Introduction

Summary Introduction Materials & Methods Results Discussion References

BZs are frequently prescribed as anxiolytics, sedatives, anticonvulsants, and muscle relaxants (1-3). It is now generally accepted that these compounds exert their therapeutic effects, at least partly, by interacting with GABA_A receptors in the brain (2-8). Thus, a substantial effort has been directed at understanding the molecular mechanism by which BZs modulate GABA_A receptor function (9-12).

Molecular cloning studies (13-15) have revealed multiple classes and isoforms of GABA_A receptor subunits in the mammalian brain (1-6, 1-4, 1-3, ). This diversity of , , and  subunits allows the expression of a vast number of structurally unique GABA_A receptor subtypes with distinct pharmacologies. Studies using exogenous expression, photoaffinity labeling, chimeric subunits, and site-directed mutagenesis have indicated that the  subunit contributes a major component of the BZ-binding site and, depending on the subtype, can confer either BZ1 or BZ2 pharmacology on the GABA_A receptor (16-23). In particular, a histidine residue at position 101 (22) and a glycine residue at position 200 (21) have been implicated in BZ binding to the GABA receptor complex (Fig. 1).

View larger version (46K): [in this window] [in a new window]   Fig. 1.   Aligned amino acid sequences of the rat GABAA 1, 2, and 2 subunits. The sequences shown extend from residue 55 ( subunit numbering) to beyond the first putative membrane spanning domain (TM1). Shaded 15-amino acid sequence, highly conserved cysteine loop postulated to play a role in subunit assembly (30). Boxed and shaded residues, implicated in BZ-mediated modulation of the GABAA receptor. Circled and shaded residues, implicated in GABA-mediated activation. Dot, amino acids mutated. *, Crucial tyrosine residues.

Although the  subunit seems to form part of the BZ-binding site, the presence of a  subunit is essential for the normal modulatory actions of BZs on GABA_A receptors (19, 24, 25; although see Ref. 26). The  subunit is photoaffinity labeled by [³H]flunitrazepam (27), suggesting that it may also contribute part of the BZ-binding site. Site-directed mutagenesis studies have identified a threonine residue at position 142 of the human 2 subunit (Fig. 1) implicated in the efficacy of BZ ligands (28).

We previously identified two tyrosines at position 157 and 205 of the 2 subunit (Fig. 1) that when mutated, dramatically impaired GABA-mediated activation of the 122 GABA_A receptor/pore complex (29). These two tyrosine residues are conserved in all , , and  subunit isoforms. Mutation of the homologous tyrosines in the 1 or 2 subunits did not alter GABA-dependent activation of the 122 GABA_A receptor (29). Here, we demonstrate that mutagenesis of these two tyrosines in the 1 (1Y159S and 1Y209S) subunit, but not in the 2 subunit (2Y172S and 2Y220S), has profound effects on BZ binding and modulation of GABA-activated currents, suggesting these amino acids may be components of the BZ-binding site.

	Materials and Methods

Summary Introduction Materials & Methods Results Discussion References

Site-directed mutagenesis and in vitro transcription. Rat 1, 2, and 2 cDNAs were cloned into the pSELECT vector (Promega, Madison, WI), and oligonucleotide-mediated site-directed mutagenesis was achieved with the Altered Sites Kit (Promega) as previously described (30). Successful mutagenesis was verified by sequencing.

Oocyte isolation and cRNA injection. Xenopus laevis (Xenopus I, Ann Arbor, MI) were anesthetized by hypothermia, and oocytes were surgically removed from the frog and placed in a solution that consisted of 82.5 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM Na₂HPO₄, 50 units/ml penicillin, and 50 µg/ml streptomycin, pH 7.5. Oocytes were dispersed in this same solution minus Ca²⁺ and plus 0.3% Collagenase A (Boehringer-Mannheim Biochemicals, Indianapolis IN). After isolation, the oocytes were thoroughly rinsed, and stage VI oocytes were separated and maintained overnight at 18°.

Recording. At 1-3 days after injection, oocytes were placed onto a 300-µm nylon mesh suspended in a small volume chamber (<100 µl). The oocytes were continuously perfused and briefly switched to the test solution containing GABA (3 µM) or GABA plus dzp. The stock dzp (Sigma Chemical, St. Louis, MO) solution was made in PEG-300 or ethanol. No differences were observed between the two vehicles.

Data analysis. The fractional potentiation (FP) was calculated for each dzp concentration as follows:

(1)

where I_dzp is the amplitude of the GABA-activated current in the presence of dzp, and I_control is the amplitude of the GABA-activated current in the absence of dzp. Thus, a fractional potentiation of 1.0 represents a 2-fold increase over the control current amplitude. To quantify dzp sensitivity, the dzp dose-potentiation relationship was fit with the following Hill equation using a nonlinear least-squares method:

(2)

where FP is the fractional potentiation as defined by eq. 1, FP_max is the maximal fractional potentiation, EC₅₀ is the concentration of dzp yielding a half-maximal enhancement of the GABA-activated current, and n_H is the Hill coefficient.

Transfection of mammalian cells. Cloned cDNAs encoding the rat wild-type or mutated subunits were subcloned into the polylinker site of appropriate expression vectors by standard recombinant DNA techniques (wild-type 1, 2, and 2: pCDM8, pRK5, and pRc/CMV, respectively; mutant subunits: pRK7). Expression plasmid DNA was prepared by CsCl gradient centrifugation. HEK 293 cells were transfected using calcium phosphate precipitation with the combinations of plasmid DNAs (20 µg/10-cm plate) indicated in the text. After 48 hr, cells were harvested, pelleted by centrifugation at 4000 × g, and frozen at 70°.

Binding assay. Cell membrane pellets were washed three times by homogenization in 20 volumes of ice-cold buffer (10 mM potassium phosphate, pH 7.2), centrifuged, and then homogenized in a mixture of 10 mM potassium phosphate and 100 mM potassium chloride, pH 7.2. Incubations contained 200-µl aliquots of membrane suspension; 25 µl of [³H]Ro 15-1788 (83.7 Ci/mmol; New England Nuclear Research Products, Boston, MA) or [³H]muscimol (16 Ci/mmol; Amersham, Arlington Heights, IL). [³H]Ro 15-1788 was used at concentrations of 0.1-10 nM, and [³H]muscimol was used at concentrations of 1.5-50 nM. After incubation for 60 min at 4°, the membranes were collected by rapid filtration on Whatman GF/C filters and immediately washed two times with 5 ml of ice-cold buffer (10 mM potassium phosphate, 100 mM potassium chloride, pH 7.2). Radioactivity was measured by liquid scintillation spectroscopy. Specific binding was defined as the difference between total binding and nonspecific binding in the presence of clonazepam or GABA. Protein concentrations were determined using the BCA Protein Assay (Pierce Chemical, Rockford, IL).

	Results

Summary Introduction Materials & Methods Results Discussion References

The dzp-mediated modulation of wild-type 122 receptors. cRNA encoding rat wild-type 1, 2, and 2 subunits were coinjected into X. laevis oocytes, and 1-2 days later, GABA-activated currents were examined using the two-electrode voltage-clamp technique. The traces in Fig. 2A are GABA-activated currents (3 µM GABA) from oocytes expressing wild-type 122 GABA_A receptors in the absence or presence of increasing concentrations of the BZ dzp. The dzp produced a concentration-dependent enhancement in the amplitude of the GABA-evoked currents. The fractional potentiation of the current is plotted as a function of dzp concentration in Fig. 2B. Note that the dose-response relationship for dzp-mediated modulation has three components (also obvious in the current traces of Fig. 2A); a potentiation that seems to plateau around 1 µM, a depression apparent at 1- 20 µM dzp, and a further potentiation at dzp concentrations of > 20 µM.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   The dzp-mediated potentiation of recombinant 122 GABAA receptors. A, GABA-activated currents (3 µM GABA) in the absence and presence of increasing concentrations of dzp (indicated above the traces). B, Plot of the fractional potentiation as a function of dzp concentration. These data represent the mean ± standard error values for 24 oocytes. Note that there is a potentiation at ~1 µM dzp, a depression obvious at 1- 20 µM dzp, and a further potentiation at > 20 µM dzp. Statistical comparison between the 1 and 5 µM data point demonstrated that the depression was statistically significant (p = 0.0017). Continuous line (extrapolated as a dashed line), from the best fit of the Hill equation for the means up to 1 µM dzp (see Discussion). This yielded an EC50 value for dzp potentiation of 66 nM, a Hill coefficient of 1.03, and a maximal fractional potentiation of 2.6. The mean ± standard error values for the fits of the Hill equation to the data for each oocyte are presented in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 The dzp-mediated modulation of wild-type and mutant GABAA receptors Parameters were determined by fitting the Hill equation to the dose-response relationship for dzp at 1 µM except for the 1Y209S mutant, which was fit to 10 µM dzp. In all cases, the fractional potentiation was examined in the presence of 3 µM GABA. Values are mean ± standard error.

Mutations in conserved domains of the 1 subunit. The tyrosine at position 159 of the 1 subunit (Fig. 1) was mutated to serine (1Y159S) and coexpressed with wild-type 2 and 2 subunits. The traces in Fig. 3A are GABA-activated currents (3 µM GABA) in the absence or presence of increasing concentrations of dzp for the 1Y159S22 receptor. Note the dramatic decrease in potentiation at lower dzp concentrations compared with that of the wild-type receptor (Fig. 2A). This mutation did not impair activation by GABA (122: EC₅₀ = 45.8 ± 3.6 µM, Hill coefficient = 1.57 ± 0.09, I_max = 381 ± 508 nA; 1Y159S22: EC₅₀ = 44.9 ± 4.5 µM, Hill coefficient = 1.62 ± 0.18, I_max = 586 ± 405 nA; see Ref. 29). Fig. 3B plots the potentiation of GABA-activated currents for 1Y159S22 (open symbols) as a function of dzp concentration. For comparison, the potentiation of the wild-type 122 receptor is also plotted (filled symbols). The 1Y159S substitution nearly abolished the dzp-mediated potentiation at < 1 µM, and therefore the Hill equation could not be reliably fitted to these data points. In contrast, the potentiation at > 20 µM greatly exceeded that of the wild-type receptor. One possible interpretation of the increased potentiation at high dzp concentrations is that the 1Y159S mutation impaired dzp sensitivity of the lower component, thereby shifting it to the right. Thus, this lower component might now overlap with the upper component, yielding the increased potentiation at high dzp concentrations (fractional potentiation of 7.5 compared with 4.4). Based on these data, however, we cannot rule out the possibility that the 1Y159S mutation enhanced the efficacy of the actions of dzp at these higher concentrations.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   The dzp-mediated potentiation of recombinant 122, 1Y159S22, and 1Y209S22 GABAA receptors. A, GABA-activated currents (3 µM GABA) from oocytes expressing 1Y159S22 GABA receptors in the absence and presence of increasing concentrations of dzp (indicated above the traces). In comparison with the wild-type receptor, the potentiation at < 1 µM dzp was greatly diminished. The potentiation at > 20 µM, however, was enhanced compared with the wild-type receptor, possibly due to a rightward shift in the more dzp-sensitive component so that it now overlaps with the upper component. B, Plot of the fractional potentiation as a function of dzp concentration for 1Y159S22 GABAA receptors (). These data represent the mean ± standard error values for 14 oocytes. The Hill equation could not be reliably fit to the initial component of the dzp dose-potentiation relationship. The wild-type data have been replotted for comparison (). C, Plot of the fractional potentiation as a function of dzp concentration for 1Y209S22 GABA receptors (). These data represent the mean ± standard error values for 11 oocytes. Continuous line (extrapolated as a dashed line), from the best fit of the Hill equation to the open symbols (see Materials and Methods) for the mean values at 10 µM dzp. This yielded an EC50 value for dzp potentiation of 412 nM, a Hill coefficient of 1.03, and a maximal fractional potentiation of 1.4. The mean ± standard error values for the fits of the Hill equation to the data from each oocyte are presented in Table 1.

Impaired dzp sensitivity is not due to the absence of the  subunit. Evidence suggests the  subunit contributes a major component of the BZ-binding site (21, 22, 31). Thus, we considered the possibility that the tyrosine substitutions impair the assembly of the mutant  subunit, resulting in a preponderance of 22 GABA_A receptors that are less affected by dzp. It has previously been shown that 22 GABA_A receptors are dzp sensitive (32-34) ,and Fig. 4 demonstrates that the dzp sensitivity of 22 GABA receptors is similar to that of 122 GABA receptors (parameters provided in Table 1). These data suggest that the impairment of dzp-mediated modulation with the 1Y159S and 1Y209S substitutions (Figs. 2 and 3) cannot be accounted for by a mutation-induced impairment in the assembly of the  subunit.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Comparison of the dzp sensitivity of 122 and 22 GABAA receptors. Plot of the fractional potentiation as a function of dzp concentration for 122 () and 22 GABA receptors (). Continuous line through  (extrapolated as a dashed line), from the best fit of the Hill equation at 1 µM dzp. This yielded an EC50 value for dzp potentiation of 37 nM, a Hill coefficient of 1.27, and a maximal fractional potentiation of 2.6. The mean ± standard error values for the fits of the Hill equation to the data from each oocyte are presented in Table 1.

More conservative substitutions at positions 159 and 209. To gain insight into the structural requirements at positions 159 and 209, more conservative substitutions with respect to the amino acid size and aromatic ring were introduced at these positions (i.e., 1Y159F and 1Y209F). Fitting the Hill equation to dose-response relationships (not shown) from the 1Y159F22 receptors (1 µM dzp) yielded an EC₅₀ value of 118.2 ± 39.4 nM, a Hill coefficient of 1.09 ± 0.06, and a fractional potentiation of 2.3 ± 0.03 (Table 1). Fitting the Hill equation to dose-response relationships (not shown) from the 1Y209F22 receptors (1 µM dzp) yielded an EC₅₀ value of 140.9 ± 3.2 nM, a Hill coefficient of 1.31 ± 0.02, and a fractional potentiation of 2.26 ± 0.24 (Table 1). Thus, in comparison with the serine substitution, the more conservative phenylalanine substitution at these two positions produced a moderate rightward shift in the dose-response relationship for dzp.

Mutation of other tyrosines in the vicinity of 1Tyr159. To test the relative importance of these two conserved tyrosines of the 1 subunit in dzp-mediated potentiation of the GABA-activated currents, we mutated other tyrosine residues in the vicinity of 1Tyr159 (positions 161 and 168). Substitution of the tyrosine at position 161 (1Y161S) with serine (just two amino acids away from the crucial 1Tyr159) had no effect on the EC₅₀ value for dzp (Table 1), although there was a slight decrease in the maximal potentiation of the initial component. Similar to Y161S, substitution of the tyrosine at position 168 with serine (1Y168S) did not alter the EC₅₀ value for dzp-mediated potentiation (Table 1). The 1Y168S mutation also induced a slight depression in the maximal potentiation at low dzp concentrations.

Mutation of a conserved threonine at position 162. Previous studies have shown that the threonine at position 160 of the 2 subunit plays a crucial role in GABA-mediated activation (29). We mutated the homologous threonine in the 1 subunit (T162A) to investigate its potential role in dzp-mediated modulation of the GABA current. 1T162A22 mutant receptors demonstrated a similar sensitivity to dzp as that of the wild-type receptor (Table 1).

Mutations in conserved domains of the 2 subunit. The  and  subunit tyrosines crucial for dzp-dependent potentiation (Figs. 2 and 3) and GABA-mediated activation (29) of the GABA_A receptor, respectively, are also conserved in the 2 subunit (Fig. 1; 2Tyr172 and 2Tyr220). Because the ₂ subunit is essential for the modulatory effects of BZs (19), we examined the potential role of these two 2 subunit tyrosines in the actions of dzp.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   The dzp sensitivity of oocytes expressing 122Y172S and 12Y220S GABAA receptors. A, Plot of the fractional potentiation as a function of dzp concentration for 122Y172S GABAA receptors (). , Wild-type data replotted for comparison. Continuous line through  (extrapolated as a dashed line), from the best fit of the Hill equation for the mean values at 1 µM dzp. This yielded an EC50 value for dzp potentiation of 138 nM, a Hill coefficient of 1.27, and a maximal fractional potentiation of 1.8. B, Plot of the fractional potentiation as a function of dzp concentration for 122Y220S GABAA receptors (). , Wild-type data replotted for comparison. Continuous line through  (extrapolated as a dashed line), from the best fit of the Hill equation for the mean value at 1 µM dzp, yielding an EC50 value for dzp potentiation of 153 nM, a Hill coefficient of 1.34, and a maximal fractional potentiation of 2.8. The mean ± standard error values for the fits of the Hill equation to the data from each oocyte are presented in Table 1. The minimal effects of these mutations suggest these tyrosine residues do not play a key role in potentiation of the GABA receptor by dzp.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   The dzp-mediated potentiation of 22Y172S and 22Y220S receptors is not impaired.  and continuous line, from the fit of the Hill equation to the potentiation of the wild-type 22 receptor. The parameters are provided in Table 1.  and , dzp-mediated potentiation of the GABA-activated current (3 µM GABA) from 22Y172S () and 22Y220S () receptors by 10, 100, and 1000 nM dzp. Values are the mean ± standard error.

Effects of tyrosine mutations on BZ binding. The observed impairment of the sensitivity of the GABA_A receptor to dzp imparted by the 1Y159S and 1Y209S mutations (Figs. 1 and 2) could be accounted for by two mechanisms: (a) impairment of dzp binding or (b) impairment of the coupling of dzp binding to receptor/channel modulation. In an effort to distinguish between these two possibilities, we compared the binding of the high affinity BZ antagonist Ro 15-1788 to wild-type and mutant receptors. Fig. 7 () is a representative Scatchard plot of [³H]Ro 15-1788 binding to a membrane preparation from HEK 293 cells expressing 122 GABA_A receptors. [³H]Ro 15-1788 bound to these receptors with a dissociation constant (K_d) of 0.98 ± 0.21 nM (Table 2), which is in agreement with previously published reports (27). Substitution of either of the two crucial tyrosines in the  subunit with serine eliminated specific binding of [³H]Ro 15-1788 to the receptor. Muscimol binding to these mutant receptors was similar to that of the wild-type receptor (Table 2). [³H]Ro 15-1788 binding to transfected receptors containing the more conservative substitution, 1Y209F was also examined. A representative Scatchard analysis is also presented in Fig. 7 (). The K_d value for 1Y209F22 was 4.07 ± 0.38 nM (Table 2), which represents a 4-fold decrease in affinity compared with the wild-type receptor (p = 0.0002). Receptors containing substitutions of the corresponding tyrosines in the 2 subunit (122Y172S, 122Y220F) had [³H]Ro 15-1788 binding that was not significantly different from the wild-type receptor (Fig. 8, Table 2).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Substitution of phenylalanine for tyrosine at position 209 of the 1 subunit results in a 5-fold decrease in BZ binding affinity. Representative Scatchard analysis showing binding affinity of the BZ antagonist [3H]Ro 15-1788 to membrane preparations from HEK 293 cells transfected with DNA encoding either 122 () or 1Y209F22 () GABAA receptors. The mean Kd value is 0.80 ± 0.12 nM for the wild-type receptor and 4.07 ± 0.38 nM for the 1Y209F22 receptor, as shown in Table 2. Parameters determined from a Scatchard analysis of the individual membrane preparations are presented in Table 2.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Substitution of the tyrosines at position 172 or 220 of the 2 subunit produces no significant change in BZ binding affinity. Representative Scatchard analysis showing binding affinity of the BZ antagonist [3H]Ro 15-1788 to membrane preparations from HEK 293 cells transfected with DNA encoding either 122Y172S () or 122Y220F () GABAA receptors. The wild-type data are replotted for comparison (). The mean Kd value is 0.80 ± 0.12 nM for the wild-type receptor, 0.73 ± 0.03 for 122Y172S, and 0.96 ± 0.10 nM for 122Y220F, as shown in Table 2. Parameters determined from a Scatchard analysis of the individual membrane preparations are presented in Table 2.

	Discussion

Summary Introduction Materials & Methods Results Discussion References

Actions of dzp on wild-type 122 and 22 GABA_A receptors. We examined the potentiation of GABA-activated currents in 122 GABA_A receptors by dzp at concentrations ranging from 5 nM to 200 µM. Three apparent components were consistently observed in these dzp dose-potentiation relationships: (a) a fractional potentiation of 2.6 in the GABA-activated current that appeared to saturate around 1 µM and demonstrated an apparent EC₅₀ value of 65 nM, (b) a slight depression evident at 1- 20 µM dzp, and (c) a further potentiation at > 20 µM dzp that imparted an additional 1.9-fold increase (with 200 µM diazepam) in the GABA-activated current over that seen at lower dzp concentrations. We examined the higher dzp concentration range based on the expectation that BZ-binding site mutants might induce rightward shifts in the dose-potentiation relationships for dzp.

1Tyr159 and 1Tyr209 may form part of the BZ-binding site. Structure-function studies of ligand-receptor interactions have typically revealed that binding sites are formed by contributions from several disparate regions of a subunit, as well as domains from neighboring subunits. Thus, the previously identified residues of the  and  subunits (21, 22) may contribute only part of the binding site. In this study, we identified two residues on the 1 subunit (positions 159 and 209) that seem to be crucial for the actions of BZs on GABA_A receptors. The mutation Y159S nearly eliminated the potentiation seen at low dzp concentrations, whereas Y209S shifted the dzp EC₅₀ value and reduced the maximal potentiation. The more conservative substitution of these tyrosines with phenylalanine produced moderate shifts in comparison with the serine substitutions.

Other studies. A putative model of the BZ-binding site is beginning to emerge from structure-function studies of the GABA_A receptor. In this model, several disparate domains of the  subunit contribute components of the BZ-binding site (Fig. 1). The histidine at position 101 is photoaffinity labeled by BZ-site ligands (23) and, when mutated to arginine, eliminates dzp binding (22) and dzp-mediated potentiation (41). A separate region associated with the glycine residue at position 200 of the  subunit (21) seems to be important for BZ affinity. More recently, Buhr et al. (31) observed that substitution of an alanine for the threonine at position 202 or tyrosine at position 161 of the 1 subunit enhanced the maximal potentiation by dzp. In the current study, we replaced this tyrosine at position 161 and did not observe an effect on dzp-dependent potentiation of the GABA-induced currents (see Table 1), suggesting a more distal role for 1Tyr202 in comparison with 1His100, 1Tyr159, and 1Gly200 in dzp-dependent modulation of GABA-induced currents.