INTRODUCTION

-Aminobutyric acid type A (GABA_A)¹ receptors are the major transducers of fast synaptic inhibition in the central nervous system and represent sites of action for anxiolytic and hypnotic drugs including benzodiazepines and barbiturates (1). There are four classes of GABA_AR subunits (, , , and ), and three of these have multiple members (1-6, 1-4, 1-4) (2, 3). Although the exact composition and stoichiometry of GABA_ARs is still uncertain, it is believed that the major form of the native protein is composed of , , and  subunits (4, 5) arranged in a pentamer that encloses a central chloride channel (6).

Prolonged occupancy of GABA_ARs by agonists evokes a series of regulatory mechanisms that control the function, subcellular distribution, and number of receptors. Such use-dependent mechanisms can be partially distinguished by their temporal order, a paradigm used to describe down-regulation of G-protein-linked receptors (7). For GABA_ARs, the most rapid of these events is desensitization, which is characterized by a decline in the frequency of channel openings occurring within a few s after GABA application (8). The kinetics of densensitization in isolated membrane patches show properties that are probably intrinsic to GABA_ARs rather than a result of extrinsic modification. Within 1-2 h of exposure of neurons to GABA or benzodiazepines, [³H]flunitrazepam binding sites (9) and GABA_AR ¹²⁵I-polypeptides (10) are sequestered from the cell surface. -carboline-evoked internalization of GABA_AR binding has also been described (11). The pathway for use-dependent GABA_AR receptor sequestration is not yet defined, but clathrin-coated vesicles have been implicated as a vehicle (12, 13). Following agonist-dependent internalization, GABA_AR polypeptides appear to be rapidly degraded (10). Much of the interest in these and related processes is due to a possible role in the development of tolerance and habituation to benzodiazepines and GABA mimetic drugs, a major problem in clinical applications (5, 13).

It is well established that chronic (several days) exposure of primary neuronal cultures to GABA reduces the density of high affinity binding sites for GABA_AR ligands (14-18) and the magnitude of GABA-gated Cl currents (14, 16), a process referred to as down-regulation. In this article we continue to use the term "down-regulation" to refer specifically to the agonist-evoked decline in the number of assembled plasma membrane receptors. Down-regulation is accompanied by reductions in GABA_AR ¹²⁵I-polypeptides (10) and in -subunit immunoreactivity (19, 20). Two distinct mechanisms have been proposed for this use-dependent GABA_AR down-regulation: (i) repression of GABA_AR subunit biosynthesis (21) and (ii) degradation of GABA_AR subunits initiated by endocytosis (10). While there is general agreement that expression of GABA_AR subunit mRNAs declines as a result of GABA exposure (19, 21-23), the relationship of this repression to receptor down-regulation is unclear. Although Montpied et al. (21) reported that GABA_AR 1- and 2-subunit transcripts were reduced coordinately with [³H]flunitrazepam binding, Baumgartner et al. (22) found that the decline in [³H]flunitrazepam binding preceded that for the 1 subunit mRNA. To examine these issues further, we have employed [³⁵S]Met/Cys labeling to measure the synthesis and degradation of nascent GABA_AR 1 subunit polypeptides in chick cortical neurons. The results are in accord with previous studies of the GABA_A receptor 1 subunit transcript in these cells (22), showing that repression of 1 subunit biosynthesis became detectable long after the GABA-evoked loss of the polypeptide.

EXPERIMENTAL PROCEDURES

A fusion protein containing a region of the chick GABA_A receptor 1 subunit, corresponding to amino acid residues 331-381 (24), was cloned in pRSET-B, expressed in Escherichia coli JM109, and purified by Ni²⁺ affinity chromatography according to the general procedures of Kroll et al. (25). This protein was used for rabbit immunizations, and the resulting RP4 antiserum was characterized by immunoprecipitation and Western blot analysis. Details of these procedures and the antibody characterization appear elsewhere (26, 27). A crude IgG fraction, prepared by elution of the antiserum from CM Affi-Gel Blue (Bio-Rad), was absorbed on Affi-Gel 10 (Bio-Rad) derivatized with the 1-(331-381) fusion protein. The antibody was eluted with 4 M MgCl₂ (pH 6.4), dialyzed against TBS (20 mM Tris-Cl, 150 mM NaCl, pH 7.4), and concentrated.

The procedures of Tehrani and Barnes (28) were used for extraction and immunoprecipitation of central [³H]flunitrazepam binding sites from chick cortical neurons. Western blotting was performed according to Calkin and Barnes (10) using 1 µg/ml purified RP4 antibody or a 1:100 dilution of RP4 antiserum.

Primary cultures of embryonic chick cortical neurons were prepared according to Thampy et al. (29). Where indicated, GABA or other test compounds were added 3 h after plating the cells. Neurons cultured for 4-7 days in 60-mm Petri dishes were washed and incubated for 15 min at 37 °C in Dulbecco's modified Eagle's medium lacking Met and Cys. The cells were pulsed by the addition of fresh medium containing 200 µCi/ml [³⁵S]Met/Cys (Translabel, NEN Life Science Products) and GABA_A receptor ligands (where indicated) and returned to the incubator for 2 h. For chase experiments, the cells were washed three times with Dulbecco's modified Eagle's medium containing 2 mg/ml each of Met and Cys and then incubated in conditioned medium containing 1 mM Met, 1 mM Cys, and 200 µM GABA (where indicated) for 3-48 h. Incubations were terminated by washing the cells with ice-cold PBS (137 mM NaCl, 2.68 mM KCl, 0.49 mM MgCl₂, 0.9 mM CaCl₂, 1.47 mM KH₂PO₄, 8.1 mM Na₂HPO₄, pH 7.4) containing 1 mM Met and 1 mM Cys. The cells were collected and homogenized in extraction buffer (2 mM EDTA, 2 mM EGTA, 1 mM Met, 1 mM Cys, 2 mM benzamidine, 0.1 mg/ml bacitracin, 0.1 mg/ml each of trypsin inhibitors II-O and II-S, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml phosphoramidon in TBS). The homogenates were centrifuged for 1 h at 100,000 × g, and the pellets were resuspended in extraction buffer containing 1% Triton X-100 and 0.1% SDS. After 30 min on ice, the extracts were clarified by centrifugation at 100,000 × g for 1 h and precleared twice with Staphylococcus aureus cells, and aliquots were withdrawn for determination of total ³⁵S incorporation by precipitation with 10% trichloroacetic acid. The trichloroacetic acid precipitates were washed with 70% ethanol and then washed with ether and counted. The remainder of the extract was used for double immunoprecipitation as described by Firestone and Winguth (30). Both immunoprecipitations utilized overnight incubations with 1 µg of affinity-purified RP4 antibody. The precipitates were analyzed on 10% polyacrylamide-SDS gels. The gels were prepared for autoradiography as described by Skinner and Griswold (31).

Autoradiographic densities from the pulse-chase and immunoblotting experiments were determined by using a laser scanner and Scan Analysis software (Biosoft, Ferguson, MO) following the procedures described by Kendrick et al. (32). The linear range of analysis for both types of experiments was determined by application of a series of sample dilutions to the gel. Linear regression analysis of the resulting calibration curves gave correlation coefficients >0.990, and all densities were quantified within this linear range of analysis. Student's two-tailed t test was used for statistical evaluation of the data.

RESULTS

Immunoblotting and Immunoprecipitation of GABA_A Receptor 1 Subunits

For identification and quantitation of GABA_A receptor 1 subunits from chick cortical neurons in culture, we utilized an affinity-purified polyclonal antibody (RP4) directed against an 1-(331-381) fusion protein. On Western blots (Fig. 1), this antibody reacted with a single 51-kDa neuronal polypeptide, the mass expected for the GABA_AR 1 subunit (33, 34). After preabsorption with the 1-(331-381) fusion protein, antibody RP4 did not give detectable labeling of the blots. Cross-reactivity with proteins in the 53-66-kDa range expected for GABA_AR 2-6 subunits (33) was not observed. Treatment of the neuronal membranes with endoglycosidase H caused a shift in the apparent mass of the immunoreactive polypeptide from 51 to 48 kDa (not shown), the latter corresponding to the value found for the unglycosylated GABA_AR 1 subunit (35). Additional experiments (not shown) established that 58 ± 3% of the [³H]flunitrazepam binding sites extracted from 100 µg of neuronal membrane protein were precipitated at a saturating level (1 µg) of RP4 antibody. Similar values were obtained with membrane extracts from chick cerebral cortex.

To estimate the molar quantities of the GABA_AR 1 subunit produced by the neurons under these conditions, we utilized 10-50 fmol of the 1-(331-381) fusion protein as a standard for quantitative Western blotting (Fig. 2). These calibration curves were then used for the analysis of densities for the native 1 subunit obtained from immunoblots similar to those in Fig. 1. When applied to gels along with the standards, membrane proteins (10-50 µg) from 4-day cultures gave signals for the native 51-kDa 1 subunit (not shown) that were within the linear range of densitometric analysis as illustrated in Fig. 2. By this approach we obtained for the neuronal 1 subunit a value of 0.65 ± 0.11 pmol/mg of membrane protein (mean ± S.E., n = 3 culture preparations, each analyzed on a separate blot).

Quantitative immunoblotting of GABA_AR 1-(331-381) fusion protein.

Use-dependent Decline in GABA_A Receptor 1 Subunit Immunoreactivity

Quantitative Western blotting was also used to examine the modulation of GABA_AR 1 subunit polypeptides during the exposure of neuronal cultures to receptor agonists. GABA (200 µM) was added 3 h after plating neurons, and the cells were cultured for 4 days. With the chick cortical preparation, similar procedures were employed previously to examine the use-dependent decline of GABA_AR ligand binding sites, ¹²⁵I-polypeptides, and subunit mRNAs (10, 14, 22). The concentration of GABA (200 µM) used in these experiments was also shown to be nearly saturating for down-regulation (14). Under these conditions, chronic exposure to GABA or to isoguvacine, a GABA_A-specific agonist, produced a decline in the level of the 51-kDa immunoreactive polypeptide (Fig. 3). Densitometric analysis of a series of such experiments revealed an attenuation of 27-32% in the presence of either ligand. These effects of GABA and isoguvacine were statistically significant (p < 0.02). Since isoguvacine is not a substrate for GABA transport or GABA transaminase, the data indicate that neuronal uptake or degradation does not limit the effectiveness of GABA in these experiments. The decline in 1 subunits produced by GABA was blocked by the addition of the GABA_A-specific antagonist R5135 (3-hydroxy-16-imino-5-17-aza-androstan-11-one), indicating the necessity of receptor occupancy. R5135 was shown previously to block the GABA-evoked sequestration and down-regulation of GABA_A receptors (10).

Repression of GABA_A Receptor 1 Subunit Biosynthesis

To measure the relative rates of GABA_AR 1 subunit biosynthesis, the neurons were pulsed for 2 h in culture with ³⁵S-Met/Cys, and extracts were prepared from 100,000 × g pellets of crude membranes. The extracts were analyzed by double immunoprecipitation with affinity-purified RP4 antibody and separation of the precipitates on SDS gels. As shown in Fig. 4, a major 51-kDa ³⁵S-polypeptide and a minor labeled protein at 56 kDa was obtained. Preabsorption of the RP4 antibody with 1-(331-381) fusion protein eliminated the 51-kDa band, while the 56-kDa peptide remained. On the other hand, pretreatment of the antibody with a GABA_AR 1-(2-202) fusion protein had little effect. The amount of the 56-kDa product in the immunoprecipitates was also much more variable than that of the 51-kDa polypeptide. This identifies the 51-kDa ³⁵S-polypeptide as the GABA_AR 1 subunit and the 56-kDa band as contaminant. It is also worth noting that coprecipitation of other GABA_AR ³⁵S-subunits, which presumably could have assembled with 1 subunits, would not be favorable using the double immunoprecipitation technique. Additional studies (Fig. 5) demonstrated that the amount of ³⁵S incorporation into the 1 subunit during a 2-h pulse was in the linear time range for labeling and that pulses longer than 2 h departed from linearity. Using [³H]flunitrazepam binding, it was established that a saturating amount of antibody RP4 was present in all immunoprecipitations.

GABA_AR 1 subunit synthesis in neurons after a 7-day exposure to GABA_A ligands.

Time course for ³⁵S incorporation into GABA_AR 1 subunits.

To significantly repress de novo synthesis of GABA_AR 1 subunits, it was necessary to expose the neurons to GABA or isoguvacine for a 7-day culture period prior to ³⁵S labeling (Fig. 4). The GABA-evoked repression observed under these conditions was prevented by R5135. Quantitation of autoradiographs from three such experiments revealed that both agonists produced a decline of 33% in the rate of 1 subunit expression (Fig. 4), and the effects of both were statistically significant (p < 0.025). The S.E. in these ³⁵S incorporation experiments was typically ±6%. These agonist treatments did not produce a detectable change in the rate of ³⁵S incorporation into the general pool of membrane proteins (not shown). It is noteworthy that, as illustrated in Fig. 6, a 4-day exposure of the cells to GABA or isoguvacine failed to significantly repress de novo synthesis of the 1 polypeptide. Although the S.E. values in Fig. 6 were similar to those in Fig. 4, p values of 0.574 and 0.548 were obtained for the levels of ³⁵S-1 polypeptides found in cells cultured for 4 days with GABA or isoguvacine, respectively, relative to the control (Fig. 6). Likewise, when neurons were cultured for 4 or 7 days in the absence of agonist, the presence of GABA during a 2-h pulse did not alter the level of ³⁵S incorporation into this subunit (not shown). This indicates the lack of an acute effect of GABA that could have faded during the chronic exposures.

GABA_AR 1 subunit synthesis in neurons after a 4-day exposure to GABA_A ligands.

Turnover of GABA_A Receptor 1 Subunits

To determine the rate of GABA_AR 1 subunit degradation, neurons from 4-day cultures were pulsed for 2 h with [³⁵S]Met/Cys and then chased in conditioned media for 3-48 h. The level of the ³⁵S-1 polypeptide declined continuously during the first 24-h period and was barely detectable thereafter (Fig. 7). Densitometric analysis of three such experiments yielded a t1/2 = 7.7 ± 0.8 h for the 1 subunit. The general pool of neuronal membrane ³⁵S-proteins was much more stable, declining with an apparent t1/2 > 24 h. Since previous studies suggested a GABA-evoked degradation of GABA_A receptor ¹²⁵I-polypeptides derived from the cell surface (10), the effect of adding GABA to the chase medium was examined. However, the presence of GABA during the chase period had no discernible effect on the rate of degradation of either the GABA_AR ³⁵S-1 subunit or the membrane protein pool.

Pulse-chase analysis of GABA_AR 1 polypeptide degradation.

DISCUSSION

Use-dependent Decline of GABA_A Receptor 1 Subunit Immunoreactivity

The 1 subunit is a major component of GABA_ARs in most brain regions and an important determinant for receptor pharmacology. It also forms part of the high affinity site for benzodiazepine binding (36, 37). Although there have been multiple reports of a decline in GABA_AR 1 subunit mRNA levels that are evoked by exposure of neurons to GABA agonists (21-23), there is little related information about the corresponding polypeptide. Although Calkin and Barnes (10) found that chronic exposure of chick cortical neurons to GABA agonists produced a decline in GABA_AR ¹²⁵I-polypeptides, the 1 subunit was only tentatively identified as part of this labile pool. In the present paper, we show that a 27-32% decline of GABA_AR 1 subunit immunoreactivity was produced by 4-day agonist treatments. This reduction is somewhat less than that found for [³H]flunitrazepam binding (31-50%) under comparable conditions (10, 14). However, at saturation with the 1 subunit antibody, 58 ± 3% of the ]³H[flunitrazepam binding sites were precipitated, suggesting that only this fraction of the binding sites contains 1 subunits. The GABA-evoked decline in 1 subunits was prevented by R5135, a GABA antagonist shown to be effective against various subtypes of native GABA_A receptors (38). Alone, R5135 had little effect, showing that endogenous GABA makes little contribution in these experiments. Isoguvacine, a GABA_A-specific agonist that is not subject to degradation or uptake by neurons, produced a decrease of 1 subunit immunoreactivity that was equivalent to that produced by GABA. Thus, the first conclusion to be drawn is that occupancy of GABA_A receptors produces a decline in the level of 1 subunits that is sufficient to account for receptor down-regulation.

Repression of GABA_A Receptor 1 Subunit Biosynthesis

To examine the mechanism of the use-dependent decline in 1 subunits, we measured rates of [³⁵S]Met/Cys incorporation by immunoprecipitation. Since these experiments were conducted with ³⁵S pulses whose duration was within the linear range of incorporation (Fig. 5), the results may be taken to reflect the initial rates of 1 subunit synthesis de novo. To our knowledge, these are the first such measurements for any GABA_AR subunit. Neither acute treatment of the cells with GABA during the 2-h ³⁵S pulse nor prior chronic treatment for 4 days had a detectable effect on 1 subunit synthesis. However, after 7 days of exposure to GABA or isoguvacine, a significant decline (33%) in the rate of 1 subunit translation was observed. This use-dependent repression was also prevented by R5135. These findings are in good agreement with previous measurements of GABA_AR 1 subunit mRNA levels under comparable conditions in this preparation (22). GABA administration to the neurons for 7 days produced a significant reduction (47 ± 8%) in the level of 1 subunit mRNA, while a 4-day treatment had no effect. That the 1 subunit mRNA represents a sensitive indicator for repression is indicated by an equivalent agonist-dependent decline in GABA_AR 1, 2, 4, and 2 subunit mRNAs (22). Therefore, our current findings support a delayed use-dependent repression of GABA_A receptor subunit biosynthesis. It is apparent that this repression is evoked only after a long delay, long after the neuronal pool of GABA_A receptor 1 subunits and assembled receptors has declined. This notion is also consistent with studies of GABA_AR agonist administration in vivo, which show a loss of receptor binding sites before detectable changes in pools of GABA_AR subunit mRNAs (39, 40). Although it is conceivable that small reductions in GABA_AR 1 subunit biosynthesis occurring within the first 4 days of GABA exposure could have escaped detection (cf. Fig. 6), the decline in the synthetic rate was readily measured after longer treatments (Fig. 4). Collectively, this leads to a second conclusion, that the biosynthesis of GABA_A receptor 1 subunits is subject to use-dependent repression, but this process was detectable only after, rather than before, receptor down-regulation.

Degradation of Nascent GABA_A Receptor 1 Subunits

To examine the turnover of GABA_AR 1 subunits, we utilized an ³⁵S pulse-chase technique. In the chick cortical neuron cultures we found a monophasic decline in the ³⁵S-1 polypeptide with a t1/2 = 7.7 h. Exposure of the neurons to GABA during the 24-h chase period had no discernible effect on the degradation of the ³⁵S-1 subunit. Thus, the third conclusion from our experiments is that nascent GABA_A receptor 1 polypeptides are degraded by an agonist-independent pathway. As discussed below, we believe that this differs from another, previously described pathway for the agonist-dependent degradation of mature GABA_A receptors derived from the cell surface (10).

Although GABA_A receptor ³⁵S-subunit turnover has not been previously measured, the degradation of receptor polypeptides photolabeled with [³H]flunitrazepam has been examined (41). Degradation of the photolabeled 48- and 51-kDa proteins in chick brain neurons was biphasic, showing t1/2 values of 3.8 and 32 h. Why do these values differ from the one obtained from our ³⁵S measurements? We presume that the t1/2 values obtained by Borden et al. (41) reflect properties of assembled receptors because coexpression of GABA_AR  and  subunits is necessary for [³H]flunitrazepam binding (4). Furthermore, GABA_ARs assembled from 1, 2, and 2 subunits are transported to the cell surface, while unassembled 1 subunits are retained in the endoplasmic reticulum (35). It therefore appears likely that the pathways for degradation of unassembled and assembled 1 subunits differ as illustrated in Fig. 8. If the ³⁵S-1 polypeptides from our labeling studies are mostly unassembled, then differences in degradation pathways could account for the discordance of our data with that of Borden et al. (41).

Does the turnover of GABA_AR 1 subunits in our pulse-chase measurements reflect unassembled or assembled subunits? Since our double immunoprecipitation procedure precludes a direct answer to this question, our argument that most of the nascent ³⁵S-1 subunits are unassembled relies on indirect evidence. From the quantitative immunoblotting experiments, we estimate that cortical neurons, 4 days in culture, contain approximately 0.65 ± 0.11 pmol of 1 subunit/mg of membrane protein. Previous determinations of [³H]flunitrazepam binding density in these preparations gave a value of 0.22 ± 0.01 pmol/mg (14, 42). Since 58% of these [³H]flunitrazepam binding sites contain 1 subunits (i.e. immunoprecipitable by RP4 antibody in the present study), the density of receptors containing coassemblies of 1 and  subunits is estimated to be 0.13 pmol/mg. By this approach, the ratio of unassembled to assembled GABA_A receptor 1 subunits is approximately 4:1. This is consistent with quantitative reverse transcriptase-polymerase chain reaction data showing that the level of GABA_AR 1 subunit mRNA greatly exceeds levels of the other subunits in these preparations (22). Thus, our data support the hypothesis that most of the 1 subunits in the cortical neuron cultures are unassembled, although further work is necessary to establish this conclusively. We believe that our chase experiments reflect the degradation of unassembled ³⁵S-1 polypeptides, which would presumably be diluted into a large pool of unlabeled 1 subunits retained in the endoplasmic reticulum.

We have previously shown that an acute (4-h) exposure of cortical neurons to 100 µM GABA in culture produces an increase in the intracellular pool of [³H]flunitrazepam binding sites (9). This phenomenon could have been due to an increase in the synthesis of new receptors or a sequestration of existing receptors. Since the presence of GABA during the ³⁵S pulse failed to influence GABA_AR 1 subunit biosynthesis, our current findings lend support to a mechanism involving agonist-dependent sequestration of binding sites from the surface (9). Further studies by Calkin and Barnes (10) demonstrated that surface GABA_AR ¹²⁵I-polypeptides, including a probable 1 subunit, were sequestered during 2-4 h of GABA exposure. In the absence of GABA, sequestration was not observed. Although small, the sequestered pool of ¹²⁵I-subunits was quite labile, having an approximate t1/2 of 2 h. However, during longer agonist exposures, the number of intracellular receptors stabilized, presumably due to replenishment from the surface, and the surface pool declined. If this decline in surface subunits cannot be accounted for by reductions in de novo synthesis, as our current findings indicate, they must have been degraded (cf. Fig. 8). Thus, the biosynthetic data described here help support the previous hypothesis that GABA_ARs are regulated by use-dependent sequestration and degradation.

If agonist-dependent degradation of surface GABA_A receptors occurs, how could our ³⁵S pulse/chase experiments fail to detect it? Our answer to this paradox is that assembly and transport of the nascent ³⁵S-1 subunits appears to be slow and inefficient. As discussed above, our data suggest that most of the 1 subunits in the neurons remain unassembled. Furthermore, of the newly synthesized/assembled sites photolabeled by [³H]flunitrazepam (Fig. 8, steps 1 and 2), only 4% reach the neuronal surface in 4 h (43). On this basis, we believe it is unlikely that a substantial fraction of nascent ³⁵S-1 subunits would have acquired a surface localization during the 24-h "window" of our chase experiments.

Two hypotheses have been proposed to account for the use-dependent decline in surface GABA_A receptors: (i) agonist-dependent sequestration and degradation of surface receptors (10) and (ii) repression of GABA_A receptor subunit biosynthesis (21). While the findings in the current report are consistent with the operation of both mechanisms, our data show that hypothesis II cannot account for the onset of down-regulation. Repression of GABA_AR 1 subunit biosynthesis was observed only after the detection of down-regulation. Following the initial binding of externally applied agonists, a small pool of surface GABA_A receptors appears to be rapidly sequestered and degraded, while repression of the synthesis of new receptor subunits is a much slower process. The nature of the intracellular signals that link gene expression to the surface binding of agonists is not understood. The temporal order of these events prompts speculation that such signals could be provided by molecules made available by the sequestration/degradation pathway.