©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Endogenous Phosphorylation of Distinct -Aminobutyric Acid Type A Receptor Polypeptides by Ser/Thr and Tyr Kinase Activities Associated with the Purified Receptor (*)

(Received for publication, May 23, 1995; and in revised form, August 11, 1995)

Michel H. Bureau (§) Jacques J. Laschet

From the Laboratory of Neurochemistry, University of Liège, B-4020 Liège, Belgium and the INSERM CJF 90-12, University of Rennes, F-35033 Rennes, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have investigated the phosphorylation of -aminobutyric acid type A (GABA(A)) receptor purified from bovine cerebral cortex in the absence of added kinases. Incubation of the affinity-purified receptor with [-P]ATP and 500 µM MnCl(2) yielded incorporation of 0.45 mol of P/mol of muscimol binding sites within 2 h at 30 °C. Mn was much more effective than Mg as activator. Phosphorylation of the receptor was observed on at least three different polypeptides of 51, 53, and 55 kDa. It was predominant on 51- and 53-kDa polypeptides that co-migrate with the [^3H]flunitrazepam photoaffinity-labeled bands, suggesting that P incorporation mainly occurs on alpha-subunits. A monoclonal antibody specific for alpha-subunits adsorbed the endogenously phosphorylated GABA(A) receptor with a stoichiometry close to 1 mol of phosphate/mol of muscimol. The phosphorylation of the 51-kDa polypeptide, corresponding to alpha(1)-subunit, exhibited a micromolar affinity for ATP and sigmoid kinetics (n(H) = 2). Major incorporation of phosphate occurred on serine and threonine residues in roughly equimolar ratio. By enzyme-linked immunosorbent assay and immunoblotting studies we also detected a minor incorporation on tyrosine residues; this was specific for a 55-kDa polypeptide. Comparison with molecular data suggests that at least alpha(1)- and alpha(2)-subunits (Ser and Thr residues) and possibly (2)-subunits (Tyr residue) are endogenously phosphorylated by multiple kinases, with a clear preference for alpha(1)-subunit. The beta-subunits were not phosphorylated in our experimental conditions. The corresponding kinase activities are closely associated to the receptor protein, indicating a new complexity in the regulation of the GABA(A) receptor.


INTRODUCTION

-Aminobutyric acid type A (GABA(A)) (^1)receptors are ligand-gated anion channels that mediate most inhibitory synaptic transmission in the central nervous system. Molecular studies have identified five distinct subunits with, for most of them, multiple subtypes; 17 genes have been characterized so far(1, 2) . Functional studies have used heterologous expression of several subunits to produce functional GABA(A) receptors (3, 4) . At the native protein level, ligand binding heterogeneity of the receptor was demonstrated by photoaffinity labeling and autoradiography(5, 6) .

Phosphorylation is a common mechanism for the regulation of receptor function. All subunit subtypes of the GABA(A) receptors contain some consensus substrate sequences for kinases such as cAMP-dependent protein kinase, protein-tyrosine kinase, and/or Ca-phospholipid-dependent protein kinase C(7) . Moreover, type 2 calcium/calmodulin-dependent protein kinase and cGMP-dependent protein kinase phosphorylate the intracellular domains of (2)L and (2)S fusion proteins(8) .

Using purified GABA(A) receptor, Browning et al.(9) reported that beta-subunits could be phosphorylated in the presence of exogenously added cAMP-dependent protein kinase and Ca-phospholipid-dependent protein kinase C. Ca-phospholipid-dependent protein kinase C and cAMP-dependent protein kinase phosphorylation have been reported to modify the amplitude of GABA-activated currents (4, 10, 11, 12, 13) .

In acutely dissociated neurons, it has been reported that favorable conditions for phosphorylation were required to prevent ``run-down'' of GABA(A) currents(14, 15, 16) . The authors excluded the possibility that Ca-phospholipid-dependent protein kinase C, cAMP-dependent protein kinase, or type 2 calcium/calmodulin-dependent protein kinase could be operative; they proposed that the maintenance of normal GABA currents required the activity of a unique kinase specific for the GABA(A) receptor.

Although many molecular and biochemical analyses have shown that beta1- (4) , beta3-(17) , and 2-subunits (18) of GABA(A) receptors are good substrates for several exogenously added kinases, there is little information on possible endogenous phosphorylation by kinase activities associated to the receptor. In GABA(A) receptor purified from rat cerebellum, it has been reported that an alpha-subunit could be phosphorylated by a receptor-associated protein kinase(19) . In the present study, we describe multiple endogenous kinase activities that co-purify with the GABA(A) receptor protein purified from cerebral cortex.


EXPERIMENTAL PROCEDURES

Materials

Radioligands were purchased from DuPont NEN Chemicals GmbH (Dreieich, Germany): [^3H]muscimol, 16 Ci/mmol; [^3H]flunitrazepam, 87 and 103.1 Ci/mmol; and [-P]ATP, 30 Ci/mmol. Hionic Fluor scintillation mixture, toluene, and soluene were from Packard (Groningen, The Netherlands); acrylamide, SDS, glycine, and the molecular weight electrophoresis calibration kit were from Pharmacia LKB (Uppsala, Sweden). N,N,N`,N`-Tetramethylethylenediamine was from Serva (Heidelberg, Germany); Coomassie Brilliant Blue R-250 was from Fluka Chemie (Buchs, Switzerland); hydrogen peroxide was from Carlo Erba (Milan, Italy); anti-GABA(A) receptor, alpha-chain (clone bd 24) monoclonal antibody, anti-mouse Ig polyclonal antibody (fluorescein labeled), and the nonradioactive tyrosine kinase assay kit were from Boehringer Mannheim. Flurazepam, diazepam, and GABA were obtained from Sigma. Other chemicals and solvents of analytical grade were obtained from E. Merck (Darmstadt, Germany).

Preparation of Membranes

Fresh cow brains were obtained from a local slaughter house. White matter was dissected out of brain samples, and the membrane fraction was prepared as described previously(6) .

Purification of the Receptor

Membranes were solubilized in a phosphate buffer (10 mM) containing a final concentration of 1% Triton X-100. The solubilized receptor was applied on an affinity column as described previously (20) . For elution, phosphate buffer was replaced by Tris buffer containing Flurazepam (10 µM) with urea at concentrations ranging from 2 to 6 M. Protein was estimated according to Lowry et al.(21) .

Binding Studies

The methods used have been previously described(6) . Briefly, the GABA(A) site was assayed by measuring the binding of 0.5-40 nM [^3H]muscimol.The benzodiazepine site was assayed by measuring the binding of [^3H]flunitrazepam for 60 min at 4 °C, with 10 µM diazepam for the blank. Activity for both radioactive ligands was determined after polyethylene glycol/bovine -globulin precipitation and centrifugation. The pellets were then dissolved overnight, and radioactivity was counted.

Phosphorylation

Receptor preparations (5-10 µg prot/ml) were incubated in the presence of 0.33 µM [-P]ATP for 30 min at 30 °C. The medium contained Hepes-Tris buffer (50 mM at pH 7.3) and different amounts of divalent cations (see figure legends). In some cases the phosphorylated receptor preparation was precipitated and washed with trichloroacetic acid at a final concentration of 10% before counting. In the other cases, after incubation under the same conditions, the preparations were subjected to SDS-polyacrylamide gel electrophoresis. The resolving gel was a linear gradient of 5-15% acrylamide. Strips of gel were cut in 1-mm slices, dissolved at 60 °C in H(2)O(2) (35% m/v) overnight, and counted(5) .

Photoaffinity Labeling

Photoaffinity labeling was performed as described previously(6) . Purified receptor was incubated with [^3H]flunitrazepam at 4 °C for 60 min. After incubation, samples were submitted to UV (360 nm) irradiation for 20 min, precipitated, and then submitted to SDS-polyacrylamide gel electrophoresis, sliced, and counted.

Immunoprecipitation

The purified receptor (50 µl) was incubated for 2 h at 4 °C with varying dilutions of the monoclonal antibody bd 24 in a final volume of 200 µl in phosphate buffer containing 0.1% Triton X-100. After addition of 200 µl of goat anti-mouse IgG conjugated to agarose with 1% bovine serum albumin, the samples were carefully shaken for 16 h at 4 °C (22) .

Phosphoamino Acid Analysis

Aliquots of receptor were phosphorylated by [-P]ATP (3.3 µM) in the presence of MnCl(2) (2 mM) and/or MgCl(2) (5 mM) and precipitated by the chloroform-methanol method(23) . Chemical digestion was performed in 6 M HCl at 110 °C for 1 h (24) . The phosphoamino acids were concentrated on Dowex 50W-X8 mini-column in a minimum volume of buffer made of formic acid (88%), acetic acid, and deionized water (50:156:1794) at pH 1.9(25) . Samples containing 0.3-0.5 µg of phosphoamino acid standards (Tyr(P), Thr(P), and Ser(P)) were subjected to two-dimensional thin layer electrophoresis(26) . After the run, unlabeled markers were located by spraying with 0.2% ninhydrin in acetone and developed for 15 min at 65 °C. Plates were then autoradiographed.

Tyrosine Kinase Assay Kit

We used the enzyme-linked immunosorbent assay kit developed by Boehringer Mannheim. This includes specific peptide substrates (called protein kinase substrates 1 and 2) that are biotinylated at the amino terminus. After the reaction, phosphorylated and dephosphorylated substrates were immobilized by binding to a streptavidin-coated microtiter plate (96 wells). The fraction of phosphorylated substrate was determined using an anti-phosphotyrosine antibody directly conjugated to peroxidase.

Western Blotting

Purified GABA(A) receptor preparation was transferred from polyacrylamide gels to polyvinylidene difluoride membranes as described by Towbin et al.(27) . After incubation with blocking reagent, the preparation was incubated overnight with 0.4 µg anti-phosphotyrosine antibody/milliliter of Tris buffer, pH 7.2, at 4 °C. Immunostaining was performed using 0.25 units of anti-mouse Ig-AP/ml buffer for 1 h at room temperature. Color reaction was obtained by incubating the samples for 20 min with 0.3 mM 4-nitroblue tetrazolium chloride/0.7 mM 5-bromo-4-chloro-3-indolyl phosphate in 0.1 M Tris chloride buffer, pH 7.2.


RESULTS

Purification of the GABA(A)Receptor

The technique previously described (20, 22) was modified in order to optimalize the yield of endogenous phosphorylation. Phosphate buffer was found more suitable than Tris buffer. Even though the solubilization yield was the same as with Tris buffer, lower concentrations of the detergent Triton X-100 could be used with phosphate buffer(28) . Higher concentrations of Triton X-100 (5%) decreased the incorporation of phosphate compared with lower concentrations (1%) (data not shown). The optimal buffer conditions for solubilization were found to be phosphate (10 mM, pH 7.4), containing 1% Triton X-100. During the affinity chromatography, the duration of exposure of the solubilized receptor to urea had to be minimized. Moreover, the highest yield of phosphate incorporation was obtained after elution with 4 M rather than 6 M urea. Extensive dialysis (48 h) was required to eliminate the urea before the assay. Finally, it was found that freezing and thawing of the purified receptor also strongly and irreversibly inhibited the endogenous kinase activities.

Stoichiometry of Endogenous Phosphorylation

The purified receptor was incubated at 30 °C in the presence of [-P]ATP and divalent cations Mg or Mn (Fig. 1). No significant level of P-incorporation was obtained in the absence of divalent cations. In a typical experiment with an incubation time of 120 min, we found an incorporation of 0.12 mol of PO(4)/mol of muscimol in the presence of Mg 5 mM and of 0.45 mol of PO(4)/mol of muscimol in the presence of Mn 500 µM. With higher concentrations of Mn (2-5 mM), the stoichiometry rose to 3 mol of PO(4)/mol of muscimol (data not shown).


Figure 1: Effect of incubation time on endogenous phosphorylation of the GABA(A) receptor. Stoichiometry was calculated as number of moles of phosphate incorporated per mole of muscimol bound in the presence of 5 mM Mg (squares) or 500 µM Mn (circles). The maximal incorporation (120 min) of phosphate in the presence of Mg and Mn was of 0.12 and 0.45 mol of P/mol of [^3H]muscimol respectively.



Electrophoretic Pattern

In the presence of 5 mM Mg, we detected a major incorporation in the molecular mass range 51-53 kDa and a minor incorporation at 43-45 kDa (Fig. 2). The doublet at 51-53 kDa corresponds to alpha-subunits; the 51-kDa band has been identified as the alpha(1)-subunit(29) . The 43-45-kDa polypeptides have been shown to be proteolytic products of major GABA(A) receptor subunits(6, 29) . We photolabeled the 51-53-kDa bands with [^3H]flunitrazepam (2 nM). Both P- and [^3H]flunitrazepam-labeled samples were subjected to the same run of SDS-polyacrylamide gel electrophoresis. We observed a co-migration of both P- and [^3H]flunitrazepam-labeled polypeptides at 51 and 53 kDa. This suggests that the endogenous phosphorylation predominates on 51-53 kDa alpha-subunits and particularly on the alpha(1)-subunit. There is no direct correlation between the amount of incorporated P and the relative protein abundance, confirming a clear preference of the endogenous phosphorylation for the 51-kDa polypeptide (Fig. 2, upper part). We also observed both [^3H]flunitrazepam labeling and P-incorporation on the 43-45 kDa proteolytic products of alpha-subunits. Minor incorporation was detected at molecular masses ranging from 55 to 58 and from 38 to 40 kDa. No incorporation of P was observed in other major subunits. Practically no radioactivity was associated with beta-subunits. Other proteins that co-purified with the receptor, notably the heavily stained 36-kDa voltage-dependent anion channel protein (22) , were not phosphorylated.


Figure 2: Co-electrophoresis in SDS of phosphorylated and flunitrazepam photoaffinity-labeled polypeptides of purified GABA(A) receptor samples. Molecular mass of standards are indicated in A. The main bands of the receptor (51, 53, 55, and 58 kDa) and the co-purified B-36 voltage-dependent anion channel (36 kDa) are indicated in B. The GABA(A) receptor was either phosphorylated without added kinases (squares) or photoaffinity labeled with [^3H]flunitrazepam (circles). Abscissa values represent slice number; ordinate values represent cpm of P (left) and [^3H]flunitrazepam (right). Arrows indicate the molecular mass of labeled peaks calculated from standards; they correspond to the 51- and 53-kDa polypeptides and to their degradation products (45 kDa)(29) .



A Monoclonal Antibody against alpha-Subunit Adsorbs theP-labeled GABA(A)Receptor Protein

In order to make sure that the P labeling did occur on GABA(A) receptor protein rather than on possible contaminants, we attempted to immunoprecipitate the purified receptor protein using an alpha-specific monoclonal antibody named bd 24(30) . Dilutions of 1:50 to 1:50000 of the antibody were incubated with the P-labeled purified protein and precipitated. Fig. 3shows that there is a linear relationship between the number of moles of bound muscimol and phosphate that immunoprecipitates with varying dilutions of the antibody. The slope gives a stoichiometry of 0.83 mol of phosphate/mol of muscimol sites. In parallel experiments, bd 24 immunoprecipitated 90% of [^3H]muscimol binding, which is in good agreement with the results of Häring et al.(30) . These data suggest that most if not all the incorporation of P is on the GABA(A) receptor protein.


Figure 3: Immunoprecipitation of the phosphorylated GABA(A) receptor by alpha-subunit-specific monoclonal antibody (bd 24). Affinity column-purified receptor was incubated with various dilutions of bd 24. After precipitation with anti-mouse IgG coupled to agarose, the pellets were assayed for both [^3H]muscimol binding (triangles) and P incorporation (squares). A nonimmune serum from rabbit was used as control. Nonspecific precipitation of activity did not vary significantly with the dilution of control serum. The inset represents a linear relationship (r^2 = 0.870) between the number of moles of muscimol (abscissa) and phosphate (ordinate) present in the pellets with different antibody dilutions. The slope gives a stoichiometry of 0.83 mol of phosphate/mol of muscimol.



Phosphorylation of the 51-kDa Polypeptide: Kinetic Properties

Fig. 4A shows the effect of ATP concentration on phosphorylation. The curve obtained was sigmoid with an estimated Hill coefficient of 2.0. The activity had a broad pH optimum around 8.0 (Fig. 4B). As shown in Fig. 4C, the activity dropped by 50% between 30 and 37 °C. This suggests that only half of the activity is thermolabile below 45 °C.


Figure 4: Effect of ATP concentration, pH, and temperature on phosphorylation of the 51-kDa polypeptide. A, phosphate incorporation was plotted as a function of ATP concentration in the presence of 5 mM Mg. Experimental conditions were as described in the legend to Fig. 1. The ATP concentration corresponding to 50% V(max) was 1.7 µM. B, incorporation of phosphate as a function of pH. C, incorporation of phosphate as a function of temperature.



Phosphoamino Acid Analyses

In the data shown in Fig. 5, the entire purified preparation was labeled by [-P]ATP in the presence of Mg alone (Fig. 5A) or of both Mg and Mn and subjected to two-dimensional phosphoamino acid thin layer electrophoresis (Fig. 5B). The presence of Mn ions strongly increased the amount of P-incorporation for both serine and threonine. Roughly the same amounts of radioactivity were visualized in autoradiograms for serine and threonine residues in the presence or absence of Mn. It has to be kept in mind that very small amounts of phosphoamino acids could not be detected by this procedure.


Figure 5: Phosphoamino acid analysis of phosphorylated GABA(A) receptor. Affinity-purified receptor was phoshorylated either in the presence of 5 mM Mg alone (A) or in the presence of both Mg (5 mM) and Mn (2 mM) (B). The preparation was hydrolyzed with HCl 6 M. The products were then separated by two-dimensional thin layer electrophoresis followed by autoradiography.



There is a single consensus sequence for tyrosine kinase in the (2)-subunit (31) that contrasts with the numerous consensus sequences for serine and threonine phosphorylation sites(4) . We therefore used more sensitive and specific immunochemical methods that can detect very low levels of phosphorylated tyrosine residues. For these experiments we used a highly specific anti-phosphotyrosine antibody. The results are presented in Fig. 6. Aliquots of purified receptor were phosphorylated with 50 µM unlabeled ATP. By enzyme-linked immunosorbent assay (Fig. 6A), we observed a positive reaction only when Mn (2 mM) was present in the incubation medium. In the presence of 5 mM Mg alone (i.e. without Mn), no phosphotyrosine could be detected by this method. In order to estimate the molecular mass of the polypeptide that is phosphorylated on tyrosine, we used the same antibody for Western blot analysis. Under the same conditions as for enzyme-linked immunosorbent assay, a single labeled band was recognized at an apparent molecular mass of 55 kDa (Fig. 6B). The intensity of labeling increased with the concentration of the antibody. No significant cross-labeling was observed, even at the highest antibody concentration. This apparent molecular mass is close to the mass of 55.2 kDa deduced from the bovine (2)-subunit sequence(31) . Interestingly, the antibody stained neither the 51-53-kDa polypeptides nor any other part of the blot.


Figure 6: Detection of a tyrosine protein kinase activity in purified GABA(A) receptor by both enzyme-linked immunosorbent assay and Western blotting. A, we used a nonradioactive tyrosine kinase assay kit from Boehringer Mannheim. This kit uses specific peptide substrates that are biotinylated at the amino terminus. The fraction of phosphorylated substrate is determined immunochemically via a highly specific anti-phosphotyrosine antibody directly conjugated to peroxidase. The use of this kit also allows determination of the rate of dephosphorylation (phosphatase activity) of the peptide substrate. The detection is shown on the purified receptor either in the presence of Mg alone (columns 1-3) or in the presence of Mn (columns 6-8). Columns 1 and 6, with ATP; columns 2 and 7, without ATP; columns 3 and 8, phosphatase assay; lane e, blank and autophosphorylation controls; lane d, standard curve; lanes a, b, and c, biotinylated peptide protein kinase substrate 2; lanes f, g, and h, biotinylated peptide protein kinase substrate 1. This experiment indicates a tyrosine kinase activity only in the presence of Mn for both peptide substrates. B, an anti-phosphotyrosine monoclonal antibody was used for Western blotting on purified receptor. Molecular mass standards are indicated in lane a (97.4, 66.0, 45.0, and 31.0 kDa). One band only was observed at 55 kDa in the presence of 50 µM ATP and 100 µM Mn. The antibody was used at different dilutions: 0.01 µg (lane b), 0.1 µg/ml (lane c), and 1.0 µg/ml (lane d).




DISCUSSION

GABA(A) receptor function is regulated by phosphorylation(4, 13, 14, 15, 16, 32) . The receptor protein contains consensus sequences for phosphorylation by protein kinases(7) . In the present work, we describe kinase activities associated to the affinity-purified GABA(A) receptor from cerebral cortex. In order to characterize endogenous activities, the purification procedure had to be improved. Indeed, we observed that the use of high detergent and urea concentrations during the purification procedure drastically decreased the rate of phosphorylation. Moreover, freezing the purified protein nearly completely abolished this activity. In contrast, phosphate buffer seems to have a protective effect during the solubilization of the protein. These technical differences might explain why a significant level of endogenous phosphorylation was not previously detected(9) .

We observed a major phosphorylation in the 51-53-kDa bands. Maximum incorporation of P was in the 51-kDa polypeptide. We also detected a minor incorporation of P in a band of higher molecular mass (55 kDa). When we studied the effect of ATP concentration on 51-kDa polypeptide phosphorylation, we obtained a sigmoid curve. This suggests that the endogenous protein kinase has allosteric properties with at least two cooperative binding sites for ATP. Similar effects were reported for the insulin-stimulated autophosphorylation of the insulin receptor (33) .

Immunoprecipitation of the phosphorylated receptor was obtained with a monoclonal antibody specific for alpha-subunits, confirming that phosphorylation occurs on these subunits. The 51- and 53-kDa bands co-migrated with two bands photoaffinity-labeled by [^3H]flunitrazepam. These two bands appear identical to those previously identified by partial sequencing and Western blotting as corresponding to the alpha(1) and alpha(2) gene products(29) . However, data currently available are not yet sufficient to provide definitive proof of their identity. In an earlier study, Sweetnam et al.(19) , using a partially purified GABA(A) receptor preparation from rat cerebellum, suggested that an alpha-subunit was the preferred substrate for endogenous phosphorylation (which was observed only on serine). These results did not rule out the possibility that endogenous phosphorylation might also occur on other types of subunits. Indeed, it was shown later (34, 35) that alpha(1)-, alpha(6)-, beta(2)-, beta(3)-, (2)-, and -subunits are expressed in cerebellum but the other subunit subtypes are not. The absence of expression of alpha(2) is in agreement with the lack of 53-kDa polypeptide in cerebellum(5, 6) . Our preparation, purified from bovine cortex, contains alpha(2)- and beta(1)-subunits, and endogenous phosphorylation was observed in alpha(2)- but not in beta-type subunits. In contrast, phosphorylation by cAMP-dependent protein kinase and Ca-phospholipid-dependent protein kinase C never occurs on alpha-subunits but only on beta-subunits(9, 36) . The nature of alpha-subunit subtypes affects both the sensitivity of the receptor to GABA and the affinity of different ligands for the benzodiazepine binding site(5, 6) . It is thus reasonable to propose that the alpha-subunits can regulate the channel conductance in two different ways: the binding of agonist ligands to the NH(2)-terminal extracellular domain (37) and the endogenous phosphorylation in the long intracellular loop. The physiological consequences of phosphorylation on beta-subunits by exogenous kinases are different from those proposed for endogenous phosphorylation(4) .

We measured a roughly equimolar P-incorporation in Thr and Ser residues in alpha-subunits, whereas a previous study (19) suggested an endogenous phosphorylation of alpha-subunit only on serine. A possible explanation for this apparent discrepancy is that the latter preparation (19) was from cerebellum. The additional phosphorylation on Thr residue might reflect phosphorylation on other alpha-subunits (alpha) expressed in the neocortex(38) .

When our preparation was incubated with ATP + Mn, we also detected the presence of phosphotyrosine on a single 55-kDa polypeptide. The identification of the band(s) labeled at 55 kDa is made difficult by the presence of several alpha-, -, and beta-subunit subtypes sharing similar molecular mass(1) . This incorporation may occur at least partially on the (2)-subunit as in the bovine brain, this subunit is the only one containing a unique consensus sequence for tyrosine phosphorylation (located on Tyr(31) ). The apparent molecular mass has been estimated at 55 kDa with a highly specific phosphotyrosine antibody. This corresponds to the deduced mass from the (2) gene. However, the apparent molecular mass of the (2) gene product is still uncertain due to post-transcriptional events; specific antibodies also recognized a broad band at 41-47 kDa(39, 40, 41) . Thus, the identification of the 55-kDa polypeptide with the (2)-subunit of the GABA(A) receptor is possible but not clearly demonstrated.

The parallels between the conditions reported previously and the present study are worth noticing. It has been suggested that GABA(A) receptor current is maintained by phosphorylation involving an unknown kinase and an unknown substrate (14) . Our results bring new insights on the complexity of the alpha- and possibly -subunit pharmacology and indicate that the nature of the subunit subtypes composing the GABA(A) receptor may also directly affect the properties of the GABA-activated chloride channel. The present work suggests an additional heterogeneity of receptor function due to modulation by multiple endogenous phosphorylations.


FOOTNOTES

*
This work was supported by Grants 1.5.082.93 (to M. H. B.) and 1.5.087.93 (to J. J. L.) from the Belgian National Funds for Scientific Research, by a grant from the Léon Fredericq Foundation (to J. J. L.), by grants from the Special Funds for Research of the University of Liège, and by Grant 3.45.11.90 from the Belgian Funds for Scientific Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Rennes I, 2, Av. Pr. Léon Bernard, F-35043 Rennes Cedex France. Tel.: 33-99336940; Fax: 33-99336208.

(^1)
The abbreviation used is: GABA(A), -aminobutyric acid type A receptor.


ACKNOWLEDGEMENTS

We thank Arlette Minet for technical assistance and Pierre Wins for critically reading the manuscript and for helpful discussion.


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