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Originally published In Press as doi:10.1074/jbc.M608910200 on December 5, 2006

J. Biol. Chem., Vol. 282, Issue 6, 3819-3828, February 9, 2007
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A {gamma}2(R43Q) Mutation, Linked to Epilepsy in Humans, Alters GABAA Receptor Assembly and Modifies Subunit Composition on the Cell Surface*Formula

Guillaume Frugier{ddagger}, Françoise Coussen§, Marie-France Giraud, Marie-Françoise Odessa{ddagger}, Michel B. Emerit||, Eric Boué-Grabot{ddagger}, and Maurice Garret{ddagger}1

From the {ddagger}Laboratoire de Neurophysiologie, CNRS-UMR 5543, Université de Bordeaux II, 33076 Bordeaux, §UMR 5091 Institut F. Magendie, 33077 Bordeaux, Institut de Biochimie et de Génétique Cellulaires du CNRS, 33077 Bordeaux, and ||Institut National de la Santé et de la Recherche Médicale U288, Pitié-Salpêtrière, 75013 Paris, France

Received for publication, September 18, 2006 , and in revised form, November 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Genetic defects leading to epilepsy have been identified in {gamma}2 GABAA receptor subunit. A {gamma}2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a {gamma}2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABAA receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant {gamma}2 constructs and showed that the {gamma} 2(R43Q) mutation prevented surface expression of the subunit, unlike {gamma}2(K289M) substitution. Several {gamma}2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with {alpha}3- and beta3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the {gamma}2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant {gamma}2(R43Q) was retained within intracellular compartments, {alpha}beta complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that {gamma}2(R43Q) was able to associate with {alpha}3- or beta3-subunits, although the stoichiometry of the complex with {alpha}3 was altered. Our data show that {gamma}2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABAA receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the {gamma}2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous {gamma}2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Aminobutyric acid (GABA)2 is the major inhibitory neurotransmitter in the central nervous system. A modification in the GABAergic systems has been postulated in many neurological and psychiatric diseases (1). For instance, alteration or modulation of GABA neurotransmission plays an important role in epilepsy mechanisms and treatment (2–4). Furthermore, several mutations of GABAA receptor genes have recently been associated with epileptic syndromes (5–8). These mutations offer an opportunity to obtain new insights into the structure and function of GABAA receptors (9, 10) and may provide clues to implication of these receptors in epilepsies (4, 11).

The ionotropic GABA receptors, mediating fast inhibitory transmission, are heteromeric structures composed of a combination of five out of at least 18 different subunits ({alpha}1–6, beta1–3, {gamma}1–3, {epsilon}, {theta}, {delta}, and {rho}1–3), grouped into several classes (12, 13). Studies have demonstrated that the subunit composition determines their affinity for GABA and the specific effect of allosteric modulators, as well as receptor trafficking. The major GABAA receptor in the brain is believed to consist of two {alpha}1-, two beta2-, and one {gamma}2-subunits (14). There is considerable evidence that the {gamma}2-subunit plays an essential role in the response to benzodiazepine modulators and receptor targeting (15).

An R43Q mutation in the {gamma}2-subunit N-terminal domain has been found to cause childhood absence epilepsy with febrile seizure (6). Analysis of the consequences of this mutation on GABAA receptors expressed in different heterologous cell types has produced conflicting data, raising the question of the subunit composition of receptors on the cell surface. It has been shown that the mutated subunit does not modify benzodiazepine sensitivity of GABAA receptors whereas most of the complexes are retained intracellularly (10, 16), in contradiction with other findings showing that {gamma}2(R43Q) specifically modifies pharmacological and functional properties (6, 9, 17, 18). Moreover, none of these experiments were performed on neurons.

To elucidate whether {gamma}2(R43Q) mutation altered subunit assembly or trafficking, we decided to study the surface expression of recombinant GABAA receptors by subunit-specific labeling. Labeling of transfected neurons showed that the {gamma}2(R43Q) mutation prevented surface targeting. Co-expression experiments in heterologous cells showed that the {gamma}2(R43Q)-subunit was retained intracellularly, as it was in neurons, whereas GABAA receptor complexes reached the cell surface membrane. Finally, immunoprecipitation experiments revealed a modification in subunit assembly induced by the {gamma}2(R43Q) mutation. Altogether, our findings indicate a relationship between the expression of GABAA receptors on the cell surface and epilepsy.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Eurobio). Cells were plated in Dulbecco's modified Eagle's medium onto 12-mm glass coverslips in 24- or 6-well plates 24 h before transfection.

Embryonic hippocampal neurons were obtained from E18 rat embryos as described earlier (19). Briefly, dissected hippocampi were incubated in trypsin and mechanically dissociated using a Pasteur pipette. The cells were counted and plated onto poly-D-lysin-coated 12-mm glass coverslips in complete Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen), 1 mM glutamine (Invitrogen), and 0.01% penicillin/streptomycin (Invitrogen). Four hours after plating, the medium was replaced with conditioned medium obtained by incubating complete Neurobasal medium on glial cultures for 24–48 h.

DNA Constructs—Rat {alpha}1, {alpha}3, beta3, and {gamma}2 GABAA subunits were subcloned in the pcDNA3 vector (Invitrogen). We used constructs that were available from previous studies (20). The 6-Myc epitope (MEQKLISEEDLNE, repeated 6 times) was inserted between amino acids 4 and 5 of the {gamma}2S- or {gamma}2L-subunits. As previously described, an insertion within this domain does not modify the functional properties of GABA- or glutamate-gated channels (19, 21–23). Receptor mutants were generated by polymerase chain reaction with the wild-type plasmid as a DNA template, using PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) to minimize artifactual mutations. Point mutations were constructed using the QuikChange site-directed mutagenesis system (Stratagene). All constructs were verified by automatic dideoxy DNA sequencing (Genome Express, Meylan, France).

Cell Transfection—COS-7 cells were transfected using the FuGENE 6 reagent (Roche Applied Science) according to manufacturer specifications. Equal amounts of {alpha}3- and beta3-subunit cDNAs (0.3 µg/well in 24-well plates for immunochemistry, 0.6 µg/well in 6-well plates for biochemistry) were mixed with different amounts (equal or 8-fold) of wild-type or mutated {gamma}2 cDNAs and then added to FuGENE 6. Cells were incubated for 24 h with cDNAs before analysis.

Hippocampal neurons were transfected at 7–11 days in vitro using the Lipofectamine 2000 reagent (Invitrogen) according to manufacturer specifications. Briefly, 0.5 µg of cDNAs, coding for wild-type or mutated {gamma}2-subunits, were mixed with 1.5 µl of Lipofectamine 2000 in Neurobasal medium. The cDNAs were then incubated with neurons in serum-free medium for 40–50 min before replacing the latter with conditioned complete Neurobasal medium (see above). Neurons appeared morphologically normal and expressed the neuronal marker NeuN (not shown). The cells were analyzed 24–60 h after transfection.

Immunocytochemistry—For living cell surface labeling, COS-7 cells and hippocampal neurons were incubated with antibodies at room temperature for 20 min in Dulbecco's modified Eagle's medium or Neurobasal medium supplemented with 10 mM HEPES, respectively. Antibodies were raised in rabbit against the {alpha}1 or {alpha}3 GABAA subunit N termini (Alomone Labs, Israel) or the Myc tag (Upstate, Charlottesville, VA). Sera were diluted 1:1000 (anti-{alpha}3), 1:500 (anti-{alpha}1), or 1:100 (anti-Myc) in medium. After incubation, cells were washed quickly by dipping coverslips in medium and fixed for 10 min in phosphate-buffered saline (PBS) containing 4% sucrose and 4% paraformaldehyde preheated to 37 °C, washed in PBS, and blocked in 0.3% bovine serum albumin and 50 mM glycine (in PBS) for 15 min. Cells were washed in PBS containing 0.3% bovine serum albumin. After cell permeabilization using 0.3% Triton X-100, intracellular tagged {gamma}2-subunits were detected by incubating the cells with a mouse anti-Myc 9E10 antibody (1:100; Roche Applied Science) for 2 h. Polyclonal and monoclonal antibodies were detected using an Alexa Fluor 568-coupled anti-rabbit antibody (1:1000; Molecular Probes) and an FITC-coupled anti-mouse antibody (1:200; Chemicon), respectively.

Quantitative Analysis of Fluorescence Signals—Fluorescence microscopy was performed using a Zeiss Axioplan 2 microscope, with a 63 x 1.4 numerical aperture oil immersion lens. Quantification of fluorescence signals and background substraction were performed using Metamorph Software (Molecular Devices Corp., Downington, PA). For each image acquired, background levels were determined using the surface and intracellular signals measured in neighboring non-transfected cells and subtracted from the values obtained in transfected cells. Numerical data are presented as mean ± S.E., and statistical significance, assessed using Kruskal-Wallis analysis of variance (Dunn's method) test (unless otherwise stated), was taken as p < 0.05 (Sigmastat; SPSS Inc.).

Coimmunoprecipitation—COS-7 cells were homogenized in buffer containing 20 mM HEPES, 0.15 mM EDTA, and 10 mM KCl, pH 8, supplemented with a mixture of protease inhibitors (Roche Applied Science). The buffer was then adjusted to 12% sucrose, and after four more strokes the cells were centrifuged at 2000 rpm for 3 min to remove genomic DNA. The supernatant was centrifuged at 15,000 rpm for 30 min. The pellet was recovered, and cell membranes were solubilized with 15 strokes in a buffer containing 20 mM Tris-HCl, 0.15 mM EDTA, 150 mM NaCl, 2% Triton X-100, and 0.5% deoxycholate, pH 8, supplemented with a mixture of protease inhibitors and then incubated for 45 min. The sample was centrifuged for 45 min at 15,000 rpm. The supernatant was incubated with 30 µl of Protein-G-Sepharose (Amersham Biosciences) for 1 h and centrifuged at 2000 rpm for 3 min. The clarified supernatant was then incubated with 5 µg of mouse anti-Myc 9E10 antibody for 2 h and then overnight with 30 µl of anti-mouse Protein-A-Sepharose (Sigma). The resin was washed with the solubilization buffer and with the same buffer containing 500 mM NaCl. All steps were performed at 4 °C. Four independent experiments were performed. The beads were resuspended in 80 µl of gel loading buffer, separated by 10% SDS-PAGE, and immunoblotted with the appropriate antibodies as previously described (19). Antibodies used were anti-{alpha}3 (1:2000; Alomone Labs), anti-beta3 (1:1000; Chemicon), and polyclonal anti-Myc (1:500; Upstate). The three antigens were detected consecutively on each Western blot membrane after stripping to selectively remove both primary and secondary antibodies (24). Each immunoprecipitate was analyzed twice. Pictures of the blots were taken with the ChemiGenius 2XE apparatus under the control of GeneSnap program. Quantitative analysis was performed using GeneTools Software (Syngene, Cambridge, UK).


Figure 1
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  		FIGURE 1.
Expression of {gamma}2-subunit in transfected hippocampal neurons. Neurons were transfected with either a wild-type subunit, {gamma}26mycWT, or one bearing point mutations, {gamma}26myc(R43Q) or {gamma}26myc(K289M), as indicated on the left. Surface labeling was obtained after immunostaining living neurons with a polyclonal antibody directed at the extracellular 6-Myc tag and Alexa Fluor 568-conjugated secondary antibodies (surface). Intracellular staining of the same cells was obtained after permeabilization and labeling with a monoclonal antibody directed at the same tag and FITC-conjugated secondary antibodies (intra). Merged images (overlay) revealed wild-type {gamma}2- and {gamma}2(K289M)-subunits at the surface of transfected neurons whereas {gamma}2(R43Q) was essentially intracellular. Scale bar, 20 µm.

 
Structural Building of GABAA Receptor Mutants—The coordinate file (GABA-A_expanded.pdb) of the GABAA receptor homology model (31) was used to generate mutant models. Mutations were created using DeepView/Swiss-PdbViewer software (25). Steepest descent energy minimization was performed using the GROMOS96 force field (26).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
{gamma}2(R43Q)-Subunit Does Not Reach the Surface of Neurons and COS-7 Cells—Analysis of {gamma}2(R43Q)-subunit expression in heterologous systems has produced controversial data (6, 9, 10, 16–18). To study this mutation in a neuronal environment, hippocampal neurons at 7 to 11 days in vitro were transfected with wild-type and mutant {gamma}26myc. Subunits expressed on the cell surface were detected on living cells to avoid any intracellular labeling that could occur with fixed cells (27). Labeling the extracellular epitope with a polyclonal antibody directed against the Myc tag revealed that expression of the wild-type {gamma}2-subunit on the surface membrane was uniformly distributed throughout the neurons (Fig. 1, top lane). After permeabilization, labeling of the Myc tag with a monoclonal antibody revealed perinuclear and reticular structures that probably correspond to the endoplasmic reticulum, where a large majority of subunits are retained during the protein folding and assembling processes (16, 23, 28, 29). When the arginine in position 43 of {gamma}26myc was replaced with a glutamine residue, neurons transfected with {gamma}26myc(R43Q) were not labeled at the surface (Fig. 1, middle lane). This effect was specific to {gamma}26myc(R43Q), as {gamma}26myc(K289M), known to be responsible for generalized epilepsy with febrile seizure plus (5), was targeted to the neuron cell surface (bottom lane). We obtained similar data with wild-type or R43Q mutant when {gamma}2S6myc (Fig. 1) or {gamma}2L6myc (not shown) constructs were expressed in transfected neurons for 24 (Fig. 1), 36, or 60 h (not shown). These experiments showed that the {gamma}2(R43Q)-subunit was not addressed to the surface when expressed in neurons.


Figure 2
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  		FIGURE 2.
Membrane delivery of {gamma}2-subunit co-expressed with {alpha}3- and beta3-subunits in COS-7 cells. Cells were transiently co-transfected with {alpha}3-, beta3-, and Myc-tagged {gamma}2-subunits (1:1:1 ratio), either wild type ({gamma}26mycWT) or bearing R43Q ({gamma}26mycR43Q) or K289M ({gamma}26mycK289M) substitutions as indicated on the left. Cell surface staining on living cells was revealed with anti-Myc polyclonal antibodies (surface), and intracellular staining of the same cells was revealed with a monoclonal antibody directed at the same tag (intra). Merged images (overlay) show intracellular retention of the {gamma}2(R43Q)-subunit. Scale bar, 10 µm.

 
To study the mechanisms behind this deficient GABAA receptor surface targeting, we co-expressed wild-type and mutated {gamma}26myc constructs with {alpha}- and beta-subunits in COS-7 cells. In {alpha}3-, beta3-, and {gamma}26myc-transfected cells, anti-Myc polyclonal surface labeling of living cells revealed clusters all over the cell plasma membrane (Fig. 2, top lane). When COS-7 cells were transfected with the {gamma}26myc(R43Q) mutant, surface staining was close to background (middle lane) whereas the {gamma}26myc(K289M) was clearly targeted on the cell surface (bottom lane). Our result for {gamma}2(K289M) was in agreement with previous data showing that this mutant expressed in heterologous cells gave rise to GABA-induced current with unaltered amplitude (10). Moreover, as previously observed in neurons, there was no difference when experiments on COS-7 cells were conducted using the {gamma}2S (Fig. 2) or {gamma}2L splice form (not shown) or when COS-7 cells were transfected with {alpha}1-instead of the {alpha}3-subunit (not shown).

{gamma}2(R43) Domain Is a Major Determinant to Drive Cell Surface Targeting of the {gamma}2-Subunit—Crystal structure analysis of a soluble acetylcholine-binding protein was used to design a model of the GABAA receptor ligand-binding domain (30, 31). A homology model of the whole GABAA receptor (Fig. 3A) was also generated by combining the extracellular domain model with a model of the membrane domain constructed from the coordinates of the acetylcholine receptor pore (31, 32). These homology models indicated that a bifurcated salt bridge may connect the {gamma}2(E178), {gamma}2(R43), and beta2(R117) residues (Fig. 3B). Using the coordinate file of the whole receptor homology model, we constructed models of several Arg-43 mutants. In the R43Q mutant (Fig. 3C), {gamma}2(E178) only interacted ionically with beta2(R117), whereas the {gamma}2(R43K) mutant model suggested that there could be a salt bridge network similar to the one found in the wild-type receptor (Fig. 3D and supplemental movie). We also used the GABAA wild-type homology model to highlight the Asp-39 position, conserved among GABA-gated receptor channels, and Pro-44, conserved among the Cys loop ligand-gated receptor channel family (Fig. 3E). We created mutants of the residues mentioned above to study their importance in receptor assembly or trafficking. Interestingly, we noted that {gamma}2(S171), whose influence in GABAA receptor subunit assembly has recently been reported (33), is located at the beginning of the loop close to beta2(R117) that ends with {gamma}2(E178) (Fig. 3, E and F). The {gamma}2K289 and {gamma}2R43 residues are located far apart (48 Å, Fig. 3A); thus, K289M substitution was not expected to affect GABAA receptor properties in the same way as mutations within the Arg-43 domain.


Figure 3
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  		FIGURE 3.
Localization of the {gamma}2(R43) region in the GABAA receptor homology model. A, top view of the GABAA receptor homology model. Subunits are shown in ribbon representation. The two {alpha}1-subunits are in blue, the two beta2-subunits in green, and the {gamma}2-subunit in magenta. The extracellular domain of the receptor is shown in the foreground and the membrane domain in the background. The {gamma}2(K289) residue is shown in sphere representation (arrow). B–D, putative salt bridge networks at the beta2-{gamma}2 interface in the wild-type, {gamma}2(R43Q), and {gamma}2(R43K) mutants. Residues are represented by sticks, and only putative salt bridges with distances between charged groups below 3.8 Å are represented (dashed lines). Nitrogen and oxygen atoms are shown in blue and red, respectively. Residues from the {gamma}2 chain are represented in magenta and beta2(R117) in green. E, close-up of the region at the {gamma}2-beta2 interface (inset in panel A). Residues are shown in stick representation. The side-chain atoms of {gamma}2 residues mutated in this study are shown as well as {gamma}2(S171), {gamma}2(E178), and beta2(R117). For the sake of clarity, residues around {gamma}2(R43) and beta2(R117) are only represented by their main chain and residues between {gamma}2(S171) and {gamma}2(E178) have been omitted. Molecular figures were generated with PyMOL (pymol.org). F, loop region model at the {gamma}2beta2 interface. The loop region containing residues {gamma}2(D39-P44) is shown in orange and the loop {gamma}2(S171-E178) in red. According to the whole GABAA model, residues {gamma}2(S171-E178) belong to a loop that connects two beta-sheets of an extended beta-sandwich of the {gamma}2-subunit. The loss of a positive charge at position 43 could change the {gamma}2(E178)-beta2(R117) interaction, and this may alter the flexibility of this loop and/or the {gamma}2beta2 interface rigidity.

 
As the {gamma}2(R43Q) mutant behaved in a similar way when it was expressed in COS-7 cells or neurons and surface labeling is an efficient way to study GABAA receptor expression (34), we used this approach, with several {gamma}26myc mutants, to study the role of the domain surrounding the Arg-43 position. Cells were transfected with {alpha}3- and beta3-subunits in combination with {gamma}2S6myc bearing different point mutations. Immunolabeling showed that, as described above for {gamma}2(R43Q), {gamma}2(R43A) mutant was not detected on the cell surface, whereas {gamma}2(R43K), {gamma}2(D39A), and {gamma}2(P44A) were consistently expressed at the surface (Fig. 4A). Quantitative analysis of intracellular {gamma}26myc revealed that there was no significant difference (p > 0.05) in expression levels of the wild-type subunit and any of the mutants in the Arg-43 domain (Fig. 4B), showing that the effect of mutations was not due to a subunit expression or stability defect. Surface targeting was analyzed by measuring the ratio of the amount of {gamma}2-subunit labeled on the cell surface versus {gamma}2 labeled within intracellular compartments (Fig. 4C). The surface versus intracellular signal ratio showed that both the R43Q and R43A mutations prevented cell surface targeting compared with wild type (0.033 ± 0.002, 0.052 ± 0.003, 1.163 ± 0.078, respectively). Cell surface labeling was detected on cells transfected with the {gamma}2S6myc(R43K) construct (Fig. 4A), showing that this conservative substitution restored {gamma}2 surface targeting (0.235 ± 0.033) to some extent. When the conserved Asp-39 or Pro-44 residues were replaced with alanine, labeling was detected on the membrane surface (Fig. 4A), although the {gamma}2-subunit ratio on the cell surface was reduced (0.396 ± 0.083 and 0.135 ± 0.011, respectively). The significantly higher level of intracellular signals for the {gamma}2(K289M) mutant (p < 0.05; Fig. 4B) may be due to a different expression level or stability. This increase did not interfere with surface targeting efficacy, because the K289M substitution had no significant effect on the surface versus intracellular signal ratio (0.84 ± 0.093) compared with the wild type, in agreement with previous studies (10). These experiments clearly showed the major impact of the Arg-43 residue on {gamma}2-subunit cell surface localization and led us to evaluate the influence of this position, either on the subunit itself or on GABAA receptor complexes associated with this subunit.


Figure 4
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  		FIGURE 4.
Effect of various substitutions within the {gamma}2R43 domain on surface expression. A, Myc-tagged {gamma}2S-subunit ({gamma}2 WT) or bearing substitutions as indicated were co-expressed with {alpha}3 and beta3 (1:1:1 ratio) in COS-7 cells. The level of {gamma}26myc cell surface expression was assessed on living cells with anti-Myc polyclonal antibodies and Alexa Fluor 568-conjugated secondary antibodies (surface). Total {gamma}26myc expression was measured by permeabilization and labeling with a monoclonal antibody directed at the Myc tag and FITC-conjugated secondary antibodies (intra). Scale bar, 10 µm. B, quantitative analysis of {gamma}26myc construct intracellular fluorescence labeling. C, the ratio of surface/total expression was measured as the ratio of surface Alexa Fluor 568 fluorescence level on living cells to FITC fluorescence on permeabilized cells. Labeling was measured in fluorescence intensity units (500–600 transfected cells for each combination, four independent experiments).

 


Figure 5
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  		FIGURE 5.
Expression of {gamma}2- and {alpha}3-subunits on the cell surface. A, COS-7 cells were transfected only with Myc-tagged {gamma}2-subunits, either wild type (WT) or bearing substitutions within the Arg-43 domain as indicated. The ratio of surface/total expression was measured as the ratio of surface Alexa Fluor 568 fluorescence level on living cells to FITC fluorescence on permeabilized cells (100–200 transfected cells, three independent experiments) as in Fig. 4. B, cells were transfected as in panel A except that {alpha}3- and beta3-subunits were co-expressed with {gamma}26myc (1:1:1 ratio). Level of {alpha}3-subunit cell surface expression was assessed on living cells (500–600 transfected cells, four independent experiments) with anti-{alpha}3 polyclonal antibodies and Alexa Fluor 568-conjugated secondary antibodies.

 
It has been shown that the {gamma}2S-subunit, expressed alone in heterologous cells without other subunit subtypes, is able to reach the cell surface, probably as a monomer (35). In agreement with that data, cells transfected solely with the {gamma}2S6myc construct were labeled in their intracellular compartments and at the surface (Fig. 5A). Note that when {gamma}26myc was expressed alone, the surface-versus intracellular-labeling ratio was six times lower than when the subunit was expressed with {alpha} and beta (0.218 ± 0.019 and 1.163 ± 0.078, respectively). Our experiments showed that any mutation within the domain encompassing Arg-43 significantly reduced the surface/intracellularlabeling ratio of monomeric {gamma}26myc (WT, 0.218 ± 0.019; R43Q, 0.026 ± 0.003; R43A, 0.015 ± 0.001; R43K, 0.036 ± 0.006; D39A, 0.034 ± 0.003; P44A, 0.023 ± 0.002). We then examined {alpha}3-subunit cell surface targeting that forms either {alpha}3beta3 or {alpha}3beta3{gamma}2 complexes to give rise to functional receptors (14). COS-7 cells were transfected with {alpha}3, beta3, and {gamma}26myc constructs (1:1:1 ratio). Surface expression of the {alpha}3 extracellular epitope on living cells and total {gamma}2S6myc subunit expression on fixed and permeabilized cells were revealed by double labeling. As stated above (Fig. 4B), mutations did not modify intracellular {gamma}2-subunit expression levels. Then, quantitative analysis (Fig. 5B) showed that, irrespective of the amino acid substitution in the {gamma}2-subunit, there was no major modification of {alpha}3-subunit cell surface targeting (mean ± S.E. surface labeling in arbitrary units was WT, 12.7 ± 0.5; R43Q, 16.4 ± 0.8; R43A, 13.2 ± 0.8; R43K, 11.7 ± 0.7; D39A, 17.0 ± 0.8; P44A, 17.9 ± 1.1). These data indicated that {gamma}2 mutants did not prevent {alpha}3beta3 complexes from reaching the cell surface.


Figure 6
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  		FIGURE 6.
{gamma}2(R43Q) does not prevent surface targeting of the {alpha}-subunit. A, COS-7 cells transiently co-transfected (1:1:8 ratio) with {alpha}3-, beta3-, and Myc-tagged {gamma}2-subunits, either wild type ({gamma}2WT) or with an R43Q substitution ({gamma}2 R43Q) as indicated on the left. Cell surface staining on living cells with specific polyclonal antibodies directed at the extracellular N-terminal domain of the {alpha}3-subunit (surface). Intracellular staining of the same cells with a monoclonal antibody directed at the 6-Myc tag (intra). Merged images (overlay) show surface targeting of the{alpha}3-subunit and total labeling of{gamma}2. Scale bar, 10 µm. B, when either wild-type {gamma}2-subunit (WT) or {gamma}2(R43Q) mutant (R43Q) were overexpressed, the level of {alpha}3-subunits detected on the surface was unchanged (statistical significance using Mann-Whitney test was p < 0.05). Labeling was measured in fluorescence intensity units (300–400 transfected cells for each combination, three independent experiments).

 
{gamma}2(R43Q) Mutant Is Not a Dominant Negative—Our data were in contradiction with previous reports suggesting that {gamma}2(R43Q) acted as a dominant negative mutant by forming a complex with {alpha}- and beta-subunits and retaining this complex within intracellular compartments (9, 10, 16). To investigate this issue, we overexpressed wild-type or R43Q-{gamma}2S6myc constructs with {alpha}3- and beta3-subunits in COS-7 cells at a 8:1:1 ratio (Fig. 6A). Compared with co-expression with wild-type {gamma}2S6myc, overexpression of {gamma}2S6myc(R43Q) mutant did not modify surface labeling of the {alpha}3-subunit (Fig. 6B). Similar data were obtained when the {alpha}3-subunit was replaced with {alpha}1 (supplemental data). As it is well established that {alpha}-subunits are addressed to the cell surface only in association with beta (with or without {gamma}), these data showed that, irrespective of the amount of mutated {gamma}2-subunit, R43Q substitution did not prevent the assembly of {alpha}3- (or {alpha}1-) and beta3-subunits or their cell surface targeting.


Figure 7
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  		FIGURE 7.
Expression of recombinant {gamma}2S6myc subunits in transfected hippocampal neurons. A, neurons were transfected with the {gamma}26myc(P44A) mutated subunit. The surface expression of {gamma}2-subunits was detected by double labeling with a polyclonal antibody directed at the extracellular 6Myc tag and Alexa Fluor 568-conjugated secondary antibodies (surface). Intracellular staining of the same cells was obtained after permeabilization and labeling with a monoclonal antibody directed at the same tag and FITC-conjugated secondary antibodies (intra). Merged images (overlay) revealed {gamma}2(P44A)-subunits on the surface of transfected neurons. B, neurons were transfected with either wild-type {gamma}2-subunit ({gamma}26mycWT) or {gamma}26myc(R43Q). Surface labeling was obtained after immunostaining of living neurons with a polyclonal antibody directed at the extracellular domain of the native {alpha}3-subunit (surface). Intracellular staining of the same neurons after permeabilization and labeling with a monoclonal antibody directed at the 6Myc tag (intra). Scale bar, 20 µm.

 
To determine whether {gamma}2(R43Q) behaved the same way in neurons, we investigated the possibility that recombinant subunit constructs co-assembled with native subunits in our experiments. Thus, we transfected hippocampal neurons with the {gamma}26myc(P44A) mutant that had been shown to be addressed to the COS-7 cell surface only when co-transfected with {alpha}- and beta-subunits (Figs. 4 and 5). Surface labeling of living neurons (Fig. 7A) clearly showed that the recombinant mutant was targeted to the neuronal cell membrane, suggesting that {gamma}26myc(P44A) co-assembled with native subunits. This was in agreement with previous findings that recombinant GABAA receptor {alpha}1-subunits were able to form functional complexes with native subunits in transfected hippocampal neurons (36). When wild-type and {gamma}2(R43Q) were expressed in neurons, there was no apparent modification in native {alpha}3-subunit surface labeling (Fig. 7B). Taken together, our experiments showed that recombinant {gamma}26myc subunit constructs co-assembled with native subunits and that expression of the {gamma}2(R43Q)-subunit did not dramatically reduce the number of GABAA receptors on the cell surface in neurons, as is also the case in COS-7 cells.


Figure 8
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  		FIGURE 8.
{gamma}2(R43Q) mutation modifies assembly with {alpha}3-subunit. A, {alpha}3-, beta3-, and {gamma}26myc-subunits were expressed in COS-7 cells (1:1:1 ratio) and analyzed by Western blot. Solubilized membrane proteins (SM) were immunoprecipitated with a monoclonal antibody directed at Myc tag, and immunoprecipitated proteins (IP) were detected with the corresponding polyclonal antibodies. B, quantities of {alpha}3 or beta3, normalized to the amount of {gamma}26myc detected in each lane, showed an increase in the amount of {alpha}3-subunit associated with {gamma}2(R43Q) as compared with wild-type {gamma}2. Four independent expression and immunopurification experiments were each analyzed twice. Statistical comparisons were assessed using Student's t test. Differences were considered significant if p < 0.005.

 
{gamma}2(R43Q) Mutation Disturbed GABAA Receptor Subunit Assembly—Our experiments showed that, when {alpha}-, beta-, and {gamma}2(R43Q)-subunits were co-expressed, {alpha} was found on the cell surface whereas {gamma}2(R43Q) was absent. These findings suggested that {gamma}2(R43Q) did not associate with the {alpha}beta complex. To challenge this hypothesis, we expressed {alpha}3- and beta3-subunits in COS-7 cells with either wild type or R43Q-{gamma}26myc (1:1:1 ratio). Western blot analysis of membrane protein extracts and labeling, using either polyclonal anti-Myc, anti-{alpha}3, or anti-beta3, detected bands corresponding to the expected size (Fig. 8A, left panel). Proteins associated with the tagged {gamma}2-subunit were co-immunoprecipitated, and analysis showed that {alpha}3- and beta3-subunits were associated with both wild type and R43Q-{gamma}26myc (right panel). The {gamma}26myc-, {alpha}3-, or beta3-subunits in each immunopurified sample were quantified (Fig. 8B). This analysis showed that the amount of beta3 associated with {gamma}26myc(R43Q) was not significantly different from the amount associated with wild-type {gamma}26myc (92 ± 4%). Surprisingly, the amount of {alpha}3 associated with {gamma}26myc(R43Q) was higher than with wild-type {gamma}26myc (186 ± 18%). These data showed that the {gamma}2(R43Q)-subunit is able to assemble with {alpha}3 and beta3 and that mutation modified the subunit assembly of GABAA receptors.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several mutations in GABAA receptor subunit genes have been implicated in epilepsy (2, 5–8, 37, 38). Functional studies of these mutations are required to elucidate the mechanisms underlying these disorders. However, analysis of the consequence of {gamma}2(R43Q) mutation, using the expression of recombinant subunits in heterologous cells, has produced controversial data (6, 9, 10, 16–18). This led us to analyze the effect of {gamma}2(R43Q) mutation in a neuronal environment and continue in-depth studies using heterologous cells.

R43Q Mutation Prevents {gamma}2-Subunit Cell Surface Targeting in Neurons—We used subunit-specific labeling to monitor receptor targeting. We expressed tagged {gamma}2-subunits and analyzed the expression of {gamma}2(R43Q) in neurons, for the first time. 1 to 3 days post-transfection, we showed that, whereas both wild-type {gamma}2S and {gamma}2L isoforms were expressed at the surface of cultured neurons, surface expression of {gamma}2(R43Q) mutants was below detection level. It was possible that we had detected the traffic of wild-type recombinant {gamma}2S-subunit to the surface of neurons in monomer form, as it has been shown in heterologous cells transfected with this subunit alone that this isoform is targeted to the cell plasma membrane (Ref. 35 and this work). However, our experiments suggest that this is unlikely. We found that the {gamma}2(P44A) mutant was not driven to the cell membrane of transfected COS-7 cells when expressed alone, whereas it was detected on the cell surface when co-expressed with {alpha}- and beta-subunits (see Figs. 4 and 5). When the {gamma}2(P44A) mutant was transfected in cultured hippocampal neurons, it was detected on the cell surface, suggesting an association of the recombinant {gamma} construct with native {alpha}- and beta-subunits (Fig. 7A). In addition, the {gamma}2L isoform, reported to have less propensity to traffic to the cell surface in monomer form than {gamma}2S (35), was also detected on the cell surface of transfected neurons.

As recombinant receptor expression in heterologous cells makes it possible to visualize individual subunits, we used this method to study the effect of the {gamma}2(R43Q) mutation on GABAA receptor subunit assembly and cell surface targeting. However, overexpression of membrane proteins in heterologous cells may alter their expression or stoichiometry (39, 40), which may account, at least in part, for the contradictory previous findings (9, 10, 17, 18). In our expression experiments in COS-7 cells, designed to avoid overexpression, when a tagged wild-type {gamma}2 was co-expressed with {alpha}3- (or {alpha}1-) and beta3-subunits, the extracellular N-terminals of either the {alpha}-or {gamma}2-subunits were detected on the cell surface. Most importantly, the {gamma}2(R43Q) mutant expressed in COS-7 cells, as in neurons, had hardly any surface expression, showing that experimental conditions were appropriate for studying the effect of mutations on GABAA receptor subunit trafficking in heterologous cells. In addition, these data are in line with biochemical analyses showing a vast decrease in {gamma}2(R43Q) at the surface of human embryonic kidney 293-transfected cells (18).

{gamma}2(R43Q) Substitution Is a Decisive Factor in the Effect on GABAA Receptor Targeting—Our experiments demonstrated that any mutation within the domain surrounding {gamma}2(R43) had an impact on the distinctive ability of the monomeric {gamma}2-subunit to traffic to the surface when expressed in heterologous cells (35). Although this property is probably not of any biological significance, the defect in cell surface targeting may reflect the influence of the Arg-43 domain on the integrity of the overall subunit structure. The {gamma}2(R43) domain is also implicated in intersubunit contacts, as elegantly demonstrated using a 15-residue peptide surrounding Arg-43 (9), in agreement with the current homology molecular model. This model localizes {gamma}2(R43) at the interface between {gamma}2- and beta2-subunits (30), where an ionic interaction takes place involving Arg-43, the conserved {gamma}2(E178), and the adjacent beta2(R117). When {gamma}2(R43) was replaced by an alanine or glutamine residue, the {gamma}2-subunit was barely detectable on the cell surface. Substituting the lysine for glutamine partially corrected the targeting defect of the {gamma}2(R43Q) mutant. However, in the {gamma}2(R43K) mutant, receptor cell surface targeting was only partially restored, showing that a lysine residue in this position cannot fully replace an arginine residue. Analysis of {gamma}2(D39A) and {gamma}2(P44A) mutants also highlighted the influence of the Arg-43 domain on {gamma}2-subunit cell surface targeting. Conversely, our findings that {gamma}2(K289M) mutation did not affect assembly and targeting of the GABAA receptor complex are complementary to previous studies indicating that this substitution modified GABAA receptor kinetics (10).

{gamma}2(R43Q) Mutant Does Not Reduce {alpha}beta Surface Targeting—Surprisingly, the various mutations of the {gamma}2(R43) domain, including R43Q substitution, did not lead to a significant reduction in {alpha}beta surface targeting in our co-expression experiments using COS-7 cells, even with an {alpha}beta3{gamma}2(R43Q) 1:1:8 ratio. These findings support previous data showing that GABAA receptor expression in heterologous cells with wild type or R43Q-{gamma}2 gave rise to GABA-induced currents of similar amplitude (17). It is noteworthy that upon co-expression of {alpha}, beta, and {gamma}2(R43Q), the kinetic properties of GABAA receptors resembled those of {alpha}beta receptors (17). Our experiments are also in agreement with electrophysiological data reported recently (9). Taken together, our experiments indicated that {gamma}2(R43Q) substitution prevented association with {alpha} or, more probably, beta, as this subunit is assumed to interact with the {gamma}2R43 domain. This has been reported for the {gamma}2(S171) residue, located in the vicinity of the Arg-43 domain in the three-dimensional structure of the GABAA receptor complex. Mutations in this position prevented association with {alpha}2- and beta1-subunits, resulting in intracellular retention of {gamma}2 and surface expression of {alpha}2beta1 oligomers when these subunits were co-expressed in human embryonic kidney 293 cells (33). On the contrary, our coimmunoprecipitation experiments demonstrated that no significantly different amounts of beta3-subunit were associated with wild type or R43Q-{gamma}2. This is in line with data showing that intersubunit contacts involve several domains along the polypeptide chains (41, 42). Another surprising finding was that the amount of {alpha}3-subunit co-immunoprecipitated with {gamma}2 was higher with R43Q mutant than wild-type subunits. This finding is difficult to interpret, as there is currently no model describing the scenario driving the assembly of ligand-gated ion channels (28). Nevertheless, it points to the intriguing possibility that the conserved Arg-43 position is implicated in controlling the order of subunit assembly and/or stoichiometry.

Functional Consequences of {gamma}2(R43Q) Mutation—Our data suggested that {gamma}2(R43Q) substitution gave rise to subunit complexes with an altered assembly, leading to the targeting of {alpha}beta oligomers of unknown stoichiometry. This may reconcile some of the controversial data, and, on this basis, we propose that the {gamma}2(R43Q) mutation modifies the subunit composition rather than the number of GABAA receptors at the neuron surface. Because the {gamma}2-subunit is implicated in synaptic localization of these receptors (43), this modification would lead to a decrease in the number of {alpha}beta{gamma}2 receptors and an increase in {alpha}beta complexes for heterozygous mutation with relevance to clinical syndromes. Thus, it is tempting to speculate that this would create an imbalance between phasic and tonic inhibition, resulting in important pathophysiological consequences for the physiology of neuronal networks (44, 45). However, preliminary data in an heterozygous knock-in model or in transfected neurons showed that synaptic currents did not decrease (46, 47). Interestingly, studies using {gamma}2 knock-out mice showed that {gamma}2-/- animals had impaired GABAA receptors and died shortly after birth, unlike {gamma}2+/- mice that apparently had normal GABAA receptors and developed as wild types (48, 49). However, a further analysis of heterozygous mice showed a limited reduction in synaptic GABAA receptor clusters in some brain areas with a parallel increase in, probably extrasynaptic, {alpha}beta receptors. This subtle change was sufficient to result in an anxiety-like behavior (50). This suggests that a molecular, physiological, and behavioral comparison of {gamma}2+/- mice with heterozygous {gamma}2(R43Q) knock-in animals may provide information on the relationship between GABAA receptor subunit composition, physiology, and physiopathological states.

This work might also provide support for future study using neuronal preparations and knock-in animals (46, 47). Indeed, preliminary data, using a {gamma}2(R43Q) knock-in mouse model, showed that, whereas mutant subunits failed to localize at the surface membrane, {alpha}1-subunits retained their ability to localize at the neuronal membrane. However, the clustered organization of {alpha}1-subunits was absent in {gamma}2(R43Q) knock-in mice, suggesting a loss of ability of this mutant {gamma}2-subunit to associate with GABAA receptors in neurons (46, 47). Our findings will shed further light on this issue.


   FOOTNOTES
 
* This work was supported by Centre National de la Recherche Scientifique, Université Bordeaux2, and Conseil Régional Aquitaine. 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 movie S1 and Fig. S1. Back

1 To whom correspondence should be addressed: CNRS UMR 5543, Université de Bordeaux II, Laboratoire de Neurophysiologie, 146 Rue Léo Saignat, 33076 Bordeaux, France. Tel.: 33-557571686; Fax: 33-556901421; E-mail: maurice.garret{at}u-bordeaux2.fr.

2 The abbreviations used are: GABA, {gamma}-aminobutyric acid; FITC, fluorescein-5-isothiocyanate; WT, wild-type. Back


   ACKNOWLEDGMENTS
 
We thank Frédéric Jaskolski and Romuald Nargeot for helpful discussion.



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