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J. Biol. Chem., Vol. 275, Issue 25, 18818-18823, June 23, 2000

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Ligand Binding and Structural Properties of Segments of GABA_A Receptor ₁ Subunit Overexpressed in Escherichia coli^*

Jun Hang, Haifeng Shi, Dongyang Li, Yinglei Liao, Dejun Lian, Yazhong Xiao, and Hong Xue

From the Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong

Received for publication, January 11, 2000, and in revised form, April 4, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The -aminobutyric acid, type A (GABA_A), receptor is the target for numerous therapeutic compounds. In the present study, the Gln²⁸-Leu²⁹⁶, Gln²⁸-Arg²⁷⁶, Gln²⁸-Arg²⁴⁸, and Gln²⁸-Glu¹⁶⁵ (numbering of bovine precursor protein) segments of its ₁ subunit were overexpressed in Escherichia coli, along with Cys¹⁶⁶-Leu²⁹⁶ produced previously, for structural analysis by circular dichroism and ligand binding studies by fluorescence spectroscopy. Results showed that the protein segments were rich in -sheet structures. Binding of the fluorescent benzodiazepine Bodipy-FL Ro-1986 was evident from fluorescence resonance energy transfer and fluorescence anisotropy measurements. The binding affinity was in the micromolar range. The binding was attributable more to Cys¹⁶⁶-Leu²⁹⁶ than to Gln²⁸-Glu¹⁶⁵ and was inhibited by known central benzodiazepine site ligands. Three point mutations, Y187A, T234A, and Y237A, were found to perturb protein secondary structures. Studies with the single Trp mutants W198Y and W273Y indicated that Trp²⁷³ was closer to the binding site than Trp¹⁹⁸.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

-Aminobutyric acid (GABA)¹ is the major inhibitory neurotransmitter in the mammalian brain. The type A receptors of GABA (GABA_A receptors) (1) are fast acting, ligand-gated chloride ion channels, which are believed to adopt a pentameric structure (2) with the regulated ion channel formed along the vertical axis of subunit interfaces. Since the cloning of the first two subunits, ₁ and ₁, in 1987 (3), as many as 18 different subunits have been identified (4). Of the myriad number of GABA_A receptor isoforms possible, ₁₂₂ is believed to represent the major adult isoform (5).

GABA_A receptors serve as the targets of many important neuroactive drugs (6-8), and substantial effort has been made to understand the structure of BZ-binding site (9). Evidence from site-directed mutagenesis, photoaffinity labeling, and pharmacological studies collectively suggest that  and  subunits contribute to the BZ-binding site (10-14). A majority of the amino acid residues suggested to be essential for BZ binding are located on the ₁ subunit and include such residues as His¹⁰¹, Tyr¹⁵⁹, Thr²⁰⁶, and Tyr²⁰⁹ (numbering of rat mature protein) (9, 14-18). These studies therefore pointed to a key role for the ₁ subunit in determining the binding properties of various BZ ligands to the GABA_A receptor.

To date, several systems have been utilized to express recombinant GABA_A receptors. Radioactive ligand binding studies have been performed with cell lines, and electrophysiological studies have been done in cell lines and in Xenopus laevis oocytes (19-22). However, these systems could not provide adequate amounts of protein for structural characterization. Without structural information, it would be difficult to ascertain whether a deleterious mutation might act directly to alter an active site residue or indirectly through disruption of protein conformation. To overcome this shortcoming and allow insight into the structure and function relationship of GABA_A receptors, a high expression system is of urgent importance.

Previously, we have reported the overexpression of the Cys¹⁶⁶-Leu²⁹⁶ segment of GABA_A receptor ₁ subunit and the delineation of a structural domain within the segment (23, 24). Segment Cys¹⁶⁶-Leu²⁹⁶ has been demonstrated to be able to form stable secondary structures (23) and rosette-like quaternary structures (25). In the present study the properties of Cys¹⁶⁶-Leu²⁹⁶ along with the Gln²⁸-Arg²⁴⁸, Gln²⁸-Arg²⁷⁶, Gln²⁸-Leu²⁹⁶, and Gln²⁸-Glu¹⁶⁵ polypeptides were characterized with respect to ligand binding and protein folding. Site-directed mutagenesis was applied to the three residues Tyr¹⁸⁷, Thr²³⁴, and Tyr²³⁷ (numbering of bovine precursor protein), previously identified to be important for ligand binding (16, 17, 18), and to the two Trp residues Trp¹⁹⁸ and Trp²⁷³ in order to gain insight into the structure-function relationships of BZ binding.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The cDNA clone, pCDM8-b₁, encoding bovine ₁ subunit was a gift from Dr. A. N. Bateson of the University of Alberta. The plasmid pTrcHis was purchased from Invitrogen; diazepam and estazolam were from Wuhan Pharmaceutical Co., China; GABA and muscimol were from Sigma; and the fluorescent BZ ligand Bodipy-FL Ro-1986 (BFR) was from Molecular Probes, Inc. Ethyl ester of -carboline-3-carboxylate (-CCE) was a gift from Prof. Richard W. Olsen of UCLA.

Construction of Expression Plasmids and Mutagenesis-- All subcloning and mutagenesis were performed with the PCR-based Mutagenesis Kit from Stratagene (La Jolla, CA) with slight modifications in which Pfu DNA polymerase was used instead, and DpnI treatment was replaced by gel purification of linear PCR products. A 2.3-kilobase pair DNA containing the full-length cDNA (GenBank^TM accession number X05717) encoding the mature peptide (Gln²⁸-Gln⁴⁵⁶) of bovine GABA_A receptor ₁ subunit was subcloned from pCDM8-b₁ to pTrcHis for expression after removal of the upstream vector sequence excepting the mini-cistron with a pair of PCR primers (P1 and P2 in Table I). Based on this initial subclone more deletional and substitutional mutants were made using PCR primers designated in Table I. Silent base substitutions were incorporated into the primers (Table I) to optimize codon usage and introduce termination codons as appropriate. All insert sequences were confirmed by DNA sequencing.

Expression and Purification-- Freshly transformed Escherichia coli cells of NovaBlue strain (Novagen, Inc., Madison, WI) were stored in 10% Me₂SO aliquots at 80 °C. After induction of the growing culture with 0.4 mM isopropyl--thiogalactoside for 6 h (23), the cells were harvested and washed once with 200 ml of solution A (50 mM Tris·Cl, 1 mM EDTA, pH 8.5), lysed in 100 ml of solution B (50 mM Tris·Cl, 10 mM EDTA, pH 8.5, 250 µg/ml lysozyme) at 37 °C for 30 min, diluted with 100 ml of solution C (50 mM Tris·Cl, 10 mM EDTA, pH 8.5), homogenized with an Ultra-Turrax T25 homogenizer (Janke & Kunkel IKA-Labortechnik, Germany), and centrifuged at 10,000 rpm for 15 min. The pellet was resuspended in 100 ml of solution D (2% deoxycholic acid, 50 mM Tris·Cl, 10 mM EDTA, pH 8.5), re-homogenized, collected at 9,500 rpm × 30 min, washed with 100 ml of solution E (10 mM Tris·Cl, pH 8.5), and centrifuged at 9,500 rpm for 20 min. The resultant pellet was resuspended in 4 M urea (10 ml per g of pellet) prior to centrifugation at 10,000 rpm for 20 min and washed in 100 ml of solution E. The pellet was solubilized in 2% SDS, 0.1 M -mercaptoethanol, pH 12, and subjected to gel filtration on a Superdex 200 HR 26/60 column (Amersham Pharmacia Biotech). The sample was eluted using a refolding buffer, containing 250 mM Arg, 100 mM Tris, and 1 mM EDTA, with 0.3 mM L-cystine and 3 mM L-cysteine added just before use, at a flow rate of 2.5 ml/min. The pooled fractions were dialyzed against 10 mM Tris·Cl, pH 9.0, for 3 days, with three buffer changes per day.

Protein Identification and Quantification-- Protein concentrations were determined photometrically (26). The molecular weight and purity of proteins were estimated using 15% SDS-PAGE. The identities of recombinant proteins were confirmed by N-terminal amino acid sequencing. Mass spectroscopic analyses were provided by Molecular Biology Resource Facility, University of Oklahoma Health Science Center, and N-terminal protein sequencing was by the Protein Service Laboratory, University of British Columbia.

CD Measurements-- All CD spectra were obtained using a JUSCO J-720 spectropolarimeter at room temperature with a 0.1-cm path length cuvette for far-UV CD or 1-cm for near-UV CD. The protein samples were 0.1 mg/ml in 10 mM Tris·Cl, pH 9.0, for far-UV and 1 mg/ml for near-UV CD. Secondary structure contents were estimated from far-UV CD (27).

Fluorescence Measurements-- All fluorescence measurements were performed at room temperature using a Perkin-Elmer model LS50B luminescence spectrometer. A stock of 1.65 mM BFR in Me₂SO at 20 °C was diluted into buffer just before use.

Fluorescence Resonance Energy Transfer (FRET)-- Protein solutions were mixed with 0.41 µM BFR in the cuvette, and 450-550 nm emission spectra at 5-nm slit were obtained at 280, 295, 340, or 490 nm excitation and 2.5-nm slit width. Light at 280 nm excited both BFR and protein intrinsic fluorophores, whereas light at 340 nm excited only BFR. This was utilized to correct for errors in BFR concentration and interference from protein scattering on fluorescence intensity. For this purpose, two sets of emission spectra, one with excitation wavelength of 280 nm and the other of 340 nm, were recorded in parallel. The ratio of respective fluorescence intensities, INT₂₈₀/INT₃₄₀, was used as an index of FRET; this was replaced by INT₂₉₅/INT₃₄₀ in the case of single Trp mutants.

Fluorescence Anisotropy (FA) and Equilibrium Dissociation Constant (K_d)-- FA was measured at an excitation wavelength of 490 nm with a 10-nm slit and an emission wavelength of 511 nm with a 10-nm slit. For saturation experiments, 0.165 µM BFR in 10 mM Tris·Cl, pH 9.0, was titrated with protein. At such a low BFR concentration, FA would be proportional to the concentration of ligand-receptor complex. Therefore, the fraction of bound ligand present at each titration point could be estimated from change in FA (A) as a function of protein concentration after correction for fluorescence intensity difference between free and bound ligands (28) and was employed to calculate K_d. The maximal FA increase (A_max) estimated from a nonlinear least square fitting of the saturation curve to a single-site binding model was Scatchard plotted as [bound]/[free] = [bound]/([protein]  [bound]) versus [bound] = [BFR]_total × A/A_max to yield the K_d of ligand-receptor complex, where bound or free refers to fraction of receptor bound to ligand or free of ligand. For competitive inhibition assay, 10 mM Tris·Cl, pH 9.0, containing 0.165 µM BFR and 10 µM Cys¹⁶⁶-Leu²⁹⁶ was titrated in turn with -CCE, estazolam, diazepam, GABA, or muscimol solution amounting to less than 4% volume of the assay mixture and monitored by FA measurements.

Fluorescence Quenching (FQ)-- To measure FQ by BFR, single Trp mutant proteins of 1.5 µM in 10 mM Tris·Cl, pH 9.0, was titrated with 1.65 mM BFR. The emission intensity at 342 nm with 7-nm slit was recorded at 295 nm excitation with 5-nm slit. The ratio of the initial fluorescence intensity versus the intensity at each titration point was plotted against the quencher concentration (29).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression, Purification, and Identification-- All subclones of GABA_A receptor ₁ subunit segments were successfully expressed. Under the current purification protocol, about 10 mg of purified product was obtained per liter of culture. The high level expression and the purity of the recovered recombinant proteins are demonstrated in Fig. 1.

View larger version (82K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE analysis of the recombinant GABAA receptor segments. Receptor proteins were purified as described under "Experimental Procedures." Lane 1, molecular mass standards; lane 2, total cell lysates of E. coli expressing the segment Gln28-Leu296; lane 3, pellet of the total cell lysates shown in lane 2; lanes 4-8, purified segments Gln28-Leu296, Gln28-Arg276, Gln28-Arg248, Gln28-Glu165, and Cys166-Leu296, respectively.

Secondary Structure and Tertiary Packing-- Secondary structure contents of recombinant receptor segments estimated from far-UV CD spectra are presented in Table II. The ratio /, the content of -strand relative to -helix, was calculated for Cys¹⁶⁶-Leu²⁹⁶, and its point mutation variants were calculated for comparison. All the polypeptides formed not only stable -rich secondary structure (Fig. 2A) but also ordered tertiary packing as evident from their near-UV CD spectra. As illustrated in Fig. 2B, the CD spectrum of Gln²⁸-Leu²⁹⁶ showed distinct signals around 270-295 nm characteristic of proteins with well packed aromatic side chains.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Representative CD spectra of receptor segments. A, far-UV spectra of Gln28-Arg248 and Gln28-Leu296. B, near-UV spectrum of Gln28-Leu296. C, far-UV spectra of Ala substitution mutants and the wild-type Cys166-Leu296. D, far-UV spectra of single Trp mutants of Cys166-Leu296 along with that of the wild type.

Fluorescent Ligand Binding-- The five overexpressed polypeptides, namely Gln²⁸-Arg²⁴⁸, Gln²⁸-Arg²⁷⁶, Gln²⁸-Leu²⁹⁶, Gln²⁸-Glu¹⁶⁵, and Cys¹⁶⁶-Leu²⁹⁶, were active in binding the fluorescent BZ ligand BFR. Analysis of the variation of FA of BFR with protein concentrations yielded the binding constants K_d for the five segments, which range from 1.96 to 9.97 µM (Fig. 3B and Table II). Cys¹⁶⁶-Leu²⁹⁶, the shortest segment among the five, was selected for further analysis.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   BFR binding by Cys166-Leu296 and its mutants as measured by FRET and FA. A and B, analysis of Cys166-Leu296 and the three putative binding-site mutants. Closed circles, Cys166-Leu296; open circles, Y187A; closed squares, T234A; open squares, Y237A; closed triangles, Cys166-Leu296 with the fluorescence dye Bodipy-FL replacing BFR. The saturation curves of FRET in A were each obtained as the line of sigmoidal (Boltzman) fit of the data. Excitation was carried out at 280 or 340 nm. INT280 = maximal fluorescence intensity of BFR excited at 280 nm. INT340 = maximal fluorescence intensity of BFR excited at 340 nm. For the saturation curves of FA and Scatchard transformations (inset B), excitation and emission were at 490 and 511 nm, respectively. C and D show the analysis of the two single Trp mutants. Closed circles, W198Y; open circles, W273Y. For the saturation curves of FRET in C, INT295 = maximal fluorescence intensity of BFR excited at 295 nm. For the saturation curves of FA and Scatchard transformations (inset in D), excitation and emission were at 490 and 511 nm, respectively.

Quenching of Ligand Fluorescence by Receptor-- When BFR was excited at 490 nm, the _max of its excitation spectrum and where no protein absorption occurred, its emission intensity was diminished by the addition of receptor protein (Fig. 4A). In addition, a red shift of the emission spectrum became evident. In comparison, no significant changes in BFR fluorescence emission were observed with the addition of the same concentration of lysozyme (14.30 kDa), which has a molecular weight similar to that of Cys¹⁶⁶-Leu²⁹⁶ (14.83 kDa). Therefore the quenching caused by the receptor protein was not nonspecific protein effects.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Changes in BFR emission upon protein titration. A, red-shift in max and decrease in intensity. Emission spectra of BFR (0.41 µM) excited at 490 nm in the absence (solid line) or presence of 10 µM Cys166-Leu296 (dashed line) or 10 µM lysozyme (dotted line). B, FRET from protein to BFR. Emission of BFR (0.41 µM) increasingly enhanced by increasing concentrations of Cys166-Leu296 (0, 0.657, 1.970, 3.284, 4.925, and 6.567 µM, from the bottom to the top). Excitation was at 280 nm. AFU, arbitrary fluorescence unit.

FRET from Receptor to Ligand-- In contrast to the quenching observed with 490 nm excitation, the fluorescence intensity of BFR increased upon addition of receptor protein in a concentration-dependent manner when the excitation wavelength was set at 280 nm to excite both ligand fluorescence and intrinsic protein Trp fluorescence (Fig. 4B). The phenomenon indicates that the energy released from protein fluorescence emission was transferred to the ligand. The hyperbolic increase of FRET index with protein concentration is shown in Fig. 3A.

Competitive Inhibition-- To assess whether the binding of BFR to the receptor segment bore any resemblance to ligand binding to the BZ-binding site in the central nervous system, the effects of competing ligands were examined. The known high affinity central BZ site ligand -CCE exerted a strong inhibitory effect on A in the FA binding assay. Diazepam and estazolam, two BZ ligands with affinities lower than -CCE, caused moderate inhibition, and the two non-BZ site ligands muscimol and GABA caused only very minor inhibition (Fig. 5).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Inhibition of BFR binding to Cys166-Leu296 by competing compounds. The solid curves represent nonlinear least squares fit of the data. Results are recorded as percentage of BFR binding to Cys166-Leu296 in the presence of -CCE (closed squares), diazepam (closed circles), estazolam (open squares), GABA (open circles), or muscimol (closed triangles).

Point Mutations-- Five point mutations were made on segment Cys¹⁶⁶-Leu²⁹⁶ at three putative binding-site residues, Tyr¹⁸⁷, Thr²³⁴, and Tyr²³⁷, and the two Trp residues, Trp¹⁹⁸ and Trp²⁷³. Their effects on protein secondary structures were examined with far-UV CD (Fig. 2, C and D; Table II). Secondary structure content and the / ratio of each of the five-point mutants were compared with that of Cys¹⁶⁶-Leu²⁹⁶ through statistical analyses by Student's t test for significance. Significant changes from the wild-type parameters (p < 0.05) are shown in bold face in Table II.

Compared with wild-type Cys¹⁶⁶-Leu²⁹⁶, the Y187A mutant showed an increase in -sheets (p = 0.0197), a decrease in -helix (p = 0.0091), and hence increased / ratio (p = 0.0087). This suggests that Tyr¹⁸⁷ in the wild-type Cys¹⁶⁶-Leu²⁹⁶ probably contributed to the maintenance of an -helical structure that was likely converted into a more extended structure upon Ala substitution of the residue. T234A showed a decrease in -sheets (p = 0.0068), an increase in -helix (p = 0.0074), and a decreased / ratio (p = 0.0031), suggesting the possibility that Thr²³⁴ was involved in maintaining a -stranded structure. Among the three "binding site" mutants, Y237A gave rise to the least change in secondary structures, which was mainly shown as a slight increase in the content of "Others" (p = 0.0089). The changes in -helix and -sheet contents caused by the two single Trp mutants were both insignificant (Fig. 2D and Table II). Nevertheless, it was noticeable that W273Y mutation reduced the content of -turn (p = 0.0073), which could be the consequence of the disruption of a -turn structure in Cys¹⁶⁶-Leu²⁹⁶ involving Trp²⁷³.

The three mutants Y187A, T234A and Y237A retained both Trp residues but exhibited a reduced FRET (Fig. 3A). They also bound BFR more weakly. Their K_d values calculated from FA were 13.83, 8.38, and 10.27 µM, respectively (Fig. 3B and Table II). Compared with 4.85 µM for the wild-type Cys¹⁶⁶-Leu²⁹⁶, these mutants caused statistically significant 2-3-fold reductions in binding affinity.

The two single Trp mutants W198Y and W273Y diminished in FRET values, as the consequence of the removal of one of the intrinsic fluorophores. However, their BFR binding K_d values of 5.97 and 4.77 µM obtained from FA were both not significantly different from that of Cys¹⁶⁶-Leu²⁹⁶ judging from the t test (Fig. 3D and Table II). The FRET was stronger for the W198Y mutant, which retained Trp²⁷³, than the W273Y mutant, which retained Trp¹⁹⁸ instead (Fig. 3C), indicating that Trp²⁷³ was situated closer to the BZ-binding site than Trp¹⁹⁸.

The relative accessibility of the two Trp residues in Cys¹⁶⁶-Leu²⁹⁶ by the ligand was also evaluated by quenching of Trp fluorescence by BFR. Addition of BFR caused decreases in protein intrinsic Trp fluorescence in both mutants (Fig. 6). However, the fluorescence of Trp²⁷³ in the W198Y mutant was more severely quenched by BFR than that of Trp¹⁹⁸ in W273Y, indicating that Trp²⁷³ was more accessible to the ligand than Trp¹⁹⁸, in agreement with the FRET results.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Stern-Volmer plot for ligand quenching of intrinsic Trp fluorescence. The emission was scanned from 300 to 400 nm with the excitation wavelength set at 295 nm. F0 = fluorescence intensity at 342 nm in the absence of BFR; F = fluorescence intensity at 342 nm in the presence of BFR at the indicated concentration. Closed circles, W198Y; open circles, W273Y.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The overexpression of the Cys¹⁶⁶-Leu²⁹⁶ segment of GABA_A receptor ₁ subunit was previously found to generate a protein domain with stable folding and rich in -structures (23, 24). In the present study the three overlapping long segments obtained so far containing an extension to the N terminus, namely Gln²⁸-Arg²⁴⁸, Gln²⁸-Arg²⁷⁶, and Gln²⁸-Leu²⁹⁶, also were all rich in -strands, ranging from 45.7 to 51.9% (Table II). Segment Gln²⁸-Arg²⁴⁸ would comprise the entire putative extracellular domain except for two amino acids, Gln²⁸-Arg²⁷⁶ additionally almost the entire first transmembrane region, and Gln²⁸-Leu²⁹⁶ additionally almost the entire second transmembrane region (3). Since the three segments were identical at the N terminus portion up to Arg²⁴⁸, their differences in secondary structure contents resulted from differences at the C terminus portion. Dividing the longest protein Gln²⁸-Leu²⁹⁶ into two gave rise to the shorter Gln²⁸-Glu¹⁶⁵ and Cys¹⁶⁶-Leu²⁹⁶. The latter was more enriched than the former in -strands, whereas the former contained more -helix. These results were consistent with the secondary structure predictions reported earlier (24). The higher -helical content of Gln²⁸-Arg²⁷⁶ than Gln²⁸-Leu²⁹⁶ suggests that residues Glu²⁷⁷-Leu²⁹⁶, a part of the putative second transmembrane region, did not form an -helical structure within segment Gln²⁸-Leu²⁹⁶. However, it is not ruled out that these residues could participate in an -helix within the full-length receptor.

In this regard, these GABA_{A 1} N terminus segments resemble the nicotinic acetylcholine receptor, the prototype of the fast-acting ligand-gated ion channel receptor superfamily (30). The N-terminal extracellular domain of the Torpedo nicotinic acetylcholine receptor -subunit, approximately equivalent to Gln²⁸-Arg²⁴⁸ of GABA_A receptor ₁ subunit, was expressed in Xenopus oocytes, transfected Chinese hamster ovary cells (31), and E. coli (32), respectively. It was also -rich, containing 51% -sheet (31) or 45% -stranded structures (32), levels that were comparable with the 51.9% -strand content of Gln²⁸-Arg²⁴⁸ recorded in Table II. Accordingly, a -rich extracellular N terminus portion might represent a general occurrence among this superfamily of ligand-gated ion channel receptors.

Fluorescent ligand binding assays (33, 34) were particularly advantageous for recombinant GABA_A receptors. They offer sensitivities comparable to that of radio-ligand assays, and fluorescent BZ derivatives such as BFR exhibit high affinity and selectivity for the GABA_A receptor (35-37). Unlike radio-ligand assays, fluorescence measurements do not require a separation of the bound and free forms of ligand and thus make possible the analysis of even relatively weak receptor-ligand interactions by means of homogenous phase assays such as FRET and FA. Furthermore, measurements such as FRET could provide structural insight into the ligand-binding site.

Several lines of evidence supported a specific binding of BFR to the purified GABA_{A 1} receptor Cys¹⁶⁶-Leu²⁹⁶ segment. First, the emission of BFR was changed in both intensity and _max by addition of the segment but not by the control protein lysozyme (Fig. 4). Second, the change in BFR fluorescence was dependent on protein concentration and was saturable (Fig. 3). Third, the inhibition of BFR binding exerted by potential competitors (Fig. 6) was strongest with -CCE, a GABA_A receptor reverse agonist selective for BZ type I receptors, K_i 0.3 nM (14), more modest with ligands with lower BZ affinities, namely diazepam (K_i 16.1 nM) (14) and estazolam (K_i 17.0 nM) (38), and minimal with the non-BZ ligands muscimol and GABA. These experimental results suggested that the observed BZ ligand binding to Cys¹⁶⁶-Leu²⁹⁶ is likely to be biologically relevant.

It is noteworthy, however, that the K_d displayed by the various receptor segments for BFR in the micromolar range (Table II) were considerably higher than those reported for native GABA_A receptors in the nanomolar range. This points to the existence of specific binding sites with only low affinity in these recombinant proteins. Previous studies have shown that co-expression of combinations of , , and  subunits was required to produce GABA_A receptors with pharmacological properties resembling that of native receptors (14) and suggested that the BZ-binding site was located at the interface of ₁ and ₂ subunits (6, 9). Therefore, the low affinity BFR binding displayed by the purified GABA_{A 1} subunit segments could be the consequence of the lack of contributions not only from the rest of the ₁ subunit but also from the ₂ subunit.

Since the homo-oligomeric GABA_A receptors formed by  subunits were found not to be modulated by BZ (14), it was somewhat unexpected that a segment of ₁ subunit alone could bind specifically a BZ ligand. A possible explanation could be that the weak interaction between BZ ligand and ₁ subunit would be difficult to detect by heterogeneous phase assays that require a separation of the bound and free ligand. The FRET and FA assays employed in the present study, because they do not require such separation, are intrinsically more sensitive for the detection of weaker receptor-ligand interactions. Both FRET and FA involve homogeneous assay systems and are ideal with purified proteins but not membranous preparations of receptors. This is especially true with FA, which is a relatively insensitive method and requires high receptor expression and high membrane volume per well of assay so that FA is not a method of choice for homomeric GABA_A receptors that express only poorly.

The finding that the 131-residue Cys¹⁶⁶-Leu²⁹⁶ was more active than its immediate N-terminal neighbor Gln²⁸-Glu¹⁶⁵ in BZ ligand binding was consistent with previous reports on the residues important for BZ modulation. Except for His¹⁰¹, all such reported residues were located within the Cys¹⁶⁶-Leu²⁹⁶ segment, including the residues Tyr¹⁵⁹, Tyr¹⁶¹, Thr¹⁶², Gly²⁰⁰, Ser²⁰⁴, Thr²⁰⁶, Tyr²⁰⁹, and Val²¹¹ (numbering of rat mature protein) (16-18, 39-41). Three of these residues, Tyr¹⁵⁹/Tyr¹⁸⁷, Thr²⁰⁶/Thr²³⁴, and Tyr²⁰⁹/Tyr²³⁷ in ₁ subunit (numbering of rat mature/bovine precursor polypeptides), proposed to be involved in receptor-BZ interaction are also homologous to residues in  subunits believed to be responsible for receptor-GABA interaction. In view of this, Cys¹⁶⁶-Leu²⁹⁶ was mutated at these three residues in order to evaluate their individual contributions to protein structure and BFR binding. The three mutations caused varying degrees of secondary structure alterations (Fig. 2C and Table II) and decrease in FRET (Fig. 3A) in addition to 2-3-fold reduction in the binding affinity as measured by the dissociation constant (Table II). Although the extent of decrease in binding affinity caused by the three-point mutations deviated from previous reports by others (16-18) using different experimental systems, the simultaneous changes in secondary structure and in ligand binding affinity caused by the three mutations suggest that the functional effects of mutations at these residues (14, 16-18) could be mediated at least in part by structural effects of these residues on the receptor. This possibility is also highlighted by the fact that these residues are conserved in both  and  subunits, even though  subunits are believed not to be involved in BZ binding (14).

The fluorescence of BFR was reduced and red-shifted in the presence of Cys¹⁶⁶-Leu²⁹⁶ (Fig. 4). This suggests that the BZ-binding site on this polypeptide was relatively polar in microenvironment or surrounded by fluorescence-quenching element(s). The two Trp residues in Cys¹⁶⁶-Leu²⁹⁶ were both non-essential to BFR binding (Fig. 3D and Table II). The FRET measurements indicated that Trp²⁷³ was located closer to the BFR-binding site than Trp¹⁹⁸, and this was further supported by FQ experiments, in which Trp²⁷³ was found to be more accessible to interaction with BFR than Trp¹⁹⁸ (Fig. 6).

In conclusion, the lack of an abundant supply of recombinant proteins has for many years limited the structure-function characterization of the GABA_A receptor. The present study shows that overexpression of segments of the receptor makes possible experimental analysis that could contribute usefully to a deeper understanding of GABA_A receptor. The ready availability of these fragments has also permitted the initiation in our laboratory of their crystallization trials aimed toward a three-dimensional structural analysis of the receptor.

	ACKNOWLEDGEMENTS

We thank Professors R. Wayne Davies and J. Tze-Fei Wong for helpful discussions and Dr. David Miller-Martini for critical reading of the manuscript. Prof. R. W. Olsen and Dr. A. N. Bateson are sincerely acknowledged for generous supplies of -CCE and bovine GABA_A receptor ₁ subunit cDNA clone, respectively. Technical assistance from Hui Zheng, Peggy Lee, and Ruiai Chu is acknowledged.

	FOOTNOTES

^* This work was supported by the Research Grant Council of Hong Kong.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 852-23588707; Fax: 852-23581552; E-mail: hxue@ust.hk.

Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M000193200

	ABBREVIATIONS

The abbreviations used are: GABA, -aminobutyric acid; GABA_A, -aminobutyric acid, type A; BZ, benzodiazepine; BFR, Bodipy-FL Ro-1986; FQ, fluorescence quenching; FRET, fluorescence resonance energy transfer; FA, fluorescence anisotropy; PAGE, polyacrylamide gel electrophoresis; -CCE, ethyl ester of -carboline-3-carboxylate; PCR, polymerase chain reaction.

	REFERENCES

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