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Protein Science (2003), 12:2642-2646.
Copyright © 2003 The Protein Society

FOR THE RECORD

Recombinant extracellular domain of the three major subunits of GABAA receptor show comparable secondary structure and benzodiazepine binding properties

Haifeng Shi, Shui Ying Tsang, Man Kit Tse, Zhiwen Xu and Hong Xue

Department of Biochemistry and Cooperative Center for Soluble Receptor Biology, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong

Reprint requests to: Hong Xue, Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong; e-mail: hxue{at}ust.hk; fax: 852-23581552.

(RECEIVED June 7, 2003; FINAL REVISION July 20, 2003; ACCEPTED July 22, 2003)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03240203.


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and methods
 References
 
The three most widely expressed subunits of the GABAA receptor are {alpha}1, ß2, and {gamma}2 subunits, and the major isoform in the human brain is a pentameric receptor composed of 2{alpha}12ß21{gamma}2. Previously, we overexpressed the extracellular domain Q28-R248 of GABAA receptor {alpha}1 subunit. In the present study, the homologous extracellular domains Q25-G243 of GABAA receptor ß2 subunit and Q40-G273 of {gamma}2 subunit were also obtained through overexpression in Escherichia coli. Successful production of recombinant ß2 and {gamma}2 subunit receptor protein domains facilitates the comparison of structural and functional properties of the three subunits. To this end, the secondary structures of the three fragments were measured using CD spectroscopy and the ß-strand contents calculated to be >30%, indicating a ß-rich structure for all three fragments. In addition, the benzodiazepine (BZ)-binding affinity of the recombinant fragments were measured using fluorescence polarization to be 2.16 µM, 3.63 µM, and 1.34 µM for the {alpha}1, ß2, and {gamma}2 subunit fragments, respectively, indicating that all three homomeric assemblies, including that of the ß2 subunit, generally not associated with BZ binding, can bind BZ in the micromolar range. The finding that the BZ binding affinity of these recombinant domains was highest for the {gamma}2 subunit and lowest for the ß2 subunit is consistent with results from previous binding studies using hetero-oligomeric receptors. The present results exemplify the effective approach to characterize and compare the three major subunits of the GABAA receptor, for two of which the overexpression in E. coli is reported for the first time.

Keywords: Recombinant protein; circular dichroism; secondary structure; deletion mutagenesis; benzodiazepine binding; fluorescence polarization


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and methods
 References
 
Type A {gamma}-aminobutyric acid (GABAA) receptors are fast-acting ligand-gated ion channels whose functions are allosterically regulated by many important neuroactive drugs, including benzodiazepines (BZ), barbiturates, and anesthetics (MacDonald and Olsen 1994; Smith and Olsen 1995; Leite and Cascio 2001). GABAA receptors are multiple subunit pentameric proteins, a major isoform of which consists of {alpha}1, ß2, and {gamma}2 subunits (Fritschy et al. 1992; Sigel 2002). Each subunit contains an amino-terminal extracellular domain (~200 residues), with a signature 15-residue Cys-loop sequence, followed by four putative transmembrane (TM) regions. The high-affinity GABA-binding site is situated at the extracellular {alpha}/ß subunits interface and the BZ-binding site is situated at the {alpha}/{gamma} subunits interface (Sigel and Buhr 1997; Cromer et al. 2002).

One approach to preparing samples from integral-membrane proteins for structural analysis is to express recombinant fragments corresponding to extracellular or cytoplasmic domains of functional relevance before reconstitution of the image of the whole protein. Previously, three fragments containing extracellular domain Q28-R248 (numbering based on the precursor protein), Q28-R276 (including TM1), and Q28-L296 (including TM2) of bovine GABAA receptor {alpha}1 subunit were overexpressed in Escherichia coli (Hang et al. 2000). The three fragments have been demonstrated to be able to form stable ß-rich secondary structures. It was also shown that the fragments could bind the fluorescent benzodiazepine Bodipy-FL Ro-1986 (BFR) with affinity in the micromolar range (Hang et al. 2000).

In the present study, using the previously reported E. coli expression system (Xue et al. 1998; Hang et al. 2000), the extracellular domains of human GABAA receptor ß2 and {gamma}2 subunits were overexpressed. The purified proteins were characterized by circular dichroism (CD) for secondary structure and fluorescence spectroscopy for ligand binding.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and methods
 References
 
Overexpression of the extracellular domains of the major GABAA receptor subunits
The extracellular domain of the GABAA receptor {alpha}1 subunit spans residues Q28 to R248 and the homologous fragments for the ß2 and {gamma}2 subunits are Q25-G243 and Q40-G273, respectively (Fig. 1Go). The previously reported expression of the {alpha}1 subunit fragment was based on a cDNA clone encoding the bovine {alpha}1 subunit sequence (Hang et al. 2000). Here, in the absence of corresponding clones for the ß2 and {gamma}2 subunits, the genes for the homologous fragments of ß2 and {gamma}2 subunits were assembled using sets of overlapping oligonucleotides, as detailed in Materials and Methods. The expression plasmids thus constructed were then used for the overexpression of the extracellular domains of these two subunits. Thus, in addition to the earlier expression of the {alpha}1 subunit domain, the homologous fragments of the ß2 and {gamma}2 subunits have now been successfully overexpressed (Fig. 2AGo). The availability of recombinant domains of the major GABAA receptor subunits presents the opportunity to characterize each individual subunit and to compare their structural and functional properties.



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  		Figure 1. Aligned amino acid sequence of the GABAA receptor {alpha}1, ß2, and {gamma}2 subunits. The alignment is shown for the extracellular domain of human GABAA {alpha}1, ß2, and {gamma}2 subunits. There is >25% sequence identity. The 15-residue, signature Cys-loop sequence is boxed. (*) Conserved residues; (:) semiconserved residues.

 


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  		Figure 2. (A) SDS-PAGE analysis of extracellular domain of GABAA receptor {alpha}1, ß2, and {gamma}2 subunits. Expression and purification of receptor fragments were as described in Materials and Methods. (Lane 1) Protein markers; (lane 2) {alpha}1Q28-R248; (lane 3) ß2Q25-G243; (lane 4) {gamma}2Q40-G273. (B) Hexagonal plate-like crystals of the Q28-E165 fragment of GABAA receptor {alpha}1 subunit. The protein was concentrated to 16 mg/mL in the presence of 0.5% Triton X-100 and crystallized in 25% 1, 2-propanediol in 0.1 M Na/K phosphate buffer at pH 6.2 using the sitting drop vapor diffusion method.

 
The extracellular domains have stable, well-ordered, ß-rich structures
Due to its lower molecular mass, the fragment Q28-E165 of {alpha}1 subunit, expressed and purified using the same protocol as that used for the extracellular domains and previously characterized as an independent folding unit (Shi et al. 2002), was chosen over the larger domains for crystallization studies. Preliminary trials have yielded protein crystals of hexagonal plate-like appearance (Fig. 2BGo), strongly indicating that the fragment and, by inference, the extracellular domains have been purified to homogeneity and have stable, well-ordered structures. In addition, the secondary structures of the three fragments were analyzed using CD spectroscopy, and the far-UV CD spectra of all three fragments (Fig. 3AGo) have the characteristics of a typical spectrum for all-ß proteins. The ß-strand contents of the fragments were >30 % (Table 1Go), as calculated using the CDPro package (Sreerama and Woody 2000). Of note, the fragment {alpha}1Q28-R248 showed a slight but significant increase in the content of unordered structure, compared to the ß2 and {gamma}2 fragments (Table 1Go; P < 0.05), consistent with the earlier observation that this fragment was susceptible to degradation (Shi et al. 2002).




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  		Figure 3. CD spectra and fluorescent benzodiazepine ligand binding of extracellular domain of GABAA receptor {alpha}1, ß2, and {gamma}2 subunits. (A) Far-UV CD spectra of {alpha}1Q28-R248 (solid line), ß2Q25-G243 (dashed line), and {gamma}2Q40-G273 (dotted line) in 10 mM glycine-NaOH at pH 9.6. (B,C) BFR binding by the extracellular domain as measured by FA and FRET. (Squares) {alpha}1Q28-R248; (open circles) ß2Q25-G243; (triangles) {gamma}2Q40-G273. (B) Saturation curves of FA and Scatchard transformations (inset) of BFR binding to recombinant proteins. FA was measured with excitation and emission wavelength at 490 and 511 nm, respectively. (C) The saturation curves of FRET were obtained as the line of sigmoidal fit of the data. INT280= fluorescence intensity of BFR at 511 nm excited at 280 nm. INT340 = fluorescence intensity of BFR at 511 nm, excited at 340 nm.

 

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  		Table 1. Secondary structure and BZ binding of recombinant GABAA receptor fragments
 
The extracellular domains have slightly different BZ-binding affinities
The BZ binding on the {alpha}1, ß2, and {gamma}2 subunit fragments was detected using the fluorescent BZ ligand Bodipy-FL Ro-1986 (BFR) as described previously (Hang et al. 2000). The BZ binding dissociation constants (Kd) of the three overexpressed fragments {alpha}1Q28-R248, ß2Q25-G243, and {gamma}2Q40-G273 were 2.16 µM, 3.63 µM, and 1.34 µM, respectively, as estimated by fluorescence anisotropy (FA) analysis (Fig. 3BGo and Table 1Go). Although the BZ-binding affinity of the {gamma}2 fragment was slightly higher than that of the {alpha}1 fragment, the difference was not significant (P = 0.299). On the other hand, albeit still within a comparable micromolar range, the BZ-binding affinity of ß2 subunit fragment was significantly lower than the {alpha}1 and {gamma}2 fragments (P < 0.05). The binding of BFR is also evident from fluorescence energy transfer (FRET; Fig. 3CGo). It should be noted that the higher FRET index at the plateau for {gamma}2Q40-G273 also reflects the presence of two more energy donors (Trp) in the protein sequence compared to the other two fragments.

The BZ-binding affinities of GABAA receptors vary with their subunit compositions, whereas {alpha}ß{gamma} hetero-oligomers containing {alpha}1 subunits show high BZ affinity; those containing the {alpha}5 subunit show low BZ affinity (Mehta and Ticku 1999). The high affinity BZ sites have not been detected in any of the {alpha}, ß, or {gamma} homo-oligomers and, as such, the micromolar affinity seen here for all three homo-oligomers may correspond to low affinity binding. Such low affinity is detectable using sensitive fluorescent methods but would not be detectable using the conventional radioligand assays. Unlike radioligand assays, fluorescence measurements such as FA and FRET do not require a separation of the bound and free forms of ligand, which would otherwise disturb the equilibrium of weak receptor–ligand interactions. The present results indicate that the {alpha}1 and {gamma}2 subunits displayed higher BZ-binding affinity than the ß2 subunit. This is consistent with the finding that the BZ-binding site of the {alpha}ß{gamma} hetero-oligomeric receptor, of which the major subtype consists of {alpha}1, ß2, and {gamma}2 subunits, is located at {alpha}/{gamma} subunit interface (Cromer et al. 2002; Sigel 2002). Interestingly, the ß2 subunit exhibited BZ affinity of the same order of magnitude as the other two subunits. The relatively high sequence homology and similar secondary structure of the three subunits suggest that the folding of the three fragments are similar, all giving rise to similar BZ-binding site structures, as in the six-loop agonist binding model (Cromer et al. 2002), thus explaining the observed BZ-binding affinities for the three fragments. It is likely that the requirements for nanomolar BZ binding of native receptor at subunits interface would be more stringent, requiring defined tertiary structure and binding residues, hence readily differentiating between the subunits.

Conclusion
In conclusion, the present study reports the overexpression in E. coli of the extracellular domains of the three most widely expressed subunits of GABAA receptor, making possible the comparison of structural and functional properties of these individual subunits. The overexpression of recombinant protein fragments of the ß2 and {gamma}2 subunits is reported for the first time and the fragments showed defined ß-rich secondary structure similar to that previously reported for the homologous fragment of the {alpha}1 subunit (Hang et al. 2000). The ability to form crystals strongly supports a stable, well-ordered structure. In addition, the BZ-binding capacity of recombinant extracellular domains of GABAA receptor {alpha}1, ß2, and {gamma}2 subunits was compared: the BZ-binding affinity of the recombinant {gamma}2 subunit fragment was the highest of the three and that of the ß2 subunit was the lowest, all with affinities in the micromolar range. These results exemplify the effective approach to characterize and compare the three major subunits of the GABAA receptor and further studies are in progress to extract structural information and to identify BZ-binding residues.


   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
References
 
Materials
The plasmid pTrcHis was purchased from Invitrogen and the fluorescent benzodiazepine ligand Bodipy-FL Ro1986 was from Molecular Probes, Inc. All other chemicals were from either Sigma or USB.

Construction of expression plasmids and mutagenesis
The genes encoding fragments Q25-A159 and A159-M285 of human GABAA receptor ß2 subunit and fragments Q40-G176 and R177-M315 of human GABAA receptor {gamma}2 subunit were assembled using four sets of six overlapping oligonucleotides in recursive PCR (Casimiro et al. 1995). An XmaIII restriction site was designed and placed at the end of genes encoding ß2Q25-A159 and {gamma}2Q40-G176 without changing the amino acid sequence in synthesized oligonucleotides. The codon usage was modified to prokaryote preferable. The PCR products were double-digested with NcoI/EcoRI and ligated with the same restriction enzyme-digested pTrcHisA plasmid. An XmaIII and a HindIII restriction site were introduced to the 5'-flanking and 3'-flanking regions, respectively, of the genes encoding ß2A159-M285 and {gamma}2R177-M315 by PCR. The PCR products were double-digested with XmaIII/HindIII and ligated with the same enzyme-digested ß2Q25-A159pTrc and {gamma}2Q40-G176pTrc, respectively. The constructed expression plasmids ß2Q25-M285pTrc and {gamma}2Q40-M315pTrc were used to make C-end deletion mutants with the PCR-based Site-directed Mutagenesis Kit from Strategene (La Jolla, CA). All insert sequences were confirmed by DNA sequencing. The expression plasmid bovine-{alpha}1Q28-R248pTrc was constructed as in Hang et al. (2000).

Expression and purification
Fragments of the GABAA receptor were expressed and purified as described in Hang et al. (2000). The molecular mass and purity of the proteins were estimated using SDS-PAGE.

Crystallization of fragment Q28-E165
The purified protein was refolded in the presence of 0.5% Triton X-100, the nonionic detergent acting to stabilize the hydrophobic protein. The protein was then concentrated to 16 mg/mL and crystallized in 25% 1, 2-propanediol in 0.1 M Na/K phosphate buffer at pH 6.2 using the sitting drop vapor diffusion method.

CD spectroscopy
All CD measurements were obtained using a JASCO J-720 spectrophotometer at room temperature with a 0.1-cm path length for far-UV CD . The protein samples were 0.1 mg/mL in 10 mM glycine-NaOH at pH 9.6 for far-UV. Secondary structure contents were estimated from the far-UV CD spectra using the program CDPro (Sreerama and Woody 2000).

Fluorescence spectroscopy
All fluorescence measurements were performed at room temperature using a Perkin-Elmer LS50B luminescence spectrometer. Fluorescence anisotropy (FA) of 20 nM BFR in 10 mM glycine-NaOH at pH 9.6 with various protein concentrations was measured at an excitation wavelength of 490 nm with a 5-nm slit and an emission wavelength of 511 nm with a 10-nm slit. The fraction of bound ligand was estimated from the increase in FA with increment of protein (van den Elsen et al. 1997). The dissociation constant Kd was estimated from FA saturation curves by nonlinear least-squares fit to a single-site binding model and linear fit of the Scatchard transformation. FRET was measured as the ratio of the fluorescence intensity at 511 nm of 0.248 µM BFR in various protein concentrations excited at 280 nm and 340 nm. FRET measurements are semiquantitative and are not used for calculation of Kd.


   Acknowledgments
 
We thank Professor J. Tze-Fei Wong for helpful discussions. Technical assistance from Peggy Lee and Hui Zheng are acknowledged. This work was supported by the Research Grants Council, Hong Kong (Project No. CRC98/01.SC04).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
 References
 
Casimiro, D.R., Toy-Palmer, A., Blake 2nd, R.C., and Dyson, H.J. 1995. Gene synthesis, high-level expression, and mutagenesis of Thiobacillus ferrooxidans rusticyanin: His 85 is a ligand to the blue copper center. Biochemistry 34: 6640–6648.[CrossRef][Medline]

Cromer, B.A., Morton, C.J., and Parker, M.W. 2002. Anxiety over GABA(A) receptor structure relieved by AChBP. Trends Biochem. Sci. 27: 280–287.[CrossRef][Medline]

Fritschy, J.M., Benke, D., Mertens, S., Oertel, W.H., Bachi, T., and Mohler, H. 1992. Five subtypes of type A {gamma}-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies. Proc. Natl. Acad. Sci. 89: 6726–6730.[Abstract/Free Full Text]

Hang, J., Shi, H., Li, D., Liao, Y., Lian, D., Xiao, Y., and Xue, H. 2000. Ligand binding and structural properties of segments of GABAA receptor {alpha} 1 subunit overexpressed in Escherichia coli. J. Biol. Chem. 275: 18818–18823.[Abstract/Free Full Text]

Leite, J.F. and Cascio, M. 2001. Structure of ligand-gated ion channels: Critical assessment of biochemical data supports novel topology. Mol. Cell Neurosci. 17: 777–792.[CrossRef][Medline]

MacDonald, R.L. and Olsen, R.W. 1994. GABAA receptor channels. Annu. Rev. Neurosci. 17: 569–602.[Medline]

Mehta, A.K. and Ticku, M.K. 1999. An update on GABAA receptors. Brain Res. Rev. 29: 196–217.[CrossRef][Medline]

Shi, H., Tsang, S.Y., Zheng, H., Sturgis, J.N., and Xue, H. 2002. Two ß-rich structural domains in GABA(A) receptor {alpha}(1) subunit with different physical properties: Evidence for multidomain nature of the receptor. Protein Sci. 11: 2052–2058.[Abstract/Free Full Text]

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Sigel, E. and Buhr, A. 1997. The benzodiazepine binding site of GABAA receptors. Trends Pharmacol. Sci. 18: 425–429.[Medline]

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van den Elsen, J.M., van Pomeren, E., Poolman, J.T., Wilting, J., Herron, J.N., and Crommelin, D.J. 1997. On the interaction between a bactericidal antibody and a PorA epitope of Neisseria meningitidis in outer membrane vesicles: A competitive fluorescence polarization immunoassay. Anal. Biochem. 247: 382–388.[CrossRef][Medline]

Xue, H., Chu, R., Hang, J., Lee, P., and Zheng, H. 1998. Fragment of GABAA receptor containing key ligand-binding residues overexpressed in Escherichia coli. Protein Sci. 7: 216–219.


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