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Originally published In Press as doi:10.1074/jbc.M707233200 on October 2, 2007 Originally published In Press as doi:10.1074/jbc.M707233200 on September 17, 2007

J. Biol. Chem., Vol. 282, Issue 45, 33052-33063, November 9, 2007
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Protein Kinase C{epsilon} Regulates {gamma}-Aminobutyrate Type A Receptor Sensitivity to Ethanol and Benzodiazepines through Phosphorylation of {gamma}2 Subunits*Formula

Zhan-Heng Qi{ddagger}1, Maengseok Song{ddagger}1, Melisa J. Wallace{ddagger}, Dan Wang{ddagger}, Philip M. Newton{ddagger}, Thomas McMahon{ddagger}, Wen-Hai Chou{ddagger}, Chao Zhang§, Kevan M. Shokat§, and Robert O. Messing{ddagger}2

From the {ddagger}Ernest Gallo Clinic and Research Center, Department of Neurology, University of California San Francisco, Emeryville, California 94608 and the §Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94143

Received for publication, August 28, 2007 , and in revised form, September 17, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ethanol enhances {gamma}-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABAA receptors, especially at low concentrations achieved during social drinking. We postulated that the {epsilon} isoform of protein kinase C (PKC{epsilon}) regulates the ethanol sensitivity of GABAA receptors, as mice lacking PKC{epsilon} show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKC{epsilon} mutant to selectively inhibit the catalytic activity of PKC{epsilon}. We used this mutant and PKC{epsilon}-/- mice to determine that PKC{epsilon} phosphorylates {gamma}2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at {alpha}1beta2{gamma}2 receptors, which is the most abundant GABAA receptor subtype in the brain. Our findings indicate that PKC{epsilon} phosphorylation of {gamma}2 regulates the response of GABAA receptors to specific allosteric modulators, and, in particular, PKC{epsilon} inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Aminobutyrate type A (GABAA)3 receptors mediate the majority of rapid inhibitory neurotransmission in the brain and are an important target for ethanol, the most widely abused drug (1). Ethanol modulation of GABAA receptors was first identified in synaptosomal preparations where intoxicating concentrations (10-30 mM) enhanced receptor function as measured by 36Cl uptake assays (2, 3). However, after 30 years of investigation, it is apparent that ethanol enhancement of synaptic GABAA receptors is variable and in some preparations cannot be detected even at anesthetic concentrations (1, 4).

GABAA receptors are pentameric protein complexes of subunits from eight classes ({alpha}1-6, beta1-3, {gamma}1-3, {delta}, {epsilon}, {pi}, {theta}, and {rho}1-3) (5). Most receptors are composed of two {alpha} subunits and two beta subunits that co-assemble with one {gamma}2 subunit, which anchors these receptors at synapses where they mediate phasic inhibition (6). A minority contain a {delta} subunit instead of {gamma}2; these receptors are extrasynaptic and mediate tonic inhibition in neurons (7). Because earlier ethanol studies that focused on GABA currents carried by {gamma}2-containing receptors produced variable results, recent attention has turned to ethanol effects at receptors containing {delta} subunits. Reports from three laboratories found these receptors enhanced by low (≤30 mM) intoxicating concentrations of ethanol (8-10). However, two recent studies were unable to demonstrate low dose ethanol sensitivity of {delta}-containing GABAA receptors (11, 12), indicating that, like synaptic GABAA receptors, ethanol modulation of extrasynaptic receptors is also variable.

The reasons for this high degree of variability are unknown. One possibility is that intracellular signaling pathways may regulate ethanol sensitivity of GABAA receptors, and the activity of such pathways was not controlled for in these studies. This hypothesis is consistent with our findings in mice lacking protein kinase C{epsilon} (PKC{epsilon}), which show an increased behavioral response to ethanol (13). Ethanol modulation of GABAA receptors is also increased in PKC{epsilon}-/- mice compared with wild-type mice (13, 14). These results suggest that activation of PKC{epsilon} suppresses potentiation of GABAA receptors by ethanol.

The mechanism by which PKC{epsilon} regulates the ethanol sensitivity of GABAA receptors is unknown. Here, we examined this mechanism using receptors composed of {alpha}1, beta2, and {gamma}2 subunits, which is the most abundant receptor subtype in the brain (15). Because there are no selective inhibitors of PKC{epsilon} catalytic activity available, we used a chemical-genetics approach to generate an ATP analog-sensitive mutant, as-PKC{epsilon}, that could be potently and selectively inhibited by a cell-permeant, small molecule inhibitor. Our findings indicate that PKC{epsilon} regulates the sensitivity of {alpha}1beta2{gamma}2 receptors to ethanol and benzodiazepines through phosphorylation of serine 327 in the large intracellular loop of {gamma}2.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Care and Behavior—PKC{epsilon}-/- mice were generated by homologous recombination in J1 embryonic stem cells as described (16). Male and female PKC{epsilon}+/- mice were maintained on inbred 129S4/SvJae and C57BL6/J backgrounds and were crossed to produce PKC{epsilon}+/- C57BL6/J x129S4/SvJae F1 hybrid mice. These mice were intercrossed to generate F2 hybrid PKC{epsilon}+/+ and PKC{epsilon}-/- littermates for experiments. Mice were genotyped by PCR of tail biopsies (5-10 mm fragment) obtained from non-anesthetized, 10-day-old pups. All animals were between 2 and 4 months old at the time of experimentation. All procedures were conducted in accordance with Ernest Gallo Clinic and Research Center Institutional Animal Care and Use Committee policies. Zolpidem-induced ataxia was measured as described previously (17). The "up and down" method (17) was used to measure initial sensitivity to zolpidem with a starting dose of 0.25 mg/kg and a log dose interval of 0.0276.

Immunoprecipitation—Frontal cortex was dissected from 8- to 10-week-old mice and homogenized with a Teflon-glass homogenizer in ice-cold buffer containing 25 mM Tris-HCl, pH 7.4, 1 mM EDTA, CompleteTM protease inhibitor mixture (Roche Applied Science), 2 mM benzamidine chloride, and 0.1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 200,000 x g for 30 min at 4 °C. The pellet was solubilized in ice-cold Tris-buffered saline (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.1% Triton X-100, and CompleteTM protease inhibitor mixture), and centrifuged again at 120,000 x g for 15 min at 4 °C. The supernatant (500 µg of protein) was incubated for 2 h at 27 °C with 1 µg of rabbit anti-GABAA-{alpha}1 polyclonal antibody 993-2, generated against the C-terminal decapeptide sequence common to rat, mouse, and human GABAA-{alpha}1 (PQLKAPTPHQ), or with purified rabbit IgG (Sigma-Aldrich). For the reverse immunoprecipitation, lysates were incubated with 1 µg of rabbit anti-PKC{epsilon} (18) or purified rabbit IgG. Protein A beads (100 µl, Roche Applied Science) were added, and the suspension was incubated overnight at 4 °C. Beads were washed four times with ice-cold PBS, and immune complexes were eluted with 100 µl of sample buffer. After being heated for 7 min at 90 °C, samples were centrifuged at 6,000 x g for 30 s, and proteins were separated in 4-12% polyacrylamide gels (Invitrogen). Proteins were analyzed using Western blots with goat anti-PKC{epsilon} (Santa Cruz Biotechnology, Santa Cruz, CA, 1:500 dilution) or goat anti-{alpha}1 subunit antibody (Santa Cruz Biotechnology, 1:500 dilution) followed by peroxidase-conjugated rabbit anti-goat IgG (Roche Applied Science, 1:1000 dilution) and enhanced chemiluminescence (Pierce).

Primary Neuronal Culture—Primary cultures of cortical and hippocampal neurons were prepared from 1- to 3-day-old mice as described (19). Briefly, cells were dissociated from dissected cerebral cortex or hippocampus by treatment with papain (20 units/ml) for 45 min at 37 °C, followed by trituration through a 1-ml plastic pipette. For immunostaining, hippocampal neurons were plated at a density of 105 cells on 200-mm2 chamber slides coated with poly-L-lysine in Neurobasal A media containing B27 and Glutamax (Invitrogen) and maintained at 37 °C in a humidified atmosphere of 6% CO2 and 94% air. For studies of cell surface {gamma}2 subunits, cortical neurons were plated on poly-L-lysine-coated 6-well plates at 1.6 x 106 cells/well. For electrophysiology experiments, hippocampal neurons were plated on poly-L-ornithine-coated 35-mm Petri dishes. Half of the medium was changed the next day and changed every 7 days for 3 weeks.

Immunofluorescence Staining of Neurons—Mature (14-21 days in vitro) neurons were immunostained as described (20). To detect GABAA receptors on the cell surface, living cultures were incubated with rabbit anti-GABAA receptor {alpha}1 N-terminal peptide (1:100, Chemicon, Temecula, CA) diluted in Ringer's solution containing 0.5 µM tetrodotoxin for 90 min at 27 °C. After three 10-min washes in Ringer's solution, the cells were fixed with methanol for 10 min at -20 °C. Fixed cells were washed with PBS and incubated for 90 min at 27 °C with goat anti-PKC{epsilon} antibody (1:100, Santa Cruz Biotechnology) diluted in PBS containing 10% normal donkey serum. After three washes with PBS, the cells were then incubated with donkey fluorescein isothiocyanate-conjugated anti-goat and Cy3-conjugated anti-rabbit antibodies (1:200, Jackson ImmunoResearch, West Grove, PA) and covered with coverslips in mounting media containing 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Images (1.5-µm optical sections) were acquired using a Zeiss LSM 510 META laser confocal microscope with a Plan-Apochromat 63x/numerical aperture 1.40 oil immersion objective.

Generation of as-PKC{epsilon}—A pCDNA3 plasmid containing N-terminal FLAG epitope-tagged, human PKC{epsilon} cDNA was provided by Dr. Alex Toker (Harvard University, Boston, MA) and used as a template to replace Met-486 with Ala by site-directed mutagenesis (QuikChange mutagenesis kit, Stratagene, La Jolla, CA) using the following primers: 5'-GGACCGCCTCTTTTTCGTCGCCGAATATGTAAATGGTGGAGACC-3' as the forward primer and 5'-GGTCTCCACCATTTACATATTCGGCGACGAAAAAGAGGCGGTCC-3' as the reverse primer. The PCR product was treated with DpnI for 1 h at 37 °C and then amplified in Escherichia coli XL 1-Blue. The presence of the M486A mutation was confirmed by DNA sequencing.

Plasmid DNA (10 µg) was transfected into COS-7 cells using the SuperFect Transfection Reagent (Qiagen). After culture for 72 h, cells were rinsed twice with 2 ml of ice-cold PBS and incubated for 20 min at 4 °C in lysis buffer containing 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4), 1% Triton X-100, and Complete Protease Inhibitor Mixture (Roche Applied Science). Lysates from 50 10-cm plates were pooled and centrifuged at 12,000 x g for 10 min at 4 °C. The supernatant was incubated for 3 h at 4 °C with 1 ml of anti-FLAG M2-conjugated agarose (Sigma), previously equilibrated with lysis buffer. Agarose beads were transferred to a column and washed sequentially with 15 ml of lysis buffer, 15 ml of buffer B (150 mM NaCl, 50 mM Tris-HCl, pH 7.4) and 5 ml of PKC storage buffer (0.1 mM EGTA, 20 mM HEPES, pH 7.4, 25% glycerol, and 0.03% Triton X-100). To elute FLAG-tagged proteins, the beads were incubated with 1.75 ml of 0.15 mg/ml 3x FLAG peptide solution (Sigma-Aldrich) in PKC storage buffer for 30 min at 4 °C and then centrifuged for 10 s at 10,000 x g. Aliquots of the supernatant were stored at -80 °C. The concentration of PKC{epsilon} in the eluate was measured in triplicate by enzyme-linked immunosorbent assay using recombinant PKC{epsilon} (Invitrogen) as a standard, rabbit anti-PKC{epsilon} SN134 (18) diluted 1:1,000 as the primary antibody, and horseradish peroxidase-conjugated anti-rabbit IgG as the secondary antibody (1:3,500 dilution, Chemicon). Immunoreactivity was detected using 3,3',5,5'-tetramethylbenzidine as the substrate with absorbance measured at 450 nm.

Characterization of as-PKC{epsilon}—Enzyme activity was measured by fluorescence polarization using the Protein Kinase C Assay Kit from Invitrogen. PKC{epsilon} (0.5 ng) or PKC{epsilon}-M486A (3 ng) was added to kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 0.02% Nonidet P-40, 0.03% Triton X-100, 0.1 µg phosphatidylserine, and 0.05 mM sodium vanadate) with 250 nM substrate peptide (RFARKGSLRQKNV) and 12.5 nM phorbol 12-myristate 13-acetate in a final volume of 40 µl. The mixture was incubated at 22 °C for 5 min in the dark. The kinase reaction was initiated by addition of ATP (1-100 µM) in 10 µl and was stopped after 0-20 min by addition of 1 µl of 0.5 mM staurosporine (Calbiochem) in Me2SO. A 50-µl aliquot of the reaction mixture was transferred to a well of a dark-sided 96-well plate and mixed with 50 µl of a solution containing 2x fluorescein-labeled phosphopeptide and 2x anti-phosphoserine antibody, according to the manufacturer's instructions. After incubation for 30 min at 22 °C in the dark, plane-polarized fluorescence was measured using an Analyst HT (Molecular Devices, Sunnyvale, CA) with excitation at 485 nm and emission at 530 nm. Polarization (P) was calculated by the equation, P = (Ipara - Iperp)/(Ipara + Iperp), where Ipara is the parallel emission fluorescence intensity and Iperp is perpendicular emission fluorescence intensity. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) were calculated from the equation 1/VO = Km/Vmax[S] + 1/Vmax, where VO is the initial velocity of the reaction and [S] is the initial concentration of substrate. The catalytic constant (kcat) was calculated from the equation kcat = Vmax/[E]O, where [E]O is the concentration of enzyme added to the reaction.

Stable Transfection of HEK293 Cells with as-PKC{epsilon}—HEK293 cells were obtained from the American Type Culture Collection (Manassas, VA). HEK293 cells that stably express rat {alpha}1beta2{gamma}2S GABAA receptors (21) were provided by G. H. Dillon, (University of N. Texas Health Science Center, Fort Worth, TX). Human PKC{epsilon} M486A was amplified by PCR and then subcloned without the FLAG-epitope tag into pIRESpuro2 (Clontech, Palo Alto, CA). Cells were plated on poly-L-ornithine-coated 6-well plates at a density of 105 cells per well in 2 ml of growth medium (minimal essential medium with 10% fetal bovine serum, 0.2 mM l-{alpha}-glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin). The cells were incubated overnight at 37 °C in a humidified atmosphere of 5% CO2:95% air before transfection. Transfection complexes were prepared by mixing 2 µg of plasmid DNA with 6 µl of TransIT-293 reagent (Mirus, Madison, WI) in 100 µl of serum-free medium. Complexes were added dropwise to each well, and the cells were incubated for 24 h at 37 °C in 5% CO2:95% air. Clones were selected 48 h later in 5 µg/ml puromycin. Medium was replaced daily, and the concentration of puromycin was reduced to 3 µg/ml after 1 week. Expression of PKC{epsilon} or PKC{epsilon} M486A in surviving cells was confirmed by Western blot analysis.

Generation of HEK293 Cells Expressing Mutant GABAA Receptor Subunits—Plasmids containing inserts encoding human {alpha}1 and {gamma}2L in pCDM8 were provided by Dr. P. B. Wingrove (Merck Sharp & Dohme, United Kingdom). A pCIS2 plasmid encoding rat beta2 was provided by Dr. N. Harrison (Cornell University, New York, NY). Phosphorylation site mutants of beta2 (S410A) and {gamma}2L (S327A and S343A) were generated by site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) and the following primers: 5'-CGCCTGAGGAGACGTGCCGCCCAACTGAAAATCACC-3' as the forward primer and 5'-GGTGATTTTCAGTTGGGCGGCACGTCTCCTCAGGCG-3' as the reverse primer for beta2S410A; 5'-GTCAGCAACCGGAAACCAGCCAAGGACAAAGATAAAAAGAAGAAAAACCC-3' as the forward primer and 5'-GGGTTTTTCTTCTTTTTATCTTTGTCCTTGGCTGGTTTCCGGTTGCTGAC-3' as the reverse primer for {gamma}2LS327A; 5'-CCTCTTCTTCGGATGTTTGCCTTCAAGGCCCCTACC-3' as the forward primer and 5'-GGTAGGGGCCTTGAAGGCAAACATCCGAAGAAGAGG-3' as the reverse primer for {gamma}2LS343A. Plasmids were transiently transfected using calcium phosphate into HEK293 cells that stably expressed PKC{epsilon} M486A using the TransIT-293 reagent. Cells that expressed functional GABAA receptors were identified by measuring currents evoked by GABA and flunitrazepam using whole cell patch clamp electrophysiology.

Electrophysiology of Transfected HEK293 Cells—GABAA receptor currents were measured in HEK293 cells stably transfected with as-PKC{epsilon} and native or mutant GABAA receptors using a conventional whole cell patch clamp technique. The recording electrodes were pulled from borosilicate glass capillary tubing (World Precision Instruments, Sarasota, FL) using a micropipette puller (P-97, Sutter Instruments, Novato, CA), to achieve a resistance of 5-8 M{Omega} when filled with pipette solution containing (in mM): 145 N-methyl-D-glucamine-Cl, 2 MgCl2, 0.1 CaCl2, 5 EGTA, 10 HEPES, and 2 Mg2+-ATP (pH 7.3, adjusted with HCl). The external solution used to perfuse the cells continuously during the experiment contained (in mM): 145 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 d-glucose (pH 7.4, adjusted with NaOH).

To test the effect of inhibiting PKC{epsilon} on GABAA receptor function, cells were incubated with external solution containing 1 µM 1-naphthyl-PP1 (1Na) or vehicle (0.1% Me2SO) for 30 min prior to stimulation with GABA. Cells were then voltage-clamped at -75 mV, and the GABA dose-response relationship was determined in the presence or absence of 1 µM 1Na. The response to allosteric modulators was then assessed by co-application of the modulator with an EC20 concentration of GABA. Flunitrazepam was applied 5 s prior to co-application with GABA, and ethanol was co-applied with GABA by local perfusion using a Perfusion Fast-Step SF-77B system (Warner Instruments, Inc., Hamden, CT) driven by pClamp 9 software (Axon Instruments, Union City, CA). Unless otherwise stated, there was a 1-min washout period between each drug application. Whole cell currents were recorded using an Axopatch 200B patch amplifier (Axon CNS-Molecular Devices, Union City, CA), filtered at 2 kHz, and digitized at 5 kHz with a Digidata 1322A interface and pClamp 9 software (Axon CNS). The serial resistance was monitored continuously during each experiment, and data from cells that showed a >30% change in resistance were discarded. All recordings were obtained at 27 °C.

Electrophysiology of Primary Hippocampal Neurons—Cultures of hippocampal neurons were prepared as described above and used at 3-10 days in vitro. The pipette solution contained (in mM): 145 N-methyl-D-glucamine-Cl, 2 MgCl2, 10 HEPES, 2 Mg2+-ATP, and 2 Na+-ATP (pH 7.3, adjusted with HCl, 280 mosM). The external solution contained (in mM): 145 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 d-glucose (pH 7.4, adjusted with NaOH). 1 µM tetrodotoxin and 10 µM 6,7-dinitroquinoxaline-2,3-dione were freshly added to the external solution each day. All recordings were made at 32 °C.

Phosphorylation of GST-{gamma}2 Intracellular Loop Fusion Proteins—A plasmid (provided by R. Olsen, UCLA) containing the cDNA sequence encoding the mouse {gamma}2S large intracellular loop (amino acids 318-404) in pGEX-2T (GE Healthcare, Piscataway, NJ) was used to generate an alanine mutation at Ser-327 with a QuikChange site-directed mutagenesis kit (Stratagene) and the primers: 5'-GTCAGCAACCGGAAGCCAGCCAAGGATAAAGACAAAAAGAAGAAAAACCC-3' as the forward primer and 5'-GGGTTTTTCTTCTTTTTGTCTTTATCCTTGGCTGGCTTCCGGTTGCTGAC-3' as the reverse primer. BamHI and EcoRI fragments encoding the native and S327A mutant loops were subcloned into pGEX-6P-2 (GE Healthcare).

Fusion proteins were produced in E. coli BL21(DE3)pLysS cells (Invitrogen) and purified by affinity chromatography using glutathione-Sepharose 4B (GE Healthcare). Phosphorylation was performed using 0.1 µM human recombinant PKC{epsilon} (Invitrogen) in 10 µl of kinase buffer containing 20 mM HEPES, pH 7.4, 0.1 mM EGTA, 0.03% Triton X-100, 10 mM MgCl2, 0.48 µg/µl L-{alpha}-phosphatidylserine (Avanti%20Polar%20Lipids">Avanti Polar Lipids, Alabaster, AL), 1 µM phorbol 12-myristate 13-acetate, and 0.5 mM ATP. PKC{epsilon} was preincubated with kinase buffer for 3 h at 27 °C to stimulate autophosphorylation and thereby maximize the activity of PKC{epsilon}. Then 1.25 pmol of purified GST-{gamma}2S loop or GST-{gamma}2SS327A loop and 10 µCi of [{gamma}-32P]ATP were added to the reaction mixture, which was incubated at 37 °C for the indicated times. At each time point, 10 µl of reaction mixture were removed and mixed with 2.5 µl of 5x SDS sample buffer to stop the reaction. Proteins were separated by SDS-PAGE on 4-12% gradient gels (Invitrogen), and the amount of radioactivity incorporated was quantified by phosphorimaging (Typhoon 9410, GE Healthcare). Specific radioactivity per mole of [{gamma}-32P]ATP was determined by counting diluted aliquots of the stock [{gamma}-32P]ATP solution to obtain a conversion value for calculating the molar ratio of 32P incorporated into each fusion protein substrate.

Detection of {gamma}2S327 Phosphorylation by Western Analysis—GST-{gamma}2S loop proteins were phosphorylated in vitro in the presence of non-radioactive ATP. Samples of frontal cortex and hippocampus were homogenized in lysis buffer containing 25 mM Tris-HCl, pH 7.4, 1 mM EDTA, and CompleteTM protease inhibitor mixture (Roche Applied Science). Samples were adjusted to a concentration of 2 mg/ml and some were incubated with 8,000 units of lambda protein phosphatase (Upstate%20Biotechnology">Upstate Biotechnology, Lake Placid, NY) at 30 °C for 30 min. Control samples not treated with phosphatase were incubated with Phosphatase Inhibitor Mixture 1 (Sigma-Aldrich).

Brain lysates, cell lysates, or GST-{gamma}2S loop proteins were subjected to SDS-PAGE using 4-12% gradient gels (Invitrogen). The proteins were detected by Western blot analysis using affinity-purified, polyclonal rabbit anti-{gamma}2-S(P)327 antibody (1:1000 dilution) generated against the phosphopeptide 317CLHYFVSNRKPS(P)KDKDK332 (PhosphoSolutions, Aurora, CO), followed by horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:1000 dilution, Chemicon) for 1 h at 27 °C. Immunoreactivity was detected by enhanced chemiluminescence and quantified by scanning densitometry.

Statistical Analysis—Data shown are mean ± S.E. values. Data were first tested for normality using the Kolmogorov-Smirnov test. Because all data were normally distributed, mean values were compared by either two-tailed, unpaired t-tests or ANOVA with indicated post-hoc tests. Differences between pairs of means were considered significant where p < 0.05. Except where indicated, dose-response relationships were analyzed by non-linear regression analysis using Prism 4 (GraphPad Software, Inc., San Diego, CA). Potentiation of the GABA response by allosteric modulators was calculated as the ratio of the current elicited by modulator plus GABA divided by the current elicited by GABA alone.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Increased Response to Zolpidem in PKC{epsilon}-/- Mice—To determine whether PKC{epsilon} regulates GABAA receptors that contain {alpha}1 and {gamma}2 subunits, we first examined behavioral responses in mice that lack PKC{epsilon}. Because behavioral responses to benzodiazepines are increased in these mice (13), we tested their response to zolpidem, an imidazopyridine that selectively acts at the benzodiazepine site on GABAA receptors that contain {alpha}1 and {gamma}2 subunits (22). We found that PKC{epsilon}-/- mice showed greater rotarod ataxia in response to 1 mg/kg zolpidem than wild-type littermates (Fig. 1A). Two-way repeated measures ANOVA showed main effects of genotype (F1,48 = 5.66, p = 0.044) and time (F6,48 = 11.19, p < 0.001) with an interaction between these factors (F6,48 = 2,87, p = 0.018). The threshold dose of zolpidem for producing ataxia was also lower in PKC{epsilon}-/- mice compared with wild-type controls (Fig. 1B). These differences in behavior were not associated with altered distribution of {alpha}1 subunits in the brain (supplemental Fig. S1A), abundance of {alpha}1or {gamma}2 subunits in frontal cortex, cerebellum, striatum, or amygdala (supplemental Fig. S1B), or altered cell surface abundance of {gamma}2 subunits (supplemental Fig. S1C) in PKC{epsilon}-/- mice when compared with wild-type littermates. These findings suggest that the function, rather than the abundance, of zolpidem-sensitive GABAA receptors is increased in PKC{epsilon}-/- mice. Consistent with this conclusion, PKC{epsilon} and {alpha}1 subunits could be co-immunoprecipitated from frontal cortex of wild-type mice (Fig. 1, C and D) and were co-localized at the cell membrane in cultured hippocampal neurons from wild-type animals (Fig. 1E), indicating that PKC{epsilon} and {alpha}1-containing GABAA receptors physically interact in a complex.

Generation of an ATP Analog-sensitive Mutant of PKC{epsilon}—PKC{epsilon} can transduce signals by phosphorylation of substrates or by protein-protein interaction. The latter has been demonstrated for PKC{epsilon}-stimulated neurite outgrowth in human neuroblastoma cells (23). The {epsilon}V1-2 peptide (EAVSLKPT), which was designed as an inhibitor of PKC{epsilon} translocation (24), can be used to selectively inhibit PKC{epsilon}. However, because {epsilon}V1-2 does not bind the catalytic domain or inhibit the catalytic activity of PKC{epsilon}, this peptide cannot be used to distinguish between PKC{epsilon}-mediated effects due to protein-protein interactions or phosphorylation. Furthermore, no small molecules are known that specifically inhibit the catalytic activity of PKC{epsilon} over other PKC isozymes, due to the highly conserved ATP binding sites of protein kinases. Therefore, to examine whether PKC{epsilon}-mediated phosphorylation is required for modulation of GABAA receptors, we used a chemical-genetics approach to develop a selective small molecule inhibitor. We generated a single amino acid substitution at methionine 486 in the ATP binding pocket of PKC{epsilon}, which we predicted would permit binding of ATP analogs with bulky side groups attached to the purine base. Such a strategy has been used to successfully generate ATP analog-sensitive kinase mutants, which can also be selectively inhibited by analogs of the kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) at concentrations that do not affect native kinases (25).


Figure 1
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  		FIGURE 1.
PKC{epsilon}-/- mice show increased sensitivity to zolpidem, and PKC{epsilon} interacts with {alpha}1 subunits of GABAA receptors. A, compared with wild-type mice (+/+, n = 5), PKC{epsilon}-/- mice (-/-, n = 5) showed a shorter latency to fall from a constant velocity rotarod 15, 30, and 45 min after injection of 1 mg/kg zolpidem (*, p < 0.05 compared with wild-type mice at the same time by post-hoc Newman-Keuls test). B, the zolpidem ED50 for inducing rotarod ataxia was decreased in PKC{epsilon}-/- mice (-/-) compared with wild-type (+/+) littermates. Data are mean values ± 95% confidence intervals from six mice per genotype. C, {alpha}1 subunit immunoreactivity was detected by Western analysis of whole brain lysates from PKC{epsilon}+/+ mice immunoprecipitated with anti-PKC{epsilon} antibody. No {alpha}1 subunit immunoreactivity was detected in immunoprecipitates from PKC{epsilon}-/- brain lysates or when rabbit IgG was used instead of anti-PKC{epsilon} antibody. D, PKC{epsilon} immunoreactivity was detected by Western analysis of whole brain lysates from PKC{epsilon}+/+ mice immunoprecipitated with anti-{alpha}1 antibody. No PKC{epsilon} immunoreactivity was seen when lysates were immunoprecipitated with rabbit IgG. E, PKC{epsilon} (left, green) and{alpha}1 subunits (middle, red) co-localize (right, merge) at the cell surface of cultured hippocampal neurons from wild-type mice. Boxes refer to enlarged regions shown in the bottom panels. Scale bar = 20 µm.

 
We generated human PKC{epsilon} M486A (as-PKC{epsilon}) containing a FLAG-epitope tag at the amino terminus using site-directed mutagenesis. Kinetic analysis using different concentrations of ATP revealed the Km for as-PKC{epsilon} (241.3 ± 39.1 µM) to be 12-fold greater than the Km for native PKC{epsilon} (20.4 ± 4.0 µM). However, both enzymes had similar turnover (kcat) values (170,600 ± 16,500 min-1 for as-PKC{epsilon} and 156,700 ± 3,700 min-1 for PKC{epsilon}). The similarity in kcat values indicates that as-PKC{epsilon} can utilize ATP as efficiently as native PKC{epsilon} at the high ATP concentrations (2-10 mM) found in cells.

We next examined whether commercially available kinase inhibitors inhibit as-PKC{epsilon} and whether analogs of PP1 selectively inhibit as-PKC{epsilon}. The IC50 for the nonspecific kinase inhibitor staurosporine was 14-fold higher and for the general PKC inhibitor bisindolylmaleimide-I was 2-fold higher for as-PKC{epsilon} compared with native PKC{epsilon} (Table 1). In contrast, 1Na and 1-naphthylmethyl-PP1 inhibited as-PKC{epsilon} but not native PKC{epsilon}, with 1Na being 65-fold more potent than 1-naphthylmethyl-PP1 against as-PKC{epsilon}. 1Na did not inhibit the activity of a commercially available mixture of native PKC isozymes. In addition, 1Na did not inhibit representative members of the conventional (PKC{gamma}), novel (PKC{delta}), or atypical (PKC{iota}) PKC subfamilies (Table 1).


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  		TABLE 1
Selectivity of kinase inhibitors

Kinase activity was measured by fluorescence polarization using 0.012-2 x 105 nM inhibitor, 2.5 µM ATP, and incubation for 90 min at 27 °C. Data are from three triplicate experiments, and IC50 values (nM) were calculated by nonlinear regression analysis.

 
{gamma}2S327 Is Required for PKC{epsilon} Regulation of GABAA Receptors—Next, we examined the role of subunit phosphorylation in the regulation of receptor function by PKC{epsilon}. Prior in vitro studies have suggested that there is one phosphorylation site for serinethreonine kinases on beta2 (serine 410) and two on the long splice variant of {gamma}2, {gamma}2L (serine 327 and 343), with only one of these (serine 327) present in the short splice variant {gamma}2S (26). To investigate whether any of these sites are required for GABAA receptor modulation by PKC{epsilon}, we transiently expressed {alpha}1, beta2, and {gamma}2L receptor subunits carrying alanine substitutions at these residues into HEK293 cells that stably express as-PKC{epsilon} at a level that is 5-fold higher than endogenous PKC{epsilon} (Fig. 2A). In non-transfected HEK293 cells, levels of PKC{epsilon} were low (Fig. 2A), allowing as-PKC{epsilon} to dominate over endogenous PKC{epsilon} in these stably transfected cells.


Figure 2
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  		FIGURE 2.
1Na increases flunitrazepam potency in HEK293 cells expressing as-PKC{epsilon} and native {alpha}1beta2{gamma}2L GABAA receptors but not in cells expressing receptors containing {gamma}2LS327A. A, expression of PKC{epsilon}-like immunoreactivity in vector-transfected (V) and as-PKC{epsilon}-transfected HEK293 cells. B, current tracings from a representative experiment showing similar responses to GABA in control and 1Na-treated cells transiently transfected with native {alpha}1beta2{gamma}2L subunits. C, combined results from five cells for each condition showing that the response to GABA was not altered by 1Na in these cells. D, current tracings showing the response to 3 µM GABA and increasing concentrations of flunitrazepam in cells expressing native or mutant receptors. 1Na (1 µM) increased the response to flunitrazepam, and this effect was inhibited by bicuculline (BMI) and was completely reversed after a final 1-min washout period. E, a representative experiment showing that 1Na shifts the dose-response curve for flunitrazepam enhancement of GABA currents to the left in cells expressing native receptors. F, combined results showing that 1Na increases the potency of flunitrazepam (n = 9) when compared with cells treated with control vehicle (n = 8). G, 1Na did not affect the flunitrazepam response in receptors that contained the {gamma}2LS327A subunit (n = 8 for each condition). H, comparison of flunitrazepam dose-response curves in native {gamma}2L- and {gamma}2LS327A-containing receptors in the absence of 1Na revealed that flunitrazepam potency was greater in receptors containing {gamma}2LS327A subunits (n = 8 for each condition).

 
As expected, 1Na did not alter the response to GABA alone in cells expressing native receptors or any of the mutant receptor combinations (Fig. 2, B and C, and Table 2). This result is consistent with our prior chloride flux experiments, which showed that PKC{epsilon} does not affect the response to the direct GABAA receptor agonist muscimol (13). We next examined the response to a benzodiazepine, because PKC{epsilon}-/- mice showed an increased behavioral response to zolpidem (Fig. 1, A and B). For receptors containing native {alpha}1, beta2, and {gamma}2L subunits, flunitrazepam increased the GABA-stimulated current, which was completely blocked by 30 µM bicuculline (Fig. 2D). Treatment with 1 µM 1Na increased the potency of flunitrazepam 4.9-fold, decreasing its EC50 from 98 nM to 17 nM, without affecting efficacy (Fig. 2, E and F, and Table 2). Interestingly, all subunit combinations that included the {gamma}2LS327A mutation showed no response to 1Na, and this mutation alone was sufficient to completely block the effect of 1Na (Fig. 2G and Table 2). In addition, without 1Na treatment, flunitrazepam potency was greater in cells expressing {gamma}2LS327A when compared with cells expressing native {gamma}2L (Fig. 2H and Table 2). By contrast, receptors containing the beta2S410A mutant responded to flunitrazepam and 1Na in a manner indistinguishable from native receptors (Table 2). Like currents in cells expressing native receptors, currents elicited with mutant receptors or in the presence of 1Na were completely blocked by 30 µM bicuculline (Fig. 2D). These results indicate that the {gamma}2LS327A mutation mimics the effect of PKC{epsilon} inhibition, suggesting that PKC{epsilon} decreases benzodiazepine potency through phosphorylation of {gamma}2S327.


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  		TABLE 2
Effect of as-PKC{epsilon} inhibition on GABA and flunitrazepam potency at mutant GABAA receptors

GABA currents were recorded from HEK293 cells stably transfected with as-PKC{epsilon} and transiently transfected with indicated receptor subunits. The log EC50 values were calculated for GABA (µM) and flunitrazepam (nM) by non-linear regression analysis.

 


Figure 3
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  		FIGURE 3.
1Na increases the effect of ethanol in native receptors, but not in receptors containing {gamma}2LS327A. A, representative current tracings in cells treated with 3 µM GABA and increasing concentrations of ethanol, showing that 1Na increased current amplitude (middle tracing) when compared with control cells (top tracing), and this effect was blocked by bicuculline (BMI) and reversed by a final 2-min washout period. B, representative tracings showing that ethanol potentiation of GABA elicited current in {alpha}1beta2{gamma}2L receptors was increased by 1Na. C, combined results from 1Na-treated (n = 6) and vehicle-treated cells (n = 8) showing that 1Na increases the ethanol response by shifting the ethanol dose-response curve upwards. D, 1Na did not alter ethanol sensitivity in receptors containing {gamma}2LS327A (n = 9 for control and n = 8 for 1Na-treated cells). E, in the absence of 1Na, potentiation by ethanol was greater in {alpha}1beta2{gamma}2LS327A receptors (n = 8) compared with native receptors (n = 9). *, p < 0.05 compared with control cells (C) or native receptors (E) by post-hoc Newman-Keuls tests.

 
We next examined whether {gamma}2LS327 phosphorylation regulates ethanol sensitivity of GABAA receptors. In cells containing native {gamma}2L or mutant {gamma}2LS327A subunits, ethanol enhanced the GABA-stimulated current, which was completely inhibited by 30 µM bicuculline (Fig. 3A). Treatment with 1Na increased the effect of ethanol (Fig. 3, A-C) in cells expressing native receptors. Two-way, repeated measures ANOVA showed a main effect of ethanol concentration (F4,48 = 38.87, p < 0.001) and 1Na treatment (F1,48 = 20.26, p < 0.001) with a significant interaction between these factors (F4,48 = 4.59, p < 0.003). By contrast, 1Na did not increase the response to ethanol in cells containing {gamma}2LS327A (Fig. 3D). Moreover, without 1Na treatment, the response to ethanol was greater in cells expressing {gamma}2LS327A when compared with cells expressing native {gamma}2L (Fig. 3E). Two-way repeated measures ANOVA showed main effects of ethanol concentration (F4,60 = 35.98, p < 0.001) and subunit type (F1,60 = 8.16, p < 0.012) with a significant interaction between these factors (F4,60 = 4.54, p < 0.003). Ethanol-enhanced GABA-stimulated currents were completely blocked by 30 µM bicuculline when 1 Na was present in both native receptors and receptors with {gamma}2LS327A (Fig. 3A). These results demonstrate that the {gamma}2S327A mutation increases the response of GABAA receptors to ethanol and occludes further enhancement by a PKC{epsilon} inhibitor, strongly suggesting that PKC{epsilon} modulates the response to ethanol through phosphorylation of {gamma}2S327.


Figure 4
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  		FIGURE 4.
PKC{epsilon} phosphorylates {gamma}2S327 in vitro. A, GST fusion proteins, containing the large intracellular loop of {gamma}2S with or without the {gamma}2S327A mutation, were subjected to phosphorylation for up to 4 h using recombinant human PKC{epsilon}. PKC{epsilon} preferentially phosphorylated the non-mutant GST-{gamma}2S loop. Shown are an autoradiogram (top panel) and silver-stained gel (bottom panel) from a representative experiment. GST alone was not a substrate for PKC{epsilon} (not shown). The graph shows results from four experiments. *, p < 0.05 by Bonferroni posttests. B, an affinity-purified rabbit antibody (anti-{gamma}2-S(P)327) detected PKC{epsilon}-phosphorylated (P), but not non-phosphorylated (N) GST-{gamma}2S loop fusion protein. The antibody was not immunoreactive against PKC{epsilon}-phosphorylated GST-{gamma}2SS327A loop fusion protein.

 
The {gamma}2S327A Mutation Reduces Rates of Inactivation and Desensitization in Cells Treated with Flunitrazepam—By fitting the current traces activated by 100 µM GABA to a one-component exponential function and comparing the time constants, we found that currents activated by GABA alone showed similar rates of activation (261 ± 32 versus 252 ± 47 ms), inactivation (292 ± 50 versus 338 ± 50 ms), and desensitization (7128 ± 927 versus 7026 ± 1107 ms) in receptors comprised of native {gamma}2L (n = 5) or {gamma}2LS327A (n = 6). In contrast, we found differences in channel kinetics when currents were stimulated by GABA plus flunitrazepam. For currents activated by EC20 GABA and 300 nM flunitrazepam, receptors containing {gamma}2L or {gamma}2LS327A produced currents with similar activation rates (776 ± 26, n = 8, versus 790 ± 38 ms, n = 9), but {gamma}2L containing receptors had a significantly faster inactivation rate (117 ± 19, n = 8, versus 211 ± 16 ms, n = 9; p < 0.05 by t test). Moreover, whereas 6 of 8 {gamma}2L-transfected cells showed desensitization, only 3 of 9 {gamma}2LS327A-transfected cells desensitized. For those cells that showed desensitization, the desensitization rate was faster (p < 0.05 by two-tailed, t test) in {gamma}2L-tranfected cells (1847 ± 122 ms) when compared with {gamma}2LS327A-transfected cells (2892 ± 589 ms). For currents activated by EC20 GABA and 100 mM ethanol, the activation rates (567 ± 164, n = 8 versus 459 ± 134 ms, n = 13) and deactivation rates (215 ± 111 versus 212 ± 97 ms) were similar for {gamma}2L- and {gamma}2LS327A-containing receptors. The majority of the cells tested did not show desensitization (7 out of 8 {gamma}2L cells and 11 out of 13 {gamma}2LS327A cells). These findings indicate that the {gamma}2S237A mutation does not alter channel kinetics in response to GABA alone or with ethanol but instead selectively reduces inactivation and desensitization rates when benzodiazepines are present.

PKC{epsilon} Phosphorylates {gamma}2S327 in Vitro—To investigate whether PKC{epsilon} phosphorylates {gamma}2S327, we generated two GST fusion proteins, one containing the large intracellular loop of {gamma}2S, which includes Ser-327, and another containing the same sequence but with the S327A mutation. PKC{epsilon} phosphorylated the native sequence to a maximal stoichiometry of 1.00 ± 0.06, whereas the fusion protein containing the {gamma}2S327A mutation was a comparatively poor substrate for PKC{epsilon} (Fig. 4A). Two-factor ANOVA showed main effects of substrate (F1,30 = 119.5, p < 0.0001) and time (F4,30 = 60.55, p < 0.0001) with an interaction between these factors (F4,30 = 13.03, p < 0.0001). These findings indicate that PKC{epsilon} phosphorylates {gamma}2S327 in vitro.

To examine whether PKC{epsilon} phosphorylates {gamma}2S327 in intact tissues, we used an affinity-purified antibody against a peptide derived from the large intracellular loop of {gamma}2 that contains phospho-{gamma}2S327. This anti-{gamma}2-S(P)327 antibody detected a GST-{gamma}2 loop fusion protein phosphorylated by PKC{epsilon} in vitro, but not the non-phosphorylated fusion protein (Fig. 4B).

PKC{epsilon} Regulates Allosteric Responses in HEK Cells Containing {gamma}2S and Regulates {gamma}2S Phosphorylation at Ser-327—To examine receptor phosphorylation in intact cells we transfected a HEK293 cell line (HEKGR) that stably expresses {alpha}1, beta2, and {gamma}2S subunits (21) with as-PKC{epsilon} and selected a subclone (HEKGR-as-PKC{epsilon}) that expresses as-PKC{epsilon} at a level 3.3-fold greater than endogenous PKC{epsilon}. Because some previous studies had suggested that the long splice variant of {gamma}2({gamma}2L) is required to confer ethanol sensitivity to recombinant GABAA receptors (27-30), we first examined whether ethanol and PKC{epsilon} could regulate responses in these cells. We then used our anti-{gamma}2-S(P)327 antibody to detect {gamma}2S327 phosphorylation in these cells before and after treatment with 1Na.

As expected, 1Na did not alter the response to GABA (Fig. 5A) in HEKGR-as-PKC{epsilon} cells. However, 1Na increased flunitrazepam potency (Fig. 5B), decreasing the log EC50 value from 2.18 ± 0.09 nM in control cells (n = 10) to 1.4 ± 0.13 nM in 1Na-treated cells (n = 10). Ethanol modestly enhanced GABA-stimulated currents in these cells (Fig. 5C) (F5,40 = 6.67, p < 0.0001 by ANOVA). The effect of ethanol was further increased by treatment with 1Na (Fig. 5C), similar to what we observed in HEK293 cells that express {alpha}1beta2{gamma}2L receptors (Fig. 3). Two-way repeated measures ANOVA showed main effects of 1Na treatment (F1,64 = 26.08, p = 0.0001) and ethanol concentration (F4,64 = 40.48, p < 0.0001), with an interaction between these factors (F4,64 = 4.85, p = 0.0018). The effect of 1Na required the presence of as-PKC{epsilon}, because in the parent HEKGR cell line, 1Na had no effect on potentiation of GABAA current by ethanol or by the benzodiazepine diazepam (Fig. 5D). These results indicate that receptors containing {gamma}2L or {gamma}2S subunits are both responsive to ethanol when expressed in HEK293 cells and that receptors containing either {gamma}2L or {gamma}2S show PKC{epsilon} regulation of sensitivity to flunitrazepam and ethanol.


Figure 5
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  		FIGURE 5.
PKC{epsilon} regulates allosteric sensitivity to flunitrazepam and ethanol and phosphorylates {gamma}2S327 in HEK293 cells that stably express {alpha}1beta2{gamma}2S GABAA receptors and as-PKC{epsilon}. A, GABA elicited similar currents in control (n = 10) and 1Na-treated cells (n = 8). B, 1Na produced a leftward shift in the dose-response curve for flunitrazepam assayed in the presence of an EC20 concentration of GABA (p = 0.005 by non-linear regression analysis). C, ethanol increased GABA-elicited currents in cells incubated without 1Na ((n = 10; *, p < 0.05 compared with GABA alone by Dunnett's test). Treatment with 1 µM 1Na (n = 10) increased ethanol potentiation of GABA-stimulated current when compared with cells treated without 1Na (n = 10). {dagger}, p < 0.05 compared with control by Newman-Keuls test. D, 1Na had no effect on potentiation of EC20 GABA responses by 30 nM diazepam or 30 mM ethanol in cells that had not been transfected with as-PKC{epsilon}. E and F, {gamma}2-S(P)327 immunoreactivity was reduced after treatment for 1 h with 1 µM 1Na or 500 nM tat-{epsilon}V1-2 peptide. A representative experiment is shown in E. Results from four experiments are shown in F.*, p < 0.05 compared with control by Tukey test.

 
Using the anti-{gamma}2-S(P)327 antibody to measure {gamma}2S327 phosphorylation, we found that anti-{gamma}2-S(P)327 immunoreactivity was decreased (F2,8 = 28.1; p = 0.0002) in HEKGR-as-PKC{epsilon} cells after treatment with 1Na or with the PKC{epsilon} translocation inhibitor tat-{epsilon}V1-2 (Fig. 5, E and F). These results demonstrate that in HEK293 cells, PKC{epsilon} regulates the phosphorylation of {gamma}2S327.

PKC{epsilon} Phosphorylates {gamma}2S327 in Vivo—We next used the anti-{gamma}2-S(P)327 antibody to investigate whether {gamma}2S327 phosphorylation is decreased in PKC{epsilon}-/- mice. Anti-{gamma}2-S(P)327 immunoreactivity was decreased in the hippocampus and frontal cortex of PKC{epsilon}-/- mice compared with wild-type littermates (Fig. 6A). The presence of residual immunoreactivity in PKC{epsilon}-/- brain tissue suggested that {gamma}2S327 might also be phosphorylated by another kinase besides PKC{epsilon}. To investigate this possibility, we treated samples from frontal cortex with lambda protein phosphatase for 30 min before performing Western analysis. Phosphatase treatment of wild-type samples decreased anti-{gamma}2-S(P)327 immunoreactivity to levels found in PKC{epsilon}-/- samples, whereas treatment of samples from PKC{epsilon}-/- mice did not decrease anti-{gamma}2-S(P)327 immunoreactivity further (Fig. 6B). Analysis of these data by two-factor ANOVA showed main effects of genotype (F1,18 = 13.20, p = 0.0019) and treatment (F1,18 = 11.40, p = 0.0034) with an interaction between genotype and treatment (F1,18 = 5.51, p = 0.030). These findings suggest that the residual immunoreactivity present in samples from PKC{epsilon}-/- mice is not due to {gamma}2 phosphorylated at Ser-327 but, rather, is due to cross-reactivity of the antibody with non-phosphorylated {gamma}2 subunits. Consistent with this conclusion, we found that overnight incubation of the anti-{gamma}2-S(P)327 antibody with a dephosphorylated version of the immunizing peptide prevented its ability to detect a protein band at 48 kDa (data not shown). Taken together, these results indicate that PKC{epsilon} is the major kinase that phosphorylates {gamma}2S327 in vivo.

Ethanol Potentiation of GABAA Currents in Hippocampal Neurons—Using cultured hippocampal neurons from wild-type and PKC{epsilon}-/- mice we examined whether absence of PKC{epsilon} would increase sensitivity to ethanol. In initial experiments we were unable to demonstrate any response to ethanol (Fig. 7A). However, a portion of whole cell current in cultured hippocampal neurons is carried by receptors comprised of {alpha}beta subunits, which can be inhibited by 5 µM Zn2+ (31) and are insensitive to ethanol (28). Therefore, to eliminate currents carried by receptors containing only {alpha} and beta subunits, we measured ethanol responses in the presence of 5 µM ZnCl2. In cells from wild-type mice (n = 12), the residual GABA current was increased 75 ± 11% by 30 nM diazepam indicating that a substantial portion of the current was carried by receptors that contain {gamma}2 subunits. In neurons from PKC{epsilon} -/- mice (n = 14) diazepam produced a greater effect (p = 0.0092) enhancing the GABA-stimulated current by 127 ± 14%. In wild-type neurons, treatment with ethanol (Fig. 7, B and D) produced a small but significant increase in the Zn2+-resistant current (F5,30 = 6.94, p < 0.0001 by repeated measures ANOVA). In contrast, neurons from PKC{epsilon}-/- mice showed a much greater response to ethanol (Fig. 7, C and D). Analysis of these data by two-way repeated measures ANOVA showed main effects of genotype (F1,53 = 6.87, p = 0.02) and treatment (F4,53 = 12.01, p < 0.001) with an interaction between these factors (F4,53 = 2.72, p = 0.039). These findings indicate that Zn2+-resistant GABA currents are enhanced to a greater extent by diazepam and ethanol in PKC{epsilon}-/- hippocampal neurons when compared with wild-type neurons.


Figure 6
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  		FIGURE 6.
PKC{epsilon} phosphorylates {gamma}2S327 in vivo. A, anti-{gamma}2-S(P)327 antibody detected an immunoreactive band at 46 kDa in frontal cortex (Ctx; n = 9 for both genotypes) and hippocampus (Hip; n = 3 for both genotypes). Immunoreactivity was reduced in tissue from PKC{epsilon}-/- mice (-/-) compared with wild-type (+/+) littermates. *, p < 0.05 by two-tailed t-tests. B, lambda protein phosphatase ({lambda}Ptase) decreased anti-{gamma}2-S(P)327 immunoreactivity in cortical samples from wild-type but not from PKC{epsilon}-/- mice. Shown is a representative Western blot demonstrating a {gamma}2-S(P)327 immunoreactive band at 46 kDa. Without phosphatase treatment, immunoreactivity was greater in samples from wild-type mice (+/+; n = 10) compared with samples from PKC{epsilon}-/-mice (-/-; n = 9). Following treatment with lambda phosphatase, immunoreactivity in wild-type mice (n = 4) was decreased and was similar to immunoreactivity in PKC{epsilon}-/- (n = 3) samples. *, p < 0.05 compared with phosphatase-treated wild-type (+/+) samples; {dagger}, p < 0.05 compared with wild-type control samples but p > 0.05 compared with phosphatase-treated PKC{epsilon}-/- (-/-) samples by Tukey test.

 
Ethanol-induced Phosphorylation of {gamma}2S327 in Vivo Requires PKC{epsilon}—In mice, exposure to 4.0 g/kg ethanol in vivo produces GABAA receptor resistance to ethanol when measured 1 h later in cerebellar microsacs (3, 17). This acute tolerance of GABAA receptors to ethanol occurs in wild-type mice but not in PKC{epsilon}-/- mice (17). Because increased phosphorylation at {gamma}2S327 is associated with diminished receptor sensitivity to ethanol, we investigated whether in vivo exposure to ethanol increases phosphorylation of {gamma}2S327. Mice were injected with saline or with 4 g/kg ethanol intraperitoneally and then the cerebellum was removed and analyzed by Western blot analysis 1 h later. After saline injection, {gamma}2S(P)327 immunoreactivity was increased in the cerebellum of wild-type (n = 6) relative to PKC{epsilon}-/- mice (n = 6). In wild-type mice immunoreactivity was increased after injection of 4g/kg ethanol (Fig. 8). In contrast, ethanol did not alter {gamma}2S(P)327 immunoreactivity in cerebellar tissue from PKC{epsilon}-/- mice. Analysis of these data by two-factor ANOVA showed main effects of genotype (F1,20 = 47.64, p < 0.0001) and treatment (F1,20 = 6.46, P 0.0194) with a significant interaction between these factors (F1,20 = 4.93, p = 0.0381).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It has been difficult to identify PKC isozyme-specific targets, because the catalytic domains of PKC isozymes are very similar and there are no isozyme-selective inhibitors of catalytic activity available. Here we used a chemical-genetics approach to generate an ATP analog-sensitive mutant of PKC{epsilon} and selectively inhibit the catalytic activity of an individual PKC. Using this mutant we identified a specific mechanism by which PKC{epsilon} regulates the function of {alpha}1beta2{gamma}2 GABAA receptors. We demonstrated that PKC{epsilon} regulates the allosteric effect of benzodiazepines and ethanol at these receptors through phosphorylation rather than by a protein-protein interaction. Importantly, we found that phosphorylation at Ser-327 on {gamma}2 subunits, but not phosphorylation at other predicted PKC phosphorylation sites on beta2or {gamma}2 subunits, is required for PKC{epsilon} modulation of GABAA receptor function. Finally, we demonstrated that PKC{epsilon} directly phosphorylates {gamma}2S327 in vitro, {gamma}2S327 phosphorylation is reduced in mice lacking PKC{epsilon}, and exposure to ethanol in vivo increases {gamma}2S327 phosphorylation in the cerebellum of wild-type mice but not PKC{epsilon}-/- mice. These findings establish that PKC{epsilon} phosphorylation of Ser-327 in {gamma}2 subunits regulates the response of {alpha}1beta2{gamma}2 GABAA receptors to specific allosteric modulators. Moreover, our results show that concentrations of ethanol achieved during social drinking can increase the function of {alpha}1beta2{gamma}2 receptors by 40-60% when PKC{epsilon} phosphorylation of {gamma}2S327 is prevented.

Our results clearly identify a role for {gamma}2S327 phosphorylation in the regulation of GABAA receptor function. Prior studies investigated whether {gamma}2S327 phosphorylation modulates direct responses to GABA and showed minimal or negative results. In receptors containing {alpha}1, beta1, and {gamma}2 subunits heterologously expressed in HEK293 cells or in Xenopus oocytes, treatment for 10-60 min with phorbol esters, which activate several PKC isozymes and other proteins (32), reduced receptor activation by GABA, and this effect was absent in receptors with alanine mutations at beta1S409, {gamma}2LS327, and {gamma}2S343 (33). Phosphorylation of the beta1 subunit was responsible for this effect, because a single beta1S409A mutation was sufficient to eliminate phorbol ester-induced down-regulation. Another study using {alpha}1beta2{gamma}2S receptors expressed in Xenopus oocytes found a very short-lived and modest effect of {gamma}2SS327A expression; after 1.5 min of phorbol ester treatment the current amplitude decreased by 29% in native receptors but only by 19% in receptors carrying {gamma}2SS327A, although this 10% difference disappeared after 10 min (34). Our studies with HEK293 cells expressing {gamma}2SS327A indicate no effect of this mutation on current stimulated for up to 10 min by GABA alone (data not shown). Therefore, in contrast to its clear regulation of allosteric responses shown in our present study, {gamma}2S327 phosphorylation does not appear to play a role in regulating the direct response to GABA.


Figure 7
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  		FIGURE 7.
PKC{epsilon} regulates ethanol responses in cultured hippocampal neurons. A, current trace from wild-type hippocampal neurons showing no response to ethanol at concentrations up to 100 mM. B, addition of 5 µM Zn2+ to block {alpha}beta receptors that are not ethanol-sensitive allowed detection of a small ethanol response. C, greater enhancement of GABA currents by ethanol was seen in neurons cultured from PKC{epsilon}-/- mice assayed in 5 µM Zn2+. D, combined results from studies using 5 µM Zn2+ showing a greater effect of ethanol in neurons from wild-type mice (n = 8) when compared with neurons from PKC{epsilon}-/- mice (n = 9). *, p < 0.05 compared with wild-type cells treated with GABA alone by Dunnett's test. {dagger}, p < 0.05 compared with wild-type cells treated with the same concentration of ethanol by Tukey test.

 


Figure 8
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  		FIGURE 8.
Intraperitoneal injection of ethanol in vivo increases {gamma}2-S(P)327 immunoreactivity in wild type but not in PKC{epsilon}-/- cerebellum. Representative Western blots (A) and combined results (B) showing increased {gamma}2-S(P)327 immunoreactivity in wild-type compared with PKC{epsilon}-/- cerebellum after saline injection. Immunoreactivity increased after ethanol injection in wild-type but not in PKC{epsilon}-/- cerebellum (n = 8 for each condition). *, p < 0.05 when compared with saline-treated, PKC{epsilon}-/- samples, and {dagger}, p < 0.05 compared with saline-treated, wild-type samples by Bonferroni tests.

 
Previous expression studies using Xenopus oocytes or mouse L(tk-) cells suggested a requirement for the long splice variant of {gamma}2 subunits, {gamma}2L, in conferring PKC-mediated sensitivity to low doses (<50 mM) of ethanol (27-30). However, subsequent studies failed to observe a requirement for {gamma}2L in receptor responses to ethanol (35-37) and mice expressing {gamma}2S but not {gamma}2L showed normal behavioral responses to ethanol (38). We found that receptors containing the long or short splice variant of {gamma}2 respond similarly to ethanol and that inhibition of PKC{epsilon} enhances the effect of ethanol in both receptor types. These findings add to the body of evidence indicating that splice variation in {gamma}2 or phosphorylation at Ser-343 in {gamma}2L does not modulate GABAA receptor responses to ethanol.

In primary hippocampal neurons, we found that it was not possible to demonstrate ethanol enhancement of whole cell GABA currents. However, in cultured rat hippocampal neurons there is a benzodiazepine-insensitive tonic current that exhibits high sensitivity to Zn2+(IC50 ~ 1.89 µM), contributes to the tonic current, and is likely composed of {alpha}beta receptors (39). In stably transfected L(tk-) mouse fibroblasts, ethanol enhances GABA-stimulated chloride uptake through {alpha}1beta1{gamma}2 receptors but not {alpha}1beta1 receptors, suggesting a requirement for {gamma}2 subunits for response to ethanol (28). Therefore, we used 5 µM Zn2+ to inhibit such ethanol-insensitive currents, because in HEK293 cells expressing recombinant GABAA receptors, currents carried by {alpha}1beta3 receptors are inhibited by >90% by 5 µM Zn2+, whereas {alpha}1beta3{gamma}2 receptors are inhibited by less than 10% (31). Our finding that 5 µM Zn2+ unmasked a response to ethanol indicates the presence of an ethanol-insensitive current that contributes significantly to GABA responses in these cells and underscores the difficulty in using whole cell patch clamp recording to detect ethanol sensitivity in primary neurons that express both ethanol-sensitive and ethanol-insensitive receptors.

Our finding that PKC{epsilon} inhibition alone was sufficient to increase allosteric sensitivity indicates that there is an active endogenous phosphatase associated with {gamma}2 subunits. One candidate is calcineurin (protein phosphatase 2B), which can be co-immunoprecipitated with {gamma}2 subunits and which dephosphorylates {gamma}2S327 in mouse hippocampus (40). Therefore, PKC{epsilon} and calcineurin may act in concert to reciprocally regulate GABAA receptor phosphorylation and function in the brain.

Genetic variation in the {gamma}2 subunit plays an important role in behavioral responses to ethanol. Quantitative trait locus analysis has revealed two loci associated with severity of acute alcohol withdrawal in mice within a 3-centimorgan interval on mouse chromosome 11 that includes the GABAA receptor gene cluster encoding {alpha}1, {alpha}6, beta2, and {gamma}2 subunits (41). DBA/2J mice show particularly severe alcohol withdrawal seizures and posses a unique {gamma}2 allele resulting in an alanine to threonine substitution at residue 11 in the mature {gamma}2 protein. Moreover, {gamma}2+/- mutant mice show more severe alcohol withdrawal seizures than wild-type littermates (41). In addition to alterations in alcohol withdrawal seizure severity, allelic variation in {gamma}2is associated with susceptibility to ethanol-induced ataxia (42). It is therefore interesting that, compared with wild-type mice, PKC{epsilon}-/- mice show less severe alcohol withdrawal seizures (43) and less acute tolerance to alcohol-induced ataxia (17). Together with our present results, these findings suggest that PKC{epsilon} modulates the intensity and duration of alcohol intoxication and the severity of alcohol withdrawal through phosphorylation of GABAA receptors at {gamma}2S327.


   FOOTNOTES
 
* This work was supported by National Institutes of Health (NIH) Grant AA013588 and funds provided by the State of California for medical research on alcohol and substance abuse through the University of California at San Francisco (to R. O. M.), and by NIH Grant AI/CA44009 (to K. M. S.). 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Methods and supplemental Fig. S1. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Ernest Gallo Clinic and Research Center, 5858 Horton St., Suite 200, Emeryville, CA 94608. Tel.: 510-985-3950; Fax: 510-985-3101; E-mail: romes{at}gallo.ucsf.edu.

3 The abbreviations used are: GABAA, {gamma}-aminobutyrate, type A; PKC, protein kinase C; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; 1Na, 1-naphthyl-PP1; PBS, phosphate-buffered saline; as-PKC, ATP analog-sensitive PKC; GST, glutathione S-transferase; ANOVA, analysis of variance. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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