Abstract
Imidazobenzodiazepines such as RY-80 have been reported to exhibit both high affinity and selectivity for GABAA receptors containing an α5 subunit. A single amino acid residue (α5Ile215) has been identified that plays a critical role in the high-affinity, subtype-selective effects of RY-80 and structurally related ligands. Thus, substitution of α5Ile215 with the cognate amino acid contained in the α1 subunit (Val211) reduced the selectivity of RY-80 for α5β3γ2 receptors from ∼135- to ∼8-fold compared with α1β3γ2 receptors. This mutation produced a comparable reduction in the selectivity of RY-24 (a structural analog of RY-80) for α5β3γ2 receptors but did not markedly alter the affinities of ligands (e.g., flunitrazepam) that are not subtype-selective. Conversely, substitution of the α1subunit with the cognate amino acid contained in the α5subunit (i.e., α1V211I) increased the affinities of α5-selective ligands by a ∼20-fold and reduced by 3-fold the affinity of an α1-selective agonist (zolpidem). Increasing the lipophilicity (e.g., by substitution of Phe) of α5215 did not significantly affect the affinities (and selectivities) of RY-80 and RY-24 for α5-containing GABAA receptors. However, the effect of introducing hydrophilic and or charged residues (e.g., Lys, Asp, Thr) at this position was no greater than that produced by the α5I215V mutation. These data indicate that residue α5215 may not participate in formation of the lipophilic L2 pocket that has been proposed to contribute to the unique pharmacological properties of α5-containing GABAA receptors. RY-80 and RY-24 acted as inverse agonists in both wild-type α5β3γ2 and mutant α5I215Kβ3γ2 receptors expressed in Xenopus laevis oocytes. However, both RY-24 and RY-80 acted as antagonists at mutant α5I215Vβ3γ2 and α5I215Tβ3γ2 receptors, whereas the efficacy of flunitrazepam was similar at all three receptor isoforms. The data demonstrate that amino acid residue α5215 is a determinant of both ligand affinity and efficacy at GABAA receptors containing an α5subunit.
The principal therapeutic actions of drugs such as the benzodiazepines (e.g., diazepam), imidazopyridines (e.g., zolpidem), and triazolopyridazines (e.g., zaleplon) are effected through the family of GABAA receptors (Lüddens et al., 1995; Korpi et al., 1997; Sigel and Buhr, 1997). Based on sequence homology, 17 distinct subunits belonging to six related families (α1–6, β1–3, γ1–3, δ, ε, ρ1–2, θ) have been identified as members of this group of ligand-gated ion channels (for review, see McKernan and Whiting, 1996; Sigel and Buhr, 1997; Bonnert et al., 1999). Assuming a pentameric arrangement (Nayeem et al., 1994), there is a remarkable potential for GABAA receptor heterogeneity. Nonetheless, no more than 10 to 20 distinct GABAAreceptor isoforms have been identified in the adult rat central nervous system (Fritschy and Mohler, 1995; De Blas, 1996; McKernan and Whiting, 1996), with the majority existing as heteromers composed of α-, β-, and γ-subunits (Fritschy and Mohler, 1995; De Blas, 1996). Although the stoichiometry of native GABAA receptors has not been definitively established, several studies have proposed a GABAA receptor configuration as consisting of 2α-, 2β-, and 1γ-subunit (Chang et al., 1996; Tretter et al., 1997).
Studies using recombinant GABAA receptors have demonstrated that subunit composition defines ligand pharmacology at these ligand-gated ion channels (Pritchett and Seeburg, 1990; Hadingham et al., 1993). This principle is amply illustrated by the impact of the α-subunit on the affinities of a chemically diverse group of substances often termed benzodiazepine site ligands (for review, seeLüddens et al., 1995; Korpi et al., 1997). For example, prototypic 1,4- benzodiazepines such as diazepam and flunitrazepam possess high (nM) affinities for GABAA receptors containing α1,2,3 and α5 subunits [comprising the “diazepam-sensitive” (DS) family of GABAAreceptors], but are essentially inactive at receptors containing α4 and α6 subunits [the “diazepam-insensitive” family of GABAAreceptors] (Korpi et al., 1992; Wong et al., 1992; Wieland and Lüddens, 1994; Fritschy and Mohler, 1995). This remarkable effect on ligand affinity is determined in a large part, by a single histidine residue in homologous positions α1101, α2101, α3126, and α5105 of the DS α-subunits and the cognate arginine in position 100 on the α4 and α6 receptors (Wieland et al., 1992; Benson et al., 1998).
The affinities of 1,4-benzodiazepines are very similar among both recombinant and native DS receptors (Mohler et al., 1978; Pritchett and Seeburg, 1990; Graham et al., 1996). Several nonbenzodiazepine molecules, including CL 218,872 and zolpidem, exhibit some selectivity for recombinant GABAA receptors bearing an α1 subunit and possess higher affinities in brain regions (e.g., cerebellum) that are relatively enriched in this species (Squires et al., 1979; Pritchett and Seeburg, 1990; Hadingham et al., 1993). Only recently have very high-affinity, selective compounds been developed for less abundant GABAAreceptor isoforms. Thus, based on the ∼10-fold selectivity of Ro 15-4513 for GABAA receptors containing an α5 subunits (Hadingham et al., 1993;Lüddens et al., 1994), compounds such as RY-80, RY-24, and L-655,708 have been developed (Liu et al., 1995, 1996; Quirk et al., 1996). These imidazodiazepine derivatives exhibit high affinity and selectivity for wild-type and recombinant GABAAreceptors containing an α5 subunit (Liu et al., 1995, 1996; Skolnick et al., 1997; Sur et al., 1998, 1999). Using these compounds as probes, we now identify a single amino acid residue on the α5 subunit (Ile215) that is critical for ligand selectivity at recombinant α5β3γ2receptors.
Materials and Methods
Transfection of Recombinant GABAA Receptors and Membrane Preparation.
cDNAs encoding rat α1 and α5 subunits were subcloned into a pRc/CMV vector, as described elsewhere (Skolnick et al., 1997). The β3 and γ2S cDNAs were subcloned into pcDNA3 (Gunnersen et al., 1996). Site-directed mutagenesis was performed with QuickChange mutagenesis kit (Stratagene, La Jolla, CA). Presence of the desired mutations was verified by direct sequencing. To verify the absence of new, unwanted substitutions, the complete coding regions were sequenced for each mutant. In case of α5I215F mutant, the plasmid resulting from the QuickChange mutagenesis reaction was digested with PflMI endonuclease, and the fragment containing the desired I215F substitution was gel-purified and ligated into the similarly digested wild-type pRc/CMV/a5 vector. Human embryonic kidney 293 cells (American Type Culture Collection, Manassas, VA) were maintained at 37°C in 5% CO2 as previously described (Gunnersen et al., 1996). Cells were transfected with equal amounts (5 μg of each DNA/90-mm dish) by calcium phosphate precipitation as described previously (Gorman et al., 1990). The cells were harvested 48 h after transfection, by washing with ice-cold phosphate-buffered saline and centrifuged at 1000g. Cells were washed three times by homogenization in ice-cold 50 mM Tris-citrate buffer, pH 7.8, and centrifuged at 20000g. These membrane suspensions were stored at −70°C until needed.
Radioligand Binding.
Incubations were performed in a final volume of 600 to 1000 μl and contained resuspended cell membranes (∼0.02–0.1 mg of protein), 0.2 M NaCl, [3H]Ro 15-1788, or [3H]RY-80 (87 and 55.4 Ci/mmol, respectively; DuPont-New England Nuclear, Boston, MA), and 50 mM Tris-citrate buffer, pH 7.8, to volume. In competition experiments, 50 μl of buffer was replaced by drugs. [3H]Ro 15-1788 was used at concentrations equal to its K D values at the respective receptor subtype. Nonspecific binding was determined with Ro 15-1788 (10 μM). [3H]Muscimol binding was determined using a membrane suspension (∼0.02–0.1 mg of protein), [3H]muscimol (20 Ci/mmol; DuPont-New England Nuclear), and 50 mM Tris-citrate buffer, pH 7.8, to volume. Nonspecific binding in this case was determined in presence of 1 mM GABA. Assays were incubated at 4°C for 2 h and terminated by rapid filtration (Brandel M-48R, Gaithersburg, MD) through GF/B filters followed by two 5-ml washes with ice-cold Tris-citrate buffer. The filter-retained radioactivity was determined by liquid scintillation counting. Data were analyzed with GraphPad Prism software (GraphPad Software Inc., San Diego, CA), and K ivalues were calculated from the equation,K i = IC50/(1 + [radioligand]/K D). BothK i and K Dvalues were calculated from at least three independent experiments performed in duplicate. Statistical significance was determined using a one-way ANOVA followed by a Dunnett's multiple comparison post hoc test. Protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). RY-24 and RY-80 were synthesized at the University of Wisconsin-Milwaukee, CL 218,872 was obtained from Lederle Laboratories (Mont-St-Guibert, Belgium), and zolpidem was obtained from Synthelabo (Laboratoire Experimental Recherche Synthelabo, Paris, France). Flunitrazepam was purchased from Research Biochemicals International (Natick, MA). The structures of Ro 15-1788 and related α5-selective benzodiazepines are given in Fig. 1. All other reagents and chemicals were from standard commercial sources.
Expression in Xenopus laevis Oocytes.
X. laevis frogs were purchased from Xenopus-1 (Dexter, MI). Collagenase B was from Boehringer Mannheim (Indianapolis, IN). All other compounds were from Sigma Chemical Co. Capped cRNA was synthesized from linearized template cDNA encoding the subunits using mMESSAGE mMACHINE kits (Ambion, Austin, TX). Oocytes were injected with cRNAs encoding the specified α5 subunit variants along with the β3 and γ2 subunits in a ratio of 1:1:1 as determined by gel electrophoresis. Mature X. laevis frogs were anesthetized by submersion in 0.1% 3-aminobenzoic acid ethyl ester, and oocytes were surgically removed. Follicle cells were removed by treatment with collagenase B for 2 h. Each oocyte was injected with 5 to 25 ng of cRNA in 50 nl of water and incubated at 19°C in modified Barth's saline (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.41 mM CaCl2, 0.82 mM MgSO4, 100 μg/ml gentamicin, and 15 mM HEPES, pH 7.6). Recordings were performed 1 to 7 days post injection.
Electrophysiological Recordings
Oocytes were perfused at room temperature in a Warner Instruments oocyte recording chamber #RC-5/18 (Hamden, CT) with perfusion solution (115 mM NaCl, 1.8 mM CaCl2, 2.5 mM KCl, 10 mM HEPES, pH 7.2). Perfusion solution was gravity fed continuously at a rate of 15 ml/min. GABA was dissolved in the perfusion solution. Drugs were added as a 10 mM solution in ethanol to the perfusion solution to achieve the appropriate concentration.
Unless otherwise indicated, current responses to GABA application were measured under two-electrode voltage clamp at a holding potential of −60 mV. Data were collected using a GeneClamp 500 amplifier and Axoscope software (Axon Instruments, Foster City, CA). GABA responses were measured at concentrations of GABA equal to its EC50 values for all receptors tested. GABA responses in the presence of saturating concentrations of drugs are reported as a percentage of the response to GABA alone (“percent control response”, or “% control”). Data were fitted to a four-parameter logistic using GraphPad Prizm. Statistical significance was determined using a one-way ANOVA followed by a Bonferroni's multiple comparison post hoc test.
Results
Amino Acid Ile215 on the α5 Subunit Contributes to Ligand Selectivity at α5β3γ2Receptors.
In an attempt to define amino acid residues on the α5 subunit that are important for high affinity and selectivity to compounds such as RY-80, we considered amino acid residues that are conserved among all other DS α-subunits (Fig.2). Based on this sequence comparison, four residues in the α5 subunit N-terminal domain were selected for the initial analysis as the most likely to be involved in defining ligand selectivity. The corresponding amino acids on the other DS α-subunits were substituted on the α5 subunit, yielding α5G24R, α5P166T, α5H196D, and α5I215V variants, respectively. Wild-type and mutant α5β3γ2receptors were transiently expressed in the human embryonic kidney 293 cells. No additional mutations were found after sequencing the complete coding regions for each mutant. The screening strategy applied to identify amino acid(s) important for ligand selectivity at α5 subunit was based on the premise that at the concentrations approximating the K D value of each ligand the binding of [3H]Ro 15-1788 and [3H]RY-80 to the wild-type α5β3γ2receptor will be similar, yielding a ratio of ∼1. If a particular amino acid substitution introduced in the α5subunit altered [3H]RY-80 binding, this ratio would change. Among the amino acid substitutions tested, only α5I215V yielded a dramatically different ratio (17.8) than the value obtained (1.04) in wild-type α5β3γ2receptors (data not shown). Based on this observation, the mutant receptor α5I215Vβ3γ2was chosen for further analysis. Saturation analysis revealed a ∼16-fold decrease in affinity for [3H]RY-80 in α5I215Vβ3γ2receptor (5.6 ± 0.6 nM) compared with wild-type receptor (0.35 ± 0.02 nM) (Fig. 3A). Consistent with these data, the affinities of the α5-selective compounds RY-80 and RY-24 in mutant receptors were reduced ∼20- and ∼10-fold, respectively, in competition studies using [3H]Ro 15-1788 (Fig.3B; Table 1). In contrast, the affinity of the nonselective ligand [3H]Ro 15-1788 was not significantly affected by this mutation (Table 1; Fig. 3B).
Properties of α1V211Iβ3γ2Receptor.
The dramatic effect produced by substitution of a valine in position α5215 on the affinities of the α5-selective ligands prompted an examination of the effect of a cognate substitution (isoleucine for valine) on position α1211 (corresponding to α5215). Consistent with the previous reports (Liu et al., 1996) both RY-24 and RY-80 exhibited low affinities (56 ± 17 and 47 ± 8 nM, respectively) for wild-type α1β3γ2receptors (Table 1). This exchange (α1V211Iβ3γ2) increased the affinities of RY-24 and RY-80 by more than one order of magnitude (to 3.3 ± 0.3 and 3.7 ± 0.3 nM, respectively) (Table 1). Moreover, this mutation decreased the affinities of the α1-selective ligands zolpidem and CL 218,872 by 3-fold without affecting the affinity of [3H]Ro 15-1788 (Table 1; Fig. 3).
Properties of Mutant α5β3γ2 Receptors with Lipophilic Amino Acid Substitutions in Position α5215.
Based on the hypothesis that interaction with a lipophilic pocket is required for ligand selectivity at the α5-containing GABAAreceptors (Liu et al., 1996), isoleucine in position 215 of the wild-type α5 subunit was exchanged with alanine, leucine, or phenylalanine. The ligand binding properties of the α5I215Aβ3γ2receptor were similar to those of the α5I215Vβγ receptor. The alanine substitution decreased (by >10-fold) the affinities of both RY-24 and RY-80 without altering the affinity of [3H]Ro 15-1788. In contrast to the valine substitution, introduction of alanine in position α5215 increased the affinity of CL 218,872 by 10-fold (Table 1). Substitution of either leucine or the more lipophilic phenylalanine resulted in no significant change in the affinities of RY-24 and RY-80. Furthermore, substitution α5I215F resulted in a slightly reduced affinity of Ro 15-1788 and a lower affinity of CL 218,872. The affinity of flunitrazepam was unchanged (compared with wild-type receptors) for both α5I215Aβ3γ2and α5I215Lβ3γ2receptors (K i values of 0.6 ± 0.1 and 0.9 ± 0.2 nM, respectively), whereas the affinity for flunitrazepam at the α5I215Fβ3γ2receptors was decreased ∼7-fold (K i = 6.9 ± 1.8 nM). A decrease in the affinity of α5-selective ligands at α5I215Aβ3γ2and α5I215Vβ3γ2receptors prompted us to further reduce the size of the side chain of the residue α5215. However, introduction of glycine resulted in levels of [3H]Ro 15-1788, [3H]RY-80, or [3H]muscimol binding that were barely detectable.
Properties of Mutant α5β3γ2 Receptors with Charged or Polar Amino Acid Residues in Position α5215.
Substitution of the negatively charged aspartate residue at position 215 produced a modest decrease in affinity of [3H]Ro 15-1788 (1.7 ± 0.3 nM compared with 0.36 ± 0.04 nM for the wild-type receptor), and a similar, modest decrease in the affinities of RY-80 and RY-24 binding (Table 1). Substitution of a threonine (a more hydrophilic amino acid) for isoleucine yielded a receptor with properties similar to α5I215Vβ3γ2receptor. This receptor produced a 10-fold decrease in affinities of both RY-24 and RY-80 without significantly affecting [3H]Ro 15-1788 binding. However, isoleucine-to-threonine substitution resulted in a small (3-fold) increase in the affinity of flunitrazepam (Table 1). Substitution of a basic lysine residue for isoleucine produced a 5- to 6-fold decrease in the affinities of both RY-24 and RY-80 without affecting the affinity of [3H]Ro 15-1788. The affinity of flunitrazepam also was not substantially changed (Table 1). Additionally, α5I215Dβ3γ2, α5I215Tβ3γ2, and α5I215Kβ3γ2receptors displayed an increase in affinity of CL 218,872 (4-fold) compared with wild-type receptors; however, none of the amino acid substitutions in position α5215 yielded a receptor variant with any measurable affinity for zolpidem.
Efficacy of RY-24 and RY-80 at Wild-Type and Mutant α5β3γ2 Receptors.
Mutation of a conserved histidine residue in the N-terminal domain of all DS α-subunits (α1H101R, α2H101R, α3H126R, and α5H105R) to arginine not only confers diazepam insensitivity to the respective αxβ2/3γ2receptors but also alters the efficacies of several ligands at these receptors (Benson et al., 1998). Based on these observations, the potential role of α5215 in modulating ligand efficacy was examined. Three mutant receptors, α5I215Vβ3γ2, α5I215Kβ3γ2, and α5I215Tβ3γ2were examined. Introduction of either valine, lysine, or threonine in position α5215 did not change the potency of GABA at these receptor subtypes (EC50 = ∼30 μM for all receptors tested; see Table2, legend).
The benzodiazepine flunitrazepam potentiated GABA-mediated currents in wild-type α5β3γ2receptors as well as the α5I215Vβ3γ2, α5I215Kβ3γ2, and α5I215Tβ3γ2, mutants (Table 2). Consistent with previous results, RY-24 and RY-80 act as inverse agonists at α5β3γ2receptors (Liu et al., 1995, 1996; Skolnick et al., 1997), producing a maximum reduction in GABA-evoked currents to 75 ± 2 and 70 ± 6% of the control response, respectively, when GABA was applied at its EC50 value (Table 2). A similar reduction of the GABA-evoked currents was produced by RY-24 and RY-80 at α5I215Kβ3γ2receptors (72 ± 4 and 62 ± 4% of control response, respectively). In contrast, neither RY-24 nor RY-80 affected GABA currents on either α5I215Vβ3γ2or α5I215Tβ3γ2receptors at concentrations of up to 1 μM, sufficient to saturate receptors.
Discussion
The objective of the present study was to localize the molecular features of the α5 subunit responsible for the high affinity and selectivity of ligands such as RY-80. Because the N-terminal extracellular domain exhibits the greatest sequence divergence among α-subunits, it was hypothesized that this region was most likely to be involved in defining ligand selectivity. Four amino acid residues conserved in this region among the α1–3 subunits but different in the α5 subunit (α5G24, α5P166, α5H195, and α5I215) were considered. Substitution of each of these four residues in the α5 subunit with the corresponding amino acids conserved among the α1–3 subunits resulted in a significant reduction in [3H]RY-80 binding only in the α5I215Vβ3γ2mutant (under Results). Saturation analysis confirmed that this reduction in [3H]RY-80 binding reflects an ∼16-fold increase in the K D value of this radioligand (Table 1; Fig. 3) compared with wild-type α5I215Vβ3γ2receptors. This mutation concomitantly reduced the selectivity of RY-80 for GABAA receptors containing an α5 subunit from ∼134- to ∼8.4-fold compared with cognate receptors containing an α1subunit. This mutation also increased theK i of RY-24 by >6.0-fold and reduced its selectivity for α5-containing GABAA receptors from ∼80- to ∼12-fold (Table1; Fig. 3). Because all known α5-selective ligands are structurally related (Fig. 1), it is not known whether the affinities of other, structurally unrelated compounds exhibiting α5-subtype selectivity would be similarly affected. However, the observation that the affinity of Ro 15-1788 was not significantly altered in the α5I215Vβ3γ2mutant and that the affinity of CL 218,872 was slightly increased supports the hypothesis that this amino acid is essential for a selective interaction at α5β3γ2receptors. We hypothesized that if Ile215 is essential for ligand selectivity at α5β3γ2receptors, then substitution of this residue at this corresponding position in α1β3γ2receptors (i.e., at Val211) should produce a significantincrease in the affinity of compounds such as RY-80. Consistent with this hypothesis, the affinities of both RY-80 and RY-24 were increased ∼20-fold in α1V211Iβ3γ2, whereas the affinities of other ligands were either unchanged or slightly reduced (Fig. 4).
Based on the affinities of a structurally diverse group of ligands, an inclusive pharmacophore of the α5β2γ2receptor has been developed (Liu et al., 1996). A large lipophilic regionl (L2) appears to contribute to the unique pharmacological properties of this GABAA receptor isoform (Liu et al., 1996). Thus, although the L2descriptor is common to the pharmacophore of other GABAA receptors (e.g., α1β2γ2receptors), the larger volume of L2 in α5β2γ2receptors has been proposed to result in low affinities for ligands (e.g., zolipidem) that do not extend into this domain, and very high affinities for compounds (e.g., RY-80) capable of filling this area (Liu et al., 1996). Both the >10-fold reduction in the affinities of α5-selective ligands produced by a subtle change in the lipophilicity of residue 215 (i.e., isoleucine-to-valine) and the corresponding increases in affinity produced by the cognate (i.e., “back”) mutation in α1β3γ2receptors prompted us to hypothesize that Ile215 constitutes a portion of this L2 domain. If this hypothesis is correct, then increasing the lipophilicity of the residue at α5215 should increase the affinities of ligands such as RY-80 and RY-24 without remarkably affecting the affinities of nonselective ligands. Conversely, reducing lipophilicity, either by substituting a nonpolar amino acid with a smaller side chain or introduction of polar or charged residues at α5215 should produce a further reduction in the affinities of these compounds. Increasing the lipophilicity of this residue (e.g., leucine, phenylalanine) did not remarkably affect the affinities of RY-80 and RY-24. In contrast, 5- to 6-fold decreases in affinities of nonspecific ligands Ro 15-1788 and flunitrazepam were observed (Table 1). However, neither reducing lipophilicity by introducing an alanine residue nor substitution of polar and/or charged residues (e.g., threonine, lysine, and aspartic acid) decreased the affinities of the α5-selective ligands beyond that produced by the original α5I215V mutation (Table 1). These observations indicate that although contributing to the high affinity and selectivity of RY-80 and RY-24 at α5β3γ2receptors, residue 215 may not directly participate in the formation of L2. Thus, the original model, based on an L2 lipophilic region exerting influence over ligand selectivity at α5-containing GABAA receptors merits reconsideration. Furthermore, in the absence of crystallographic studies of ligand-bound receptor (Dingledine et al., 1999), it is possible that α5215 modulates the affinities of RY-24 and RY-80 through an allosteric mechanism rather than as an integral part of the binding pocket.
Although the present findings demonstrate that α5Ile215 substantially contributes to ligand selectivity at α5β3γ2receptors, it cannot be the sole determinant of the unique pharmacological profile of this receptor isoform. Thus, RY-80 and RY-24 retain modest selectivities (∼8–14-fold) for α5I215V, α5I215A, and α5I215Tβ3γ2receptors compared with α1β3γ2receptors. Moreover, despite a ∼20-fold increase in the affinities of RY-80 and RY-24 for α1β3γ2receptors containing a back mutation (i.e., α1V211Iβ3γ2), these compounds remain significantly (∼5-fold) more potent in wild-type α5β3γ2receptors. Finally, the very low affinity of zolpidem at wild-type α5-containing receptors is maintained through a range of mutations at this residue. Other likely candidates contributing to the pharmacological profile of α5β3γ2receptors (i.e., selectivity for RY-80 and RY-24) are one or more of the other N terminus amino acid residues that differ between the α5 and α1–3 subunits, as well as residues on the γ-subunit that may act in concert with α5I215 to produce a unique pharmacology. This latter hypothesis is consistent with both the dramatic reduction in the affinity of zolpidem produced by substitution of a γ3 for a γ2 subunit in recombinant α1-containing GABAA receptors (Lüddens et al., 1994), and the absolute requirement for a γ-subunit for high-affinity binding of benzodiazepine-site ligands (Pritchett et al., 1989; Wong et al., 1992;Boileau et al., 1998).
The γ-subunit has been closely linked to the efficacy of benzodiazepine-site ligands (Knoflach et al., 1991; Puia et al., 1991). Nonetheless, a series of conservative mutations (α1H101R, α2H101R, α3H126R, and α5H105R) that imparts diazepam insensitivity to the corresponding αxβ2/3γ2receptors (Wieland et al., 1992; Benson et al., 1998) were recently shown to affect the efficacy of several benzodiazepine-site ligands (Benson et al., 1998). This finding prompted us to examine the role of α5215 in controlling ligand efficacy at recombinant α5β3γ2receptors. Three mutations (α5I215V, α5I215T, and α5I215K) were chosen for study based on their structural divergence from the residue present in wild-type receptors and the reduced affinities of α5-selective agents. Introduction of valine, lysine, or threonine in position α5215 does not affect the potency of GABA compared with wild-type receptors. These mutations did not alter the ability of flunitrazepam to act as an agonist, increasing currents evoked by subsaturating concentrations of GABA (Fig. 5; Table 2). In contrast, the characteristic ability of RY-80 and RY-24 to reduce GABA-gated currents in wild-type α5β3γ2receptors (Fig. 5; Table 2; Liu et al., 1996)) was abolished in α5I215Vβ3γ2and α5I215Tβ3γ2receptors, but retained in the α5I215Kβ3γ2mutants. The failure to observe a change in the efficacies of RY-24 and RY-80 in the α5I215K mutants indicates this effect on ligand efficacy is independent of changes in ligand affinity because all three mutations reduced (albeit to different degrees) the affinities of these α5-selective ligands. These data demonstrate that in addition to a well described role in defining the affinities of benzodiazepine-site ligands, the α-subunit can also impact ligand efficacy. The identification of amino acid residues contributing to ligand selectivity at GABAAreceptor isoforms may provide insights resulting in compounds with a more limited spectrum of action than traditional 1,4-benzodiazepines.
Footnotes
- Received December 23, 1999.
- Accepted September 8, 2000.
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Send reprint requests to: Dr. Marina I. Strakhova, Neuroscience Discovery Research, Lilly Research Laboratories, Drop code 0510, Lilly Corporate Center, Indianapolis, IN 46285. E-mail:strakhova_marina{at}lilly.com
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↵1 Current address: Thermogen, Inc., 2225 W. Harrison Street, Chicago, IL 60521.
Abbreviations
- GABA
- γ-aminobutyric acid
- DS
- diazepam sensitive
- The American Society for Pharmacology and Experimental Therapeutics