SEARCH

SEARCH BY CITATION

Keywords:

  • benzodiazepine;
  • β-carboline;
  • chimera;
  • diazepam insensitive;
  • GABAA;
  • Ro15-4513

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

GABAA receptors that contain either the α4- or α6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including β-carbolines, bind to these diazepam-insensitive receptor subtypes. Some β-carbolines [e.g. ethyl β-carboline-3-carboxylate (β-CCE) and methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM)] display a higher affinity for α4- compared to α6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric α6/α4 subunits and co-expressed these with wild-type rat β2 and γ2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (α6N204/α4I203). Substitutions at α6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive α1 subunit (S205N) resulted in a 7–8-fold reduction in affinity for the β-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the α1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by ∼19-fold and ∼38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the α subunit in diazepam-sensitive and -insensitive receptors.

Abbreviations used
AChBP

acetylcholine binding protein

β-CCE

ethyl β-carboline-3-carboxylate

BZD

benzodiazepine

DI

diazepam insensitive

DMCM

methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate

DS

diazepam sensitive

GABAA

γ-aminobutyric acid type A

i -BZD

imidazobenzodiazepine

LGIC

ligand-gated ion channel

γ-Aminobutyric acid type A (GABAA) receptors are the major inhibitory receptors in the mammalian CNS. These receptors are members of the ligand-gated ion channel (LGIC) family that includes the nicotinic acetylcholine, glycine and serotonin type 3 (5-HT3) receptors (Ortells and Lunt 1995). Each GABAA receptor is a heteropentamer of homologous subunits that assemble to form a GABA-gated, chloride-selective ion pore. Several different classes of receptor subunits have been cloned (α1–6, β1–3, γ1–3, δ, ɛ, π and θ) (for reviews, see Barnard et al. 1998; Korpi et al. 2002) and different combinations of these subunits in the oligomer determine the pharmacology of the receptor subtype. Although the number of subtypes remains unknown, most receptors appear to be formed by α, β and γ subunits in a 2 : 2 : 1 stoichiometry (Tretter et al. 1997; Farrar et al. 1999). Allosteric modulation is a hallmark feature of the GABAA receptor and this is best exemplified by compounds that act at the benzodiazepine (BZD) binding site. Ligands of many chemical classes recognize this site and these compounds display a spectrum of efficacy from positive to negative allosteric modulation of GABA-gated chloride currents (Barnard et al. 1998).

All receptors in the LGIC family share common structural features, including a similar subunit topology and overall receptor conformation. In all members of the family, agonist binding sites are thought to lie at subunit–subunit interfaces and to be formed by six distinct stretches or ‘loops’ of amino acids; three of these domains (A–C) are contributed by one subunit, and the other three (D–F) are provided by the adjacent subunit in the pentamer (reviewed in Corringer et al. 2000). In the case of the GABAA receptor, GABA binding sites are predicted to lie at the interfaces between the β–α (see Smith and Olsen 1995) and α–β (Newell et al. 2000) subunits. The BZD binding site is predicted to be formed by homologous loops of amino acids that lie at the interface between α and γ subunits (Sigel and Buhr 1997; Cromer et al. 2002; Ernst et al. 2003). BZD pharmacology is determined by the specific α- and γ-subunit isoforms that are present in the receptor oligomer. The incorporation of the α4 or α6 subunit in the receptor, for example, bestows insensitivity to diazepam and other classical 1,4-BZDs, a property that has been ascribed to the substitution of a histidine (H101, rat α1-subunit numbering) by an arginine (Wieland et al. 1992). However, other ligands from diverse classes retain affinity for these diazepam-insensitive (DI) receptors and some display differential affinity for those containing the α4 or α6 isoform. Certain β-carboline and pyrazoloquinolinone ligands, for example, have higher affinity for DI receptors in the cortex compared with the cerebellum (Ito et al. 1994). Studies with recombinant receptors suggest that this may be due to the higher levels of expression of the α4 subunit in the cortex compared with the cerebellum, where the α6 subunit is preferentially expressed (see Yang et al. 1995; Gunnersen et al. 1996; Knoflach et al. 1996).

This study initially focused on the identification of specific residues in the α4 and α6 subunits that confer the differential affinity for certain β-carbolines. In order to identify regions of interest, we used a chimeric approach similar to that used previously in the analysis of binding requirements of BZD ligands (e.g. Boileau et al. 1998). Randomly derived chimeras from the α4 and α6 subunits were created and, based on their observed binding characteristics, individual residues were targeted for site-directed mutagenesis. This led to the identification of a specific residue (α6N204/α4I203) that bestows the differential affinity for β-carbolines. By mutating the homologous residue in the α1 subunit to that found in α6 (S205N), we showed that, although this residue has little influence on binding of the classical agonist flunitrazepam, it has significant effects on the binding of both β-carbolines and the imidazobenzodiazepines (i-BZD) Ro15-1788 and Ro15-4513.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

Ro15-4513 and Ro15-1788 were gifts from Hoffmann-La Roche and Co. (Basel, Switzerland). Ethyl β-carboline-3-carboxylate (β-CCE) was a generous gift from Dr Brian Jones (GlaxoSmithKline, Harlow, UK). Methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) was obtained from RBI/Sigma (Natick, MA, USA). [3H]Ro15-4513 (23.06 Ci/mmol) and [3H]flunitrazepam (84.5 Ci/mmol) were obtained from Perkin-Elmer (Boston, MA, USA).

Production of α-subunit chimeras

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

Several randomly derived α-subunit chimeras (χ) were created following the protocol of Moore and Blakely (1994). Sequences of both the rat α6- and α4-subunit cDNA were subcloned into the same pcDNA3.1(+) expression vector (Invitrogen, San Diego, CA, USA) (Fig. 1). The complete α6 cDNA was inserted (as a HindIII fragment) upstream of a partial α4 cDNA sequence (an EcoRI–XbaI fragment containing an N-terminally truncated α4 sequence). A BamHI restriction site was left in the polylinker between the inserts. Restriction digestion was used to excise the intervening polylinker sequence as well as a portion of the α6 insert, thereby limiting the amount of α6 insert that was homologous to the α4 cDNA. Digestion with BamHI and HpaI or with EcoRI and BstXI limited the region of homology with the α4 insert to a sequence in the α6 cDNA that represents the extracellular N-terminus of the mature α6 subunit. In this manner, random crossover events were targeted to occur in regions of the cDNA that represent the extracellular, ligand binding domain of the α subunits, a technique that has been used previously to produce targeted random chimeras of GABAA receptor subunits (Boileau et al. 1998). Linearized plasmid DNA was transformed into Library Efficiency® competent DH5αEscherichia coli cells (Life Technologies, Gaithersburg, MD, USA) where random crossover events occurred at regions of homology in the α-subunit cDNA sequences, producing in-frame hybrid cDNA. Individual colonies were isolated and the extracted plasmid DNA was assessed for size to indicate a potential recombinant crossover event. For those plasmids showing a chimeric crossover, the switch point was assessed by restriction digestion analysis and confirmed by DNA sequencing. Chimeras are named based on the point of crossover; the nomenclature represents the last residue of N-terminal α6-subunit contribution (rat subunit numbering) before the in-frame switch. Individual chimeras were used for transfection with rat β2 and γ2L plasmid constructs (see below).

image

Figure 1. (a) Schematic of the vector construct that was used to create targeted random chimeras from rat GABA A α6- and α4-subunit cDNA. The entire α6-subunit cDNA was subcloned into pcDNA3.1(+) upstream of a partial α4 sequence (that excludes the signal sequence and the first seven amino acids). This dual plasmid was digested with restriction enzymes to limit the amount of homologous cDNA that was capable of participating in a recombination event between the subunit sequences (see Materials and methods ). (b)  Two chimeric α subunits (χ) were isolated and named according to the last residue of α6 sequence before the switch point. A linear depiction of the subunits is presented. White, α4 sequence; black, α6 sequence; gray, putative transmembrane domains.

Download figure to PowerPoint

Mutagenesis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

Site-directed mutagenesis was performed using the QuikChange protocol (Stratagene, La Jolla, CA, USA). Wild-type rat α6 or α1 cDNA inserts were subcloned into pcDNA3.1 (Invitrogen) and were used as the templates in separate PCR-mediated mutagenesis protocols. Complementary 27-mer mutagenic oligonucleotide primer pairs were synthesized (Department of Biochemistry, DNA Core Laboratory, University of Alberta, Edmonton, Alberta, Canada) for each substitution reaction. PCR amplification with Pfu polymerase (Stratagene) was carried out under the recommended conditions. Plasmid DNA was digested with DpnI endonuclease to degrade methylated template DNA, and was subsequently transformed into competent E. coli cells for propagation of the plasmid. Individual colonies were isolated and plasmid DNA extracted. Successful substitutions within the cDNA insert of the plasmid were confirmed by DNA sequencing.

Transient transfection

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

Recombinant expression of GABAA receptor subtypes was carried out in tsA201 cells. Cells were maintained in low-glucose Dulbecco's modified Eagle's medium (GibcoBRL, Grand Island, NY, USA) supplemented with 10% (v/v) Fetal Clone III bovine serum (HyClone, Logan, UT, USA) and were provided with fresh medium before transient transfection (Davies et al. 2000). Briefly, subunit cDNAs were subcloned into the pcDNA3.1 expression vector (Invitrogen). Plates of cells were transiently co-transfected with 10 µg each of wild-type, mutant or chimeric α-subunit plasmid together with wild-type β2 and γ2L vector constructs in a 1 : 1 : 1 ratio. The DNA was added to an appropriate volume of 250 mm CaCl2, to which was added an equal volume of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer (pH 7.02). Solutions were mixed and added dropwise to plates of cells. Cells were incubated at 37°C in a 3% CO2 incubator for 48 h. After incubation, cells were scraped into ice-cold harvesting buffer (50 mm Tris, 250 mm KCl, pH 7.4) containing protease inhibitors (1 mm benzamidine, 0.1 mg/mL bacitracin, 0.01 mg/mL chicken egg white trypsin inhibitor and 0.5 mm phenylmethylsulfonyl fluoride). Harvested cells were homogenized with two pulses (10 s, 13 500 r.p.m.) of an Ultra Turrax homogenizer (IKA Labortechnik, Staufen, Germany). Homogenates were recovered by centrifugation, resuspended in ice-cold harvesting buffer and stored at − 80°C until the day of experiments.

Radioligand binding

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

Radioligand binding was performed as described previously (Davies et al. 2000). In brief, for equilibrium binding assays, aliquots of cell homogenates were incubated in duplicate with increasing concentrations (0.5 nm−80 nm) of [3H]Ro15-4513 or [3H]flunitrazepam in binding assay buffer (50 mm Tris, 250 mm KCl, 0.02% NaN3, pH 7.4) at 4°C for 60 min. Non-specific binding was determined in the presence of excess unlabeled Ro15-4513 or flunitrazepam as appropriate. For competition binding assays, aliquots of cell homogenates were incubated with a constant concentration of radioligand (at a concentration equal to its Kd for the receptor subtype) and increasing concentrations of displacing ligand. Parallel samples were filtered through GF/B filters (Whatman, Maidstone, UK) using a cell harvester (Brandel, Gaithersburg, MD, USA). The filters were immediately washed three times with 3 mL ice-cold assay buffer before scintillation counting.

Data and statistical analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

Saturation and competition ligand binding data were analyzed using the least-squares non-linear regression curve-fitting programs of GraphPad Prism 3.0 (GraphPad, San Diego, CA, USA). Binding parameters (Kd or Ki) were determined as the mean ± SEM of at least three independent experiments. Data were analyzed by one-way anova and levels of significance were determined by the Dunnett post-test for multiple comparisons or by a two-tailed t-test between two groups.

Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

The α4 and α6 GABAA receptor subunits were each recombinantly expressed with the β2 and γ2L subunits in tsA201 cells for radioligand binding assays. Each receptor subtype recognized [3H]Ro15-4513 with nanomolar affinity (Table 1). This ligand was therefore used in competition studies to determine β-carboline affinities for these receptors. Both β-CCE and DMCM displayed a greater affinity (approximately 6- and 9-fold higher respectively) for the α4-containing subtype (Table 1, see also Fig. 3).

Table 1.  Binding affinities of BZD site ligands at GABA A receptor subtypes that incorporate wild-type, mutant and chimeric α subunits with β2 and γ2 L subunits
α SubunitAffinity (nM)
β-CCEaDMCMaRo15-4513b
  1. Data shown are mean ± SEM from at least three independent experiments performed in duplicate. aAffinity values for β-CCE and DMCM are obtained from studies of the displacement of [3H]Ro15-4513 and are represented as Ki. bAffinity of Ro15-4513 from saturation analysis is given as Kd. ND, not determined. *p < 0.05, ***p < 0.001 versus wild-type α6β2γ2; p < 0.05, †††p < 0.001 versus wild-type α4β2γ2 (one-way anova followed by the Dunnett post-test).

α4347 ± 28*24 ± 5***6.4 ± 0.4
α62270 ± 189222 ± 27†††5.7 ± 0.7
χ141540 ± 59*ND4.1 ± 0.1
χ2075446 ± 1042***,†††ND21.0 ± 3.7***
α6(N143D)2231 ± 176ND3.8 ± 0.1
α6(N204I)210 ± 17*30 ± 7***1.8 ± 0.5
α6(N204S)442 ± 27*41 ± 11***3.3 ± 1.1
image

Figure 3. Competition binding curves of the displacement of [ 3 H]Ro15-4513 by β-CCE. The β-carboline was used to displace the radioligand from membranes prepared from cells expressing either wild-type α4β2γ2 (○) or α6β2γ2 (•) GABA A receptors, receptors incorporating the α-subunit chimeras χ141 (▵) or χ207 (▴), or from receptors including the α6(N204I)β2γ2 (□) or α6(N143D)β2γ2 (▪) mutant α subunits. [ 3 H]Ro15-4513 was present at a concentration equal to its Kd value for each receptor subtype. High-affinity binding of the radioligand to the chimera- and mutant-containing receptors was retained as shown by saturation binding (see Table 1 ). Data shown represent the mean ± SEM of three independent experiments performed in duplicate.

Download figure to PowerPoint

Chimeric α6/α4 subunits

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

Chimeric α subunits were created from random recombination events at the cDNA level. Three chimeras were analyzed initially and, of these, two were identical. The two chimeric subunits with different crossover points (χ141 and χ207) were isolated for transient transfection with β2 and γ2L subunits (Fig. 1b). Expression of receptors containing the chimeric α subunits was verified by the retention of high-affinity binding for [3H]Ro15-4513 (Table 1) although, in the case of the χ207-containing receptor, the affinity for this ligand was reduced by ∼ 4-fold compared with the wild-type α6β2γ2L receptor (p < 0.001).

Competition assays were used to screen for the effect of the chimeric α subunit on β-CCE affinity. The isolation of the initial two chimeras was fortunate, as inclusion of the χ141 or χ207 subunit produced receptor subtypes with an affinity for β-CCE that was α4-like or α6-like respectively (Fig. 3; Table 1). Thus the stretch of amino acids between the two crossover points, α6(L141–E207), was implicated as containing residues that may confer the differential affinities of the α4- and α6-containing receptor subtypes.

Effect of substitution mutations in the α6 subunit

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

The α6(L141–E207) region has a high degree of sequence homology with the same domain in the wild-type α4 subunit; there are only eight divergent residues. Of these eight amino acids, one (α6N143) lies within the characteristic cys-loop region and another (α6N204) lies within the Loop C segment previously implicated in BZD binding (Fig. 2). To investigate their potential roles in β-carboline recognition, these residues in the α6 isoform were individually substituted by their homologues in the α4 subunit, i.e. α6N143D and α6N204I. Expression of receptors bearing these α-subunit mutations was again verified by the retention of high-affinity binding of [3H]Ro15-4513 (Table 1). Although the α6N143D substitution had no significant effect on the affinity for β-CCE, the α6N204I substitution resulted in a greater than 10-fold increase in its apparent affinity (Fig. 3; Table 1), i.e. this mutation bestowed α4-like higher affinity to the wild-type α6 subtype. This substitution also conferred higher-affinity α4-like binding of another β-carboline, DMCM (Fig. 4, Table 1). Thus a single residue in Loop C can account for the differences in β-CCE and DMCM affinities for the α4- and α6-receptor subtypes.

image

Figure 2. Sequence alignment of the α1-, α4- and α6-subunit Loop C region of the extracellular N-terminus. Numbering above the figure represents the rat α1 sequence whereas the number below refers to the rat α6 subunit. Letters in boldface type indicate residues that have been implicated previously in BZD binding (see Sigel 2002 ). The boxed letters indicate the α-subunit residues that are the focus of this present study. The boxes above the sequence represent the structural features proposed by comparative modeling of the GABA A receptor ( Ernst et al. 2003 ) based on the structure of the AChBP ( Brejc et al. 2001 ). White, putative β-strands, β9 and β10; black, structural loop 10 (L10). gray, sequence corresponding to the Loop C domain.

Download figure to PowerPoint

image

Figure 4. Competition binding curves of the displacement of [ 3 H]Ro15-4513 by DMCM. The competition curves show DMCM displacement of the radioligand at wild-type α4β2γ2 (○), α6β2γ2 (•) and α6(N204I)β2γ2 (□) GABA A receptors. [ 3 H]Ro15-4513 was present at a concentration equivalent to its Kd value for each receptor subtype. High-affinity binding of the radioligand to the mutant receptors was retained as shown by saturation binding (see Table 1 ). Data shown represent the mean ± SEM of three independent experiments performed in duplicate.

Download figure to PowerPoint

In the other α subunits (α1–3,5) the residue in the equivalent position to α6N204 is a serine (Fig. 2). A further substitution was therefore the α6N204S mutation. Receptors carrying this mutation again retained high-affinity binding of [3H]Ro15-4513 (Table 1). The affinity of this mutant receptor for both β-CCE and DMCM was increased approximately 5-fold compared with their respective affinities for wild-type α6-containing receptors, again implicating this residue in β-carboline recognition.

Effect of substitution mutations in the α1 subunit

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

In additional experiments, we characterized the binding of β-CCE and DMCM to the wild-type α1β2γ2L receptor (Table 2). Within the α1 subunit, a substitution with the α6-subunit asparagine for the homologous serine residue (α1S205N) produced a mutant receptor with reduced affinity for β-CCE and DMCM (∼6.5- and ∼8-fold respectively), compared with the wild-type α1-containing receptors. Although this α1S205N substitution led to a slight increase (∼3-fold) in the affinity for [3H]flunitrazepam, there were considerable decreases in the affinities for both of the i-BZD compounds Ro15-1788 (∼ 19-fold) and Ro15-4513 ( ∼38-fold) (Fig. 5; Table 2).

Table 2.  Binding affinities of BZD site ligands at GABA A receptor subtypes that incorporate wild-type and mutant α1 subunits with β2 and γ2 L subunits
α SubunitAffinity (nM)
FNZaRo15-4513bRo15-1788bβ-CCEbDMCMb
  1. Data shown are mean ± SEM from at least three independent experiments performed in duplicate. aAffinity of flunitrazepam (FNZ) was determined by saturation binding analysis and is given as the Kd value. bAffinity values of Ro15-4513, Ro15-1788, β-CCE and DMCM are given as Ki values determined from the displacement of [3H]flunitrazepam or [3H]Ro15-4513. cData from Davies et al. (1998). *p < 0.05, **p < 0.01 versus wild-type α1β2γ2 (two-tailed t-test).

α16.1 ± 0.4c3.2 ± 0.31.3 ± 0.10.43 ± 0.0211 ± 1
α1(S205N)2.0 ± 0.5**121 ± 15**25 ± 5*2.8 ± 0.2**88 ± 13**
image

Figure 5. Displacement of [ 3 H]flunitrazepam binding by Ro15-1788 and Ro15-4513. Competition binding curves show the displacement of [ 3 H]flunitrazepam from wild-type α1β2γ2 L receptors (open symbols) and from mutant α1(S205N)β2γ2 L receptors (filled symbols) by Ro15-1788 (○,•) and Ro15-4513 (□,▪). [ 3 H]flunitrazepam was present for competition binding experiments at a concentration equal to its Kd at the receptor subtypes ( Table 2 ). Data shown represent the mean ± SEM of three independent experiments performed in duplicate. Inset shows representative curve of [ 3 H]flunitrazepam saturation binding at the α1(S205N)β2γ2 subtype ( Kd  = 2.0 ± 0.5 n m ).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References

Beyond the simple dichotomy of diazepam-sensitive (DS) and DI GABAA receptor subtypes, ligand binding to the BZD site of the DI subtypes can be pharmacologically differentiated by the inclusion of either the α4 or the α6 subunit. In agreement with previous reports (Yang et al. 1995; Gunnersen et al. 1996; Knoflach et al. 1996), we found that β-carbolines (β-CCE and DMCM) display higher affinity for receptors containing the α4 subunit. We then used two subunit chimeras randomly derived from both the α4 and α6 isoforms and site-specific mutagenesis to probe the basis of this differential affinity. Substitution of a single residue within the Loop C segment (α6N204I) was sufficient to bestow α4-like higher affinity to the lower-affinity α6-containing receptor. Several residues from the α-subunit Loop C segment have been shown previously to play a role in BZD binding (see Sigel 2002). The present results now implicate an additional residue in this domain of the α4 and α6 subunits as a determinant of β-carboline recognition.

The results from additional mutagenesis studies show that the amino acid in the equivalent position in the α1 subunit also influences β-carboline binding. Introduction of a serine (conserved in α1–3,5) into the α6 isoform (α6N204S) resulted in an approximately 5-fold increase in the affinity for both β-CCE and DMCM. The reciprocal substitution at the homologous position in the α1 subunit (α1S205N) reduced the affinity (∼ 7–8-fold) of these ligands compared with that of wild-type α1-containing receptors. Thus structural determinants for the binding of these ligands occur in similar domains of both DI and DS receptors. These results are consistent with previous reports of altered β-carboline pharmacology upon mutation of other residues within the Loop C domain of the α1 subunit. Substitutions at the neighbouring α1T206 (T206A, Buhr et al. 1997; T206V, Schaerer et al. 1998) increase DMCM affinity by 5- and 40-fold respectively.

The present study also demonstrates the importance of Ser205 in the α1 subunit in the binding of different classes of BZD site ligands. In addition to the effects on β-carboline affinity, the α1S205N substitution led to a modest increase (∼ 3-fold) in the affinity for [3H]flunitrazepam, consistent with a previous report in which the α1S205A mutation did not compromise the ability of classical 1,4-BZDs to potentiate GABA-gated chloride currents (Buhr et al. 1996). In contrast, the α1S205N substitution significantly decreased the affinity for the i-BZDs Ro15-1788 and Ro15-4513, by ∼ 19- and ∼38-fold respectively. The large reduction in the affinity for Ro15-4513 was unexpected as homologous mutations in the α6 subunit (α6N204I, α6N204S) had no appreciable effect on the binding of this ligand, permitting its use as a reporter ligand in displacement studies of β-carboline binding. In previous studies we have found that the binding of Ro15-4513 is much more resistant to other α1-subunit mutations than the binding of flunitrazepam and Ro15-1788 (Davies et al. 1998). Additionally, several mutations within the α1-subunit Loop C domain have smaller effects on the binding of Ro15-1788 and Ro15-4513; substitutions at the adjacent α1S204 and α1T206 residues (Buhr et al. 1997; Schaerer et al. 1998; Casula et al. 2001) alter the affinity of Ro15-1788 and Ro15-4513 by only ∼ 2–4-fold. The considerable effect of the α1S205N substitution on i-BZD affinity suggests a specific interaction of the α1Ser205 residue with these ligands. As mutation at the homologous residue in the α6 isoform did not change the affinity of Ro15-4513, there is an apparent differential contribution of this residue to i-BZD binding at the DS and DI subtypes.

The results of the reciprocal α6/α1 mutations emphasize the divergent requirements for ligand recognition at the DI compared with the DS subtypes, in particular for Ro15-4513. Several lines of evidence, including opposite efficacies at DI and DS receptors (Hadingham et al. 1996; Dunn et al. 1999), suggest that Ro15-4513 interacts differently with each receptor subtype. Distinct domains of α subunits are photolabeled by [3H]Ro15-4513 in native bovine cortical DS receptors compared with cerebellar DI receptors (Duncalfe and Dunn 1996); however, this difference was not reproduced in recombinant receptors containing the α1 or α6 subunits (Davies et al. 1996). The substrate of [3H]Ro15-4513 labeling in cortical receptors has been mapped to the extracellular loop between transmembrane domains 2 and 3 (Davies and Dunn 1998), but more recent investigations have identified α1Y209 (and the homologous tyrosine in the α2 and α3 subunits) as a major site of Ro15-4513 labeling (Sawyer et al. 2002). The proximity of this residue to α1S205 supports a role of this domain in the recognition of Ro15-4513 at α1-containing receptors. Determination of the site for covalent incorporation of Ro15-4513 into the α6 (and α4) subunits remains to be determined.

Recent homology models of the BZD site based on the molluscan acetylcholine binding protein (AChBP) (Brejc et al. 2001) propose a specific orientation of Ro15-4513 within the binding cleft at the α1–γ2-subunit interface (Sawyer et al. 2002; Kucken et al. 2003) and predict the proximity of the photoreactive 8′-azide for interaction with α1Y209. Ligand mapping modeling of the BZD pharmacophore predicts structural differences of the DI BZD site, including a smaller binding pocket created with the α6 isoform (e.g. Huang et al. 2000; He et al. 2000). Further predictions suggest that the 8′-substituent of i-BZD ligands underlies DS/DI subtype selectivity and that the 8′-azide moiety of Ro15-4513 must adopt different diastereomeric conformations for lowest energy docking within the DS and DI binding sites (Wong et al. 1993; Huang et al. 2000). Although the present mutational results cannot be directly compared with those from pharmacophore models, disparate effects of the α1S205N/α6N204S substitutions on Ro15-4513 binding are consistent with a differential interaction of the 8′-azide with the Loop C domain of the α subunit at DS and DI subtypes.

Comparative modeling of the BZD site (Cromer et al. 2002; Ernst et al. 2003) suggests that, although there exists conservation of the overall hairpin structure of Loop C, low sequence homology with the AChBP (Brejc et al. 2001) in this region leads to high model variability. In particular, the low homology prevents the exact placement of the preceding β-strand segment (β9) and hampers the determination of the length of the bend (L10; centered on Loop C) between the β9 and β10 structural β strands of the proposed α-subunit structure (see Fig. 2; Ernst et al. 2003). As a consequence, a unified model across all α isoforms is not possible. The variable L10 region contains the identified α1S205/α6N204 divergence; structural differences in this region at DI and DS BZD sites may underlie the disparate effects of substitution on Ro15-4513 recognition. Additional residues from Loop C that have been implicated in BZD ligand selectivity (see Sigel 2002) align to the β-sheet structures that flank the L10 hairpin, indicating a more complex region that influences the structure and pharmacology of the binding site. Interestingly, inclusion of the χ207 subunit (with the crossover point in Loop C) reduced the affinity of Ro15-4513 compared with that of either wild-type DI receptor, implying that the exact structure of this region influences Ro15-4513 recognition. The reduced affinity of Ro15-4513 at χ207-containing receptors does not negate the usefulness of that chimeric subunit in identifying α6N204 as a mediator of β-carboline affinity at the DI subtypes, and emphasizes the complex contributions to ligand recognition from multiple residues at the α–γ-subunit interface. Further investigation of the differences in the binding sites produced with the different α isoforms (see Sigel 2002) is warranted to better understand ligand recognition, especially at the DI BZD binding site.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production of α-subunit chimeras
  6. Mutagenesis
  7. Transient transfection
  8. Radioligand binding
  9. Data and statistical analysis
  10. Results
  11. Binding to wild-type α4β2γ2 and α6β2γ2 GABAA receptors
  12. Chimeric α6/α4 subunits
  13. Effect of substitution mutations in the α6 subunit
  14. Effect of substitution mutations in the α1 subunit
  15. Discussion
  16. Acknowledgements
  17. References
  • Barnard E. A., Skolnick P., Olsen R. W., Mohler H., Sieghart W., Biggio G., Braestrup C., Bateson A. N. and Langer S. Z. (1998) International Union of Pharmacology XV. Subtypes of γ-aminobutyric acidA receptors: classification on the basis of subunit structure and function. Pharmacol. Rev. 50, 291313.
  • Boileau A. J., Kucken A. M., Evers A. R. and Czajkowski C. (1998) Molecular dissection of benzodiazepine binding and allosteric coupling using chimeric γ-aminobutyric acidA receptor subunits. Mol. Pharmacol. 53, 295303.
  • Brejc K., Van Dijk W. J., Klaassen R. V., Schuurmans M., Van Der Oost J., Smit A. B. and Sixma T. K. (2001) Crystal structure of an Ach-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269276.
  • Buhr A., Baur R., Malherbe P. and Sigel E. (1996) Point mutations of the α1β2γ2 γ-aminobutyric acidA receptor affecting modulation of the channel by ligands of the benzodiazepine binding site. Mol. Pharmacol. 49, 10801084.
  • Buhr A., Schaerer M. T., Baur R. and Sigel E. (1997) Residues at position 206 and 209 of the α1 subunit of γ-aminobutyric acidA receptors influence affinities for benzodiazepine site ligands. Mol. Pharmacol. 52, 676682.
  • Casula M. A., Bromidge F. A., Pillai G. V., Wingrove P. B., Martin K., Maubach K., Seabrook G. R., Whiting P. J. and Hadingham K. L. (2001) Identification of amino acid residues responsible for the α5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABAA receptor. J. Neurochem. 77, 445451.
  • Corringer P.-J., Le Novère N. and Changeux J.-P. (2000) Nicotinic receptors at the molecular level. Annu. Rev. Pharmacol. Toxicol. 40, 431458.
  • Cromer B. A., Morton C. J. and Parker M. W. (2002) Anxiety over GABAA receptor structure relieved by AChBP. Trends Biochem. Sci. 27, 280287.
  • Davies M. and Dunn S. M. J. (1998) Identification of a unique domain in bovine brain GABAA receptors that is photoaffinity labeled by [3H]Ro15-4513. Biochem. Biophys. Res. Commun. 246, 650653.
  • Davies M., Martin I. L., Bateson A. N., Hadingham K. L., Whiting P. J. and Dunn S. M. J. (1996) Identification of domains that are photoaffinity labeled by [3H]flunitrazepam and [3H]Ro15-4513. Neuropharmacology 35, 11991208.
  • Davies M., Bateson A. N. and Dunn S. M. J. (1998) Structural requirements for ligand interactions at the benzodiazepine recognition site of the GABAA receptor. J. Neurochem. 70, 21882194.
  • Davies M., Newell J. G., Derry J. M. C., Martin I. L. and Dunn S. M. J. (2000) Characterization of the interaction of zopiclone with γ-aminobutyric acid type A receptors. Mol. Pharmacol. 58, 756762.
  • Duncalfe L. L. and Dunn S. M. J. (1996) Mapping of GABAA receptor sites that are photoaffinity-labelled by [3H]flunitrazepam and [3H]Ro15-4513. Eur. J. Pharmacol. 298, 313319.
  • Dunn S. M. J., Davies M., Muntoni A. L. and Lambert J. J. (1999) Mutagenesis of the rat alpha1 subunit of the gamma-aminobutyric acid(A) receptor reveals the importance of residue 101 in determining the allosteric effects of benzodiazepine site ligands. Mol. Pharmacol. 56, 768774.
  • Ernst M., Brauchart D., Boresch S. and Sieghart W. (2003) Comparative modeling of GABAA receptors: limits, insights, future developments. Neuroscience 119, 933943.
  • Farrar S. J., Whiting P. J., Bonnert T. P. and McKernan R. M. (1999) Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer. J. Biol. Chem. 274, 1010010104.
  • Gunnersen D., Kaufman C. M. and Skolnick P. (1996) Pharmacological properties of recombinant ‘diazepam-insensitive’ GABAA receptors. Neuropharmacology 35, 13071314.
  • Hadingham K. L., Garrett E. M., Wafford K. A., Bain C., Heavens R. P., Sirinathsinghji D. J. S. and Whiting P. J. (1996) Cloning of cDNAs encoding the human γ-aminobutyric acid type A receptor α6 subunit and characterization of the pharmacology of α6-containing receptors. Mol. Pharmacol. 49, 253259.
  • He X., Huang Q., Ma C., Yu S., McKernan R. and Cook J. M. (2000) Pharmacophore/receptor models for GABAA/BzR α2β3γ2, α3β3γ2 and α4β3γ2 recombinant subtypes. Included volume analysis and comparison to α1β3γ2, α5β3γ2 and α6β3γ2 subtypes. Drug Des. Discov. 17, 131171.
  • Huang Q., He X., Ma C., Liu R., Yu S., Dayer C. A., Wenger G. R., McKernan R. and Cook J. M. (2000) Pharmacophore/receptor models for GABAA/BzR subtypes (α1β3γ2, α5β3γ2, and α6β3γ2) via a comprehensive ligand-mapping approach. J. Med. Chem. 43, 7195.
  • Ito Y., Abiko E., Mitani K. and Fukuda H. (1994) Characterization of diazepam-insensitive [3H]Ro 15-4513 binding in rodent brain and cultured cerebellar neuronal cells. Neurochem. Res. 19, 289295.
  • Knoflach F., Benke D., Wang Y., Scheurer L., Lüddens H., Hamilton B. J., Carter D. B., Mohler H. and Benson J. A. (1996) Pharmacological modulation of the diazepam-insensitive recombinant γ-aminobutyric acidA receptors α4β2γ2 and α6β2γ2. Mol. Pharmacol. 50, 12531261.
  • Korpi E. R., Gründer G. and Lüddens H. (2002) Drug interactions at GABAA receptors. Prog. Neurobiol. 67, 113159.
  • Kucken A. M., Teissére J. A., Seffinga-Clark J., Wagner D. A. and Czajkowski C. (2003) Structural requirements for imidazobenzodiazepine binding to GABAA receptors. Mol. Pharmacol. 63, 289296.
  • Moore K. R. and Blakely R. D. (1994) Restriction site-independent formation of chimeras from homologous neurotransmitter–transporter cDNAs. Biotechniques 17, 130136.
  • Newell J. G., Davies M., Bateson A. N. and Dunn S. M. J. (2000) Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding. J. Biol. Chem. 275, 1419814204.
  • Ortells M. O. and Lunt G. G. (1995) Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends Neurosci. 18, 121127.
  • Sawyer G. W., Chiara D. C., Olsen R. W. and Cohen J. B. (2002) Identification of the bovine γ-aminobutyric acid type A receptor α subunit residues photolabeled by the imidazobenzodiazepine [3H]Ro15-4513. J. Biol. Chem. 277, 5003650045.
  • Schaerer M. T., Buhr A., Baur R. and Sigel E. (1998) Amino acid residue 200 on the α1 subunit of GABAA receptors affects the interaction with selected benzodiazepine binding site ligands. Eur. J. Pharmacol. 354, 283287.
  • Sigel E. (2002) Mapping of the benzodiazepine recognition site on GABAA receptors. Curr. Top. Med. Chem. 2, 833839.
  • Sigel E. and Buhr A. (1997) The benzodiazepine binding site of GABAA receptors. Trends Pharmacol. Sci. 18, 425429.
  • Smith G. B. and Olsen R. W. (1995) Functional domains of GABAA receptors. Trends Pharmacol. Sci. 16, 162168.
  • Tretter V., Ehya N., Fuchs K. and Sieghart W. (1997) Stoichiometry and assembly of a recombinant GABAA receptor subtype. J. Neurosci. 17, 27282737.
  • Wieland H. A., Lüddens H. and Seeburg P. H. (1992) A single histidine in GABAA receptors is essential for benzodiazepine agonist binding. J. Biol. Chem. 267, 1429.
  • Wong G., Koehler K. F., Skolnick P., Gu Z.-Q., Ananthan S., Schönholzer P., Hunkeler W., Zhang W. and Cook J. M. (1993) Synthetic and computer-assisted analysis of the structural requirements for selective, high-affinity ligand binding to the diazepam-insensitive benzodiazepine receptors. J. Med. Chem. 36, 18201830.
  • Yang W., Drewe J. A. and Lan. N. C. (1995) Cloning and characterization of the human GABAA receptor α4 subunit: identification of a unique diazepam-insensitive binding site. Eur. J. Pharmacol. 291, 319325.