with and without KN-62, in the presence of wild-type or mutant {alpha}-CaMK-IIT286A. All bars represent mean ± S.E. (n = 3).

 
Prior analysis of GST fusion proteins in vitro identified the potential substrate sites for CaMK-II phosphorylation on the {gamma}2S subunit as Ser348 and Thr350. By applying {alpha}-CaMK-II to {alpha}1beta3{gamma}2SS348A,T350A receptors, GABA currents were still potentiated up to 142.3 ± 9.1% compared with controls (99 ± 6.5%; Fig. 4B;at t = 6 min). This level of potentiation was the same as that observed with CaMK-II and wild-type {alpha}1beta3{gamma}2S receptors (8), indicating that Ser348 and Thr350 are not involved in CaMK-II modulation of these receptors.

The application of {alpha}-CaMK-II to the mutant receptor, {alpha}1beta3S383,408,409A{gamma}2SS348A,T350A, where all potential CaMK-II sites of phosphorylation were now removed, also allowed a significant potentiation of GABA currents to 126.3 ± 6.3% as compared with control, 98.6 ± 2.7% (Fig. 4C, at t = 8 min). Once again, this was not significantly different from that seen with {alpha}1beta3S383,408,409A{gamma}2S receptors, further indicating that phosphorylation of Ser348 and Thr350 does not contribute to the potentiation of GABA currents by {alpha}-CaMK-II. In accord with our previous observations, the potentiation was transient and after 10 min the effect of {alpha}-CaMK on {alpha}1beta3S383,408,409A {gamma}2SS348A,T350A receptors was significantly different (114.2 ± 6.4%) from the effect on wild-type receptors (148.5 ± 12.2%) but not significantly different from control (102.7 ± 5.8%, Fig. 4C).


Figure 4
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  		FIGURE 4.
{alpha}-CaMK-II modulation of {gamma}2S subunit-containing receptors expressed in NG108-15 cells is not mediated through phosphorylation of GABAA Ser348 and Thr350. Peak amplitude GABA-activated currents recorded over 20 mins from NG108-15 cells expressing: {alpha}1beta3S383,408,409A{gamma}2S (A), {alpha}1beta3{gamma}2SS348A,T350A (B), or {alpha}1beta3S383,408,409A{gamma}2SS348A,T350A (C) GABAA receptors in the presence of pre-activated {alpha}-CaMK-II (60 nM, n = 4-8 cells). Control recordings were obtained with a normal patch pipette solution supplemented with the preactivation buffer without {alpha}-CaMK-II. Data obtained from parallel experiments using wild-type receptors are shown for comparison and are taken from Ref. 8. Representative 10 µM GABA-activated currents are shown at different time points in control conditions or in the presence of {alpha}-CaMK-II. D, representative GABA currents recorded from NG108-15 cells expressing {alpha}1beta3{gamma}2SS348A,T350A receptors, before and after 10 µM diazepam. The symbol (*) indicates significant differences between wild-type and mutant receptors in the presence of {alpha}-CaMK-II, and ({dagger}) indicates significance between mutant receptors in the presence of {alpha}-CaMK-II and in control conditions.

 
To ensure that {alpha}1beta3{gamma}2SS348A,T350A receptors were correctly assembled, we applied 10 µM diazepam which potentiated approximately EC50 GABA-activated currents, indicating that the {gamma}2SS348A,T350A subunit was indeed expressed at the cell surface (Fig. 4D). Furthermore, if the {gamma}2S subunit had been absent, the resultant receptor, {alpha}1beta3S383,408,409A, should not have been modulated by {alpha}-CaMK-II (Fig. 1A). Thus the increased GABA current in the presence of {alpha}-CaMK-II confirmed that {alpha}beta{gamma} heteromers were correctly assembled and expressed at the cell surface (Fig. 4C).

CaMK-II and Tyrosine Residues on {gamma}2S—Although Ser348 and Thr350 are the main sites of CaMK-II phosphorylation on the {gamma}2S subunit, their mutation clearly had no effect on {alpha}-CaMK-II modulation of {alpha}1beta3{gamma}2S receptors. This raised the possibility that CaMK-II was either phosphorylating new sites on the {gamma}2 subunit, or that another kinase, driven by CaMK-II activating downstream signaling pathways, was phosphorylating the GABAA receptor or an associated protein. The {gamma}2S subunit contains two sites for tyrosine kinase phosphorylation, Tyr365 and Tyr367, which after phosphorylation, can potentiate GABAA receptor function in a similar manner to that observed in the present study (12). To assess whether tyrosine kinases were involved in CaMK-II modulation of {alpha}1beta3{gamma}2S GABAA receptors, the tyrosine kinase inhibitor genistein (100 µM) was co-applied in the patch pipette solution with {alpha}-CaMK-II (60 nM). A significant increase in the amplitude of GABA currents to 120.3 ± 7.6% compared with control (97.8 ± 3%; Fig. 5A;at t = 4 min) was still evident in the presence of genistein and CaMK-II. However, this potentiation was significantly smaller compared with that observed with {alpha}-CaMK-II alone (wild-type: 145.7 ± 10.2%, + genistein 116.1 ± 6.4 at t = 6 min (8)). The application of genistein alone, had no effect on the amplitude of GABA currents recorded over 20 min (Fig. 5A). Furthermore, application of {alpha}-CaMK-II in the presence of genistein to {alpha}1beta3S383A{gamma}2S receptors failed to affect GABA current amplitude (103.6 ± 2.8%) over 20 min when compared with control (97 ± 7.1% at t = 6 min, Fig. 5B). Taken together, these results suggested that phosphorylation at Ser383 in the beta3 subunit and {alpha}-CaMK-II-dependent activation of tyrosine kinase activity is necessary for the modulation of GABAA receptors by {alpha}-CaMK-II.


Figure 5
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  		FIGURE 5.
{alpha}-CaMK-II modulation of {alpha}1beta3{gamma}2S subunit-containing GABAA receptors requires tyrosine kinase phosphorylation. Peak amplitude GABA-activated currents recorded from NG108-15 cells expressing: {alpha}1beta3{gamma}2S (A) or {alpha}1beta3S383A, {gamma}2S (B) GABAA receptors in the presence of {alpha}-CaMK-II (60 nM) and/or 100 µM genistein applied via the patch pipette. Control recordings were obtained with a normal patch pipette solution supplemented with the pre-activation buffer without{alpha}-CaMK-II and 100µM genistein (n = 4-7). Data obtained from parallel experiments with wild-type receptors are shown for comparison (8). Representative 10 µM GABA-activated currents are shown at different time points in the presence of genistein or genistein and {alpha}-CaMK-II. The symbol (*) indicates significant differences between wild-type receptors in the presence of {alpha}-CaMK-II and the presence or absence of genistein, and ({dagger}) denotes significance between wild-type receptors in the presence of genistein and the presence or absence of {alpha}-CaMK-II.

 
By mutating the {gamma}2 subunit to prevent tyrosine phosphorylation, the application of {alpha}-CaMK-II to {alpha}1beta3{gamma}2SY365F,Y367F receptors resulted in a significant potentiation of the GABA current amplitudes to 143.8 ± 4.5% as compared with control (94.5 ± 6.3%; Fig. 6A) at t = 16 min. However, as before, this potentiation was significantly less than that induced by {alpha}-CaMK-II on {alpha}1beta3{gamma}2S receptors (wild-type: 145.7 ± 10.2%; Y365F, Y367F: 119.4 ± 3.5%) at t = 6 min (8). Notably, after 8-10 min of recording, there was no significant difference in the response of the mutant {gamma}2SY365F,Y367F receptor to {alpha}-CaMK-II compared with wild-type. This correlated with the observation that the response to {alpha}-CaMK-II mediated by the {gamma}2S subunit is transient and is no longer evident after 10-15 min.

Finally, application of {alpha}-CaMK-II to {alpha}1beta3S383A{gamma}2SY365F,Y367F receptors abolished the response to {alpha}-CaMK-II on GABA current amplitudes over 20 min (103.3 ± 3.4% at t = 6 min) as compared with control (108.4 ± 4.3% at t = 6 min, Fig. 6B). This indicates that the functional effects of {alpha}-CaMK-II-dependent phosphorylation on {alpha}1beta3{gamma}2S receptors are mediated entirely via beta3 Ser383 and {gamma}2S Tyr365,367. Moreover, {alpha}-CaMK-II is likely to directly phosphorylate the receptor via the beta subunit and cause an indirect activation of a tyrosine kinase leading to phosphorylation of the {gamma}2S subunit.

Application of 10 µM diazepam to {alpha}1beta3{gamma}2SY365F,Y367F receptors resulted in a potentiation of the GABA current indicating that the {gamma}2SY365F,Y367F subunit was expressed and correctly assembled at the cell surface (Fig. 6C). It has been noted that the addition of the {gamma} subunit to {alpha}beta heteromers increases the amplitudes of the GABA currents (30). As expected, the current density recorded from NG108-15 cells expressing {alpha}1beta3 heteromers (20.2 ± 11.5 pA/pF) was significantly increased for {alpha}1beta3{gamma}2S receptors (94.1 ± 19 pA/pF (8)). By contrast, there were no significant differences in current densities between {alpha}1beta3{gamma}2S, {alpha}1beta3{gamma}2SS348A,T350A (87.1 ± 21.3 pA/pF) and {alpha}1beta3{gamma}2SY365F,Y367F (82.1 ± 15.6 pA/pF) receptors. Thus site-specific mutation of phosphorylation sites in the {gamma}2S subunit did not disrupt the surface expression of GABAA receptor subunits in NG108-15 cells.

CaMKII Induces Phosphorylation of Tyrosine Residues in {gamma}2 Subunits—Whether the {gamma}2 subunits can be phosphorylated by tyrosine kinases after activation of CaMK-II was assessed using our phosphospecific antibodies directed against Tyr365 and Tyr367 (anti-pY365/7) in the {gamma}2 subunit (25), the principal sites of tyrosine phosphorylation within the GABAA receptor (12). Using transfected NG108-15 cells expressing {alpha}1beta3{gamma}2 subunits and {alpha}-CaMK-II, phosphorylation was activated by exposing the cells to a high K+ external solution. SDS-soluble cell extracts were then immunoblotted with anti-pY365/7 and with anti-{gamma}2 directed against phosphoindependent epitopes. The {gamma}2 subunit was detected as a band of ~48 kDa in cells transfected with GABAA receptor cDNAs but not in cells expressing just GFP (Fig. 7A). A doublet of identical mass was also observed with anti-pY365/7 in cells expressing {gamma}2 subunits but not GFP. Significantly the detection of these bands was blocked by preabsorption with the phosphorylated antigen (not shown) used to produce this sera (25). The intensity of this band doubled after exposure to high K+ external solution while the level of total {gamma}2 subunits remained unaltered (Fig. 7B). Overall, these data indicate that the {gamma}2 subunits are phosphorylated at positions 365 and 367 after CaMK-II activation.


Figure 6
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  		FIGURE 6.
{alpha}-CaMK-II modulation of {alpha}1beta3{gamma}2S subunit-containing GABAA receptors requires tyrosine kinase phosphorylation of {gamma}2S Tyr365,367. Peak amplitude GABA-activated currents recorded from NG108-15 cells expressing: {alpha}1beta3{gamma}2SY365,367F (A) or {alpha}1beta3S383A{gamma}2SY365,367F (B) GABAA receptors in the presence of {alpha}-CaMK-II (60 nM) applied via the patch pipette. Control recordings were obtained with a normal patch pipette solution including the pre-activation buffer without {alpha}-CaMK-II (n = 4-7). Representative GABA currents are shown at different time points in absence or presence of {alpha}-CaMK-II. C, representative GABA currents recorded from NG108-15 cells expressing {alpha}1beta3{gamma}2SY365,367F receptors, before and after 10 µM diazepam. The symbol (*) indicates significant differences between wild-type and mutant receptors in the presence of {alpha}-CaMK-II, and ({dagger}) denotes significance between mutant receptors in the presence of {alpha}-CaMK-II and in control conditions.

 
Recombinant GABAA Receptors Are Modulated by CaMKII in Cerebellar Granule Neurons—Previously, we reported that {alpha}-CaMK-II potentiated GABA currents mediated by endogenous GABAA receptors expressed in cerebellar granule neurons and that heat-inactivated CaMK-II had no effect. Furthermore, the expression of recombinant GABAA receptors in granule neurons modified the effect of CaMK-II suggesting that the recombinant receptors functionally dominated the native receptor population (8). Thus, to assess the importance of the CaMK-II phosphorylation sites in a native environment, we expressed GABAA receptor subunits containing single mutated sites for phosphorylation, in granule neurons.

To ensure robust cell surface expression of recombinant beta subunits in cerebellar granule neurons we co-expressed them with recombinant {alpha}1 subunits (8). Co-expression of {alpha}1 with beta3S408A,S409A or with beta3S383A revealed that despite the disrupted phosphorylation sites, these recombinant subunits were capable of efficient expression at the cell surface. Co-expression of {alpha}1 and beta3 with Myc-tagged {gamma}2SY365F,Y367F also resulted in clear expression of surface {alpha}1beta3{gamma}2Y365F,Y367F receptors (Fig. 8C). As a control, EGFP only expressing neurons had similar resting membrane potentials to untransfected cells (-43.9 ± 2.8 mV; n = 8 and -39.2 ± 2.5 mV; n = 6, respectively) and displayed stable GABA current amplitudes over time with little evidence of rundown (93.7 ± 8.7% at t = 16 min) (8).

Application of 60 nM {alpha}-CaMK-II to {alpha}1beta3S408A,S409A-expressing granule cells resulted in a significant increase in the GABA current amplitude to 127.1 ± 5.1% at t = 6 min as compared with the application of {alpha}-CaMK-II to {alpha}1beta3S383A-expressing granule cells (103.1 ± 3%, at t = 6 min, Fig. 8A). The level of potentiation for {alpha}1beta3S408A,S409A, was not significantly different from that observed in parallel experiments after applying {alpha}-CaMK-II to wild-type {alpha}1beta3 heteromers (8). These results demonstrated that recombinant GABAA receptors responded similarly to {alpha}-CaMK-II whether they were expressed in granule neurons or NG108-15 cells. The mutation of Ser383 on the beta3 subunit still ablated the {alpha}-CaMK-II-dependent effect on {alpha}1beta3 heteromers in a neuronal environment.

Application of 60 nM {alpha}-CaMK-II to granule neurons expressing {alpha}1beta3{gamma}2SY365F,Y367F receptors increased whole cell GABA current amplitudes to 116.7 ± 3.6% at t = 6 min. This increase, however, was significantly smaller than the increase observed after application, in parallel experiments, of {alpha}-CaMK-II to {alpha}1beta3{gamma}2S-expressing granule cells (167.8 ± 13.7%, at t = 6 min, Fig. 8B, (8)). This indicated that, as for recombinant GABAA receptors expressed in NG108-15 (Fig. 6), the effect of {alpha}-CaMK-II on {alpha}1beta3{gamma}2S receptors in neurons could be reduced by mutating the tyrosine kinase phosphorylation sites on the {gamma}2S subunit. In addition, and similar to observations in NG108-15 cells (Fig. 6), the effect of CaMK-II was transient with no significant difference in GABA current amplitudes recorded from {alpha}1beta3{gamma}2S and {alpha}1beta3{gamma}2SY365F,Y367F receptors in the presence of {alpha}-CaMK-II after 10 min (Fig. 8B).


Figure 7
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  		FIGURE 7.
GABAA receptor {gamma}2 subunits are phosphorylated on tyrosine residues after activation of{alpha}-CaMK-II in NG108-15 cells. A, NG108-15 cells expressing either GFP and {alpha}-CaMK-II, or {alpha}1beta3{gamma}2S and {alpha}-CaMK-II were exposed to normal external solution (-) or high K+ solution (+) prior to immunoprecipitation with {gamma}2 subunit-selective antisera and Western blotting with phosphospecific antisera against Tyr365/Tyr367 and antisera selective for all {gamma}2 subunits. B, bar graph of the relative levels of tyrosine phosphorylation for {gamma}2 subunits in high K+ solution, normalized to control levels measured in other {alpha}1beta3{gamma}2S expressing NG108-15 cells in the absence of high K+ (= 100%). Values are mean ± S.E. (n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Using site-directed mutagenesis and the expression of recombinant GABAA receptors in NG108-15 cells and cerebellar granule neurons, we have identified the sites of phosphorylation that are important for {alpha}-CaMK-II-dependent modulation of {alpha}1beta3 and {alpha}1beta3{gamma}2S GABAA receptors. It appeared that phosphorylation of Ser383 on beta3 subunits by {alpha}-CaMK-II-mediated part of the potentiation of GABA currents, probably by directly phosphorylating the GABAA receptor. In addition, tyrosine phosphorylation of the {gamma}2S subunit was an important contributor to the potentiation as preventing phosphorylation of Tyr365,367 in the {gamma}2S subunit ablated CaMK-II modulation of {gamma}2 subunit-containing GABAA receptors. As {alpha}-CaMK-II was internally applied via the patch pipette it would indicate tyrosine kinase activity must be downstream of CaMK-II activation. The slower time course of the GABA current potentiation mediated by phosphorylation of the {gamma}2 subunit is in accord with downstream tyrosine kinase activity.

Serine 383 on the beta3 subunit would appear to be a unique site for {alpha}-CaMK-II-mediated phosphorylation because no other Ser/Thr kinase phosphorylated this residue when the beta3 subunit M3-4 intracellular loop was expressed as a GST fusion protein (3). The homologous residue in a beta1 subunit fusion protein, Ser384, can also be phosphorylated by CaMK-II (4); however, the beta2 subunit does not contain this site. This accorded with our previous work on recombinant GABAA receptors expressed in NG108-15 cells demonstrating that beta2 subunit-containing receptors are not apparently modulated by CaMK-II (8).

As {alpha}1beta3{gamma}2SY365F,Y367F mutants were less sensitive to {alpha}-CaMK-II modulation, it appears that {alpha}-CaMK-II is capable of directly activating a tyrosine kinase or acting indirectly, to allow increased tyrosine phosphorylation of the GABAA receptor {gamma}2S subunit. Phosphorylation of these tyrosine residues would be expected to result in an increase in the whole cell GABA current amplitude due to an increase in the single GABA channel mean open time and open probability (12).

The results of our study suggest that the tyrosine kinase phosphorylation of the {gamma}2S subunit must also be dependent on the beta subunit present in the receptor complex, as {alpha}1beta2{gamma}2S receptors expressed in NG108-15 cells are insensitive to CaMK-II-dependent modulation (8). For tyrosine kinases such as Src, binding to the NMDA receptor is thought to occur via association with PSD-95 or possibly via another adaptor protein rather than by directly interacting with the receptor itself (31, 32). If a similar situation occurs with GABAA receptors, the binding of CaMK-II to the receptors, either directly or through an adaptor protein, may be beta3 subunit-dependent and unable to occur if beta2 subunits are present within the receptor complex. This would explain the lack of tyrosine kinase-dependent modulation of {alpha}1beta2{gamma}2S receptors after applying CaMK-II (8).

Modulation of GABAA receptors by PKA and PKC has also been suggested to be dependent on the subtypes of beta and {gamma} subunits present in the receptor complex, with both being required for the full modulatory effect to be observed (33). Serine 409 on the beta1 subunit together with Ser327/343 on the {gamma}2L subunit, were found to be important for PKC modulation in HEK cells and L929 fibroblasts (33, 34). Both subunits were also found to be important for PKC modulation of {alpha}1beta2{gamma}2S receptors in Xenopus oocytes (35). However, although the beta and {gamma} subunits are the main targets for phosphorylation of GABAA receptors, little is known about how the intracellular loops of different subunits might interact during phosphorylation. An interaction seems likely and possibly crucial, given the insensitivity of beta2 subunit-containing receptors to {alpha}-CaMK-II. We can also conclude from our data that the presence of phosphorylation sites on the {gamma}2 subunits alone is insufficient to ensure maximum CaMK-II dependent modulation. It is possible that phosphorylation of the beta subunit is critical in allowing effective phosphorylation of the {gamma}2S subunit.

Overall, the most parsimonious explanation of our results is that CaMK-II activates a tyrosine kinase. Although it is also possible CaMK-II may act to inhibit a tyrosine phosphatase and so promote tyrosine phosphorylation of the {gamma}2 subunit indirectly. Interestingly, there is much more evidence that Ca2+-sensitive tyrosine kinases exist (36, 37) and that PKC can influence the activity of the non-receptor tyrosine kinase Src, implying that Ser/Thr kinases and the Src family of tyrosine kinases could interact (38, 39). It is generally considered that activation of Src through PKC is mediated by activation of PYK2 (40), a member of the focal adhesion kinase family highly expressed in the nervous system (41, 42), which responds to Ca2+ (41). Activation of PYK2 leads to autophosphorylation which creates an SH2 ligand that can bind the SH2 domain of Src, thereby activating it (39, 40).


Figure 8
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  		FIGURE 8.
Serine 383 and tyrosine 365,367 are important residues for CaMK-II modulation of GABAA receptors expressed in cerebellar granule cells. Peak amplitude GABA-activated currents recorded from cerebellar granule neurons transfected with recombinant GABAA receptor subunits: {alpha}1beta3S383A or {alpha}1beta3S408,409A (A); and {alpha}1beta3{gamma}2S (taken from Ref. 8) or {alpha}1beta3{gamma}2SY365,367F (B) in the presence of pre-activated CaMK-II (60 nM, n = 4-8). Representative GABA currents are shown at different time points in the presence of CaMK-II. The symbol (*) indicates significant differences between mutant or mutant and wild-type receptors in the presence of CaMK-II. C, rat cerebellar granule neurons were transfected with cDNAs encoding for: {alpha}1beta3S383A,myc (top); {alpha}1beta3S408,409A,myc (middle) or {alpha}1beta3{gamma}2SY365,367F (bottom) GABAA receptor subunits with EGFP. Cells were immunolabeled with an anti-Myc antibody without permeabilization so that only subunits at the cell surface were detected. Each image is representative of at least five transfected cells. The left column shows EGFP fluorescence (green); the middle column illustrates anti-Myc immunolabeling (red) with an enlarged inset showing cell surface staining on the cell body; and the right column presents an overlay of the left and middle columns (co-localization indicated by yellow) on a DIC brightfield image. Calibration bar, 10 µm.

 
Activation of PYK2, and subsequently Src, has been demonstrated in some cases to be Ca2+/calmodulin-dependent (43, 44). In this regard there is evidence for CaMK-II-dependent activation of Src in glomerular mesangial cells (45) and HEK293 cells (46), whereas, CaMK-II-dependent activation of PYK2 and Src has been demonstrated in vascular smooth muscle (47, 48), PC12 cells (49), and in hippocampal neurons following ischemia (50). There is also evidence for CaMK-II activating the related non-receptor tyrosine kinase, focal adhesion kinase (FAK) (51). It is unknown if NG108-15 cells express PYK2, but given that this protein is abundant in the CNS (41, 42) and the neuronal origin of the secondary cell line, it seems plausible.

Src activation has been implicated in the regulation of excitatory synaptic transmission by phosphorylating NMDA receptors (38, 52, 53). Moreover, PYK2-induced Src activation is thought to be necessary for certain forms of long-term potentiation (54). At the NMDA receptor, Src acts as a point of convergence for a number of different signaling pathways (52). There is also evidence that tyrosine phosphorylation of NR2B can modulate CaMK-II binding to the NMDA receptor (55). In comparison, CaMK-II phosphorylation of GABAA receptor beta3 subunits could be a prerequisite for tyrosine kinase targeting to the {gamma}2 subunit, for example, by allowing binding of another kinase or regulatory protein. Such a phenomenon of metaplasticity of kinase function has been demonstrated in the regulation of a nonspecific cation channel in Aplysia bag cell neurons where inhibition of Src promotes the interaction of this ion channel with PKC. Src phosphorylation of the channel was proposed to either prevent association of PKC with the channel, or Src may act as a scaffolding/marshalling protein (56).

There is also evidence that tyrosine kinases play a role in the modulation of inhibitory synaptic transmission. Src has been shown to mediate an increase in the amplitude of GABAA receptor-mediated IPSCs recorded from cerebellar Purkinje cells (57). This was considered to be in accord with the findings of Moss et al. (12) who reported an increased channel mean open time and open probability. Activation of PYK2 and Src in prefrontal cortical neurons has also been shown to increase the amplitude of IPSCs (58).

In conclusion, tyrosine kinase and CaMK-II signaling pathways may interact to modulate the function of GABAA receptors. Given the important role for beta3 subunits early in neuronal development (59, 60) this could have important consequences for synaptogenesis. It is early on in development when GABA has an excitatory role that CaMK-II and other Ca2+-sensitive signaling pathways may also play an important role, possibly in activity-dependent strengthening of synaptic connections.

The evidence presented in this study suggests that {alpha}-CaMK-II mediates its effects on {alpha}1beta3{gamma}2S receptors in NG108-15 cells and cerebellar granule neurons through direct phosphorylation of the beta3 subunit at Ser383 and via CaMK-II activation of a tyrosine kinase that phosphorylates {gamma}2S subunits at Tyr365,367.


   FOOTNOTES
 
* This work was supported in part by the MRC and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pharmacology, UCL, Gower St., London WC1E 6BT, United Kingdom. Tel.: 0044-207-679-2013; E-mail: t.smart{at}ucl.ac.uk.

2 The abbreviations used are: GABAA, {gamma}-aminobutyric acid receptor type A; TM, transmembrane; CaMK-II, calcium/calmodulin-dependent protein kinase type II; GST, glutathione S-transferase; PKG, protein kinase G; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; HEK, human embryonic kidney; IPSC, inhibitory post-synaptic current; CGC, cerebellar granule cell; EGFP, enhanced green fluorescent protein; PYK, proline-rich tyrosine kinase 2; FAK, focal adhesion kinase; ANOVA, analysis of variance; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; ATP{gamma}S, adenosine 5'-O-(thiotriphosphate). Back


   ACKNOWLEDGMENTS
 
We thank Helena Da Silva for technical support with site-directed mutagenesis and Ian Duguid for comments on the manuscript.



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 ABSTRACT
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 DISCUSSION
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