Introduction

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GABA_A receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and are members of a ligand-gated ion channel superfamily (Ortells and Lunt, 1995), which includes receptors for acetylcholine, glycine, and serotonin. Molecular cloning studies have identified several different classes and isoforms of GABA_A receptor subunits, including 6 , 4 , 3 ,1 , and 2  subunit subtypes (Sieghart, 1995). The majority of the GABA_A receptors in the brain are likely to consist of 1, 2, and 2 subunits (Stephenson, 1995). These receptors are pentameric proteins containing an integral chloride-selective channel with specific binding sites for GABA, BZDs, barbiturates, steroids, and picrotoxin (Sieghart, 1995; Smith and Olsen, 1995). BZDs, clinically used for their anxiolytic, muscle-relaxant, sedative, and antiepileptic actions, exert their therapeutic effects by allosterically modulating the activation of the GABA_A receptor. Because of their clinical usefulness, a substantial effort has been made to understand the structural determinants within the receptor that underlie BZD binding and allosteric coupling.

Evidence suggests that both the  and  subunits play critical roles in BZD binding and potentiation. By analogy to the agonist binding site of the nicotinic acetylcholine receptor (Karlin and Akabas, 1995), the BZD binding site of the GABA_A receptor has been modeled with a  subunit apposed to an  subunit, with adjacent faces of the subunits contributing to the binding site (Smith and Olsen, 1995). Alternatively, any subunit may bind BZD itself but have this ability enhanced by conformational changes conferred by the presence of the  subunit, which is required for high affinity BZD effects (Pritchett et al., 1989). Regardless, understanding the roles of the  and  subunits in BZD binding and modulation requires discovery of the specific structural elements involved.

In the 1 subunit, several amino acid residues have been identified that are important for BZD effects. Photoaffinity-labeling (Smith and Olsen, 1995; Duncalfe et al., 1996) and mutagenesis experiments (Wieland et al., 1992; Kleingoor et al., 1993) have identified histidine at position 101 (H101) as forming part of the BZD binding site. Experiments using 1/3 chimeras point to 1G200 as another potential site for BZD effects (Pritchett and Seeburg, 1991). Other residues in 1 implicated in BZD binding include T162 and V211 (Wieland and Luddens, 1994), Y161 and T206 (Buhr et al., 1996), and Y159 and Y209 (Amin et al., 1997). Taken together, these results suggest that three separate domains of the 1 subunit, near H101, Y159-T162, and G200-V211, are involved in BZD binding.

Less evidence has been gathered regarding the BZD-responsive regions of the  subunit. Mutagenesis experiments have identified two amino acids (F77 and T142) in the 2 subunit that may play a role in BZD effects. Mutation of Thr142 to serine (2T142S) altered the efficacy of several BZD ligands; both an antagonist (Ro15-1788) and a weak inverse agonist (Ro15-4513) took on the character of partial agonists (Mihic et al., 1994). Mutation of Phe77 to leucine (2F77L) enhanced diazepam potentiation of the GABA-mediated Cl current (Buhr et al., 1996), even though the binding affinity of diazepam was reduced. Substitution of 2F77 with other amino acids had complex effects on BZD pharmacology (Buhr et al., 1997).

Both 2F77 and 2T142 are conserved in the aligned sequence of 1. The  subunit, even though it contains the homologous phenylalanine and threonine residues, cannot substitute for a  subunit in conferring BZD effects. receptors do not bind BZDs or exhibit BZD-induced potentiation of the GABA-activated Cl current, whereas receptors do. Thus, other residues specific to the  subunit are required for BZD binding and modulation.

To determine which regions unique to the 2S subunit confer BZD binding and potentiation, we generated chimeric protein combinations of rat 2S and 1 subunits. Chimeric studies have the potential to target whole domains, which is important if we envision the drug binding site as a pocket formed by the side chains of a variety of amino acids from one or more regions of a subunit. Using this method, we identified two domains of 2S that are, in conjunction, necessary and sufficient for high affinity BZD binding. In addition, we demonstrated that the 2S regions responsible for high affinity BZD binding are distinct from the 2S regions necessary for efficient allosteric coupling of the BZD binding site to the GABA binding site. The construction of chimeric subunits that exhibit wild-type binding but reduced allosteric coupling of GABA and BZD binding sites affords new probes for elucidating the structural components of allosteric modulation.

	Materials and Methods

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Molecular cloning. Chimeras () were generated by placing the rat 2 coding region 5 to and in register with the rat 1 sequence in pBlueScript SK (Stratagene, La Jolla, CA). The dual plasmid (pTRCP, Fig. 1A) was digested, and the linearized plasmid was recircularized in bacteria by random homologous crossover events (Moore and Blakely, 1994). To create chimeric subunits containing amino-terminal domains of the 2S subunit and carboxyl-terminal domains of the 1 subunit, we cut the dual-subunit plasmid with a restriction enzyme that cuts only in each coding region of 2S and 1 (either AflII or BbsI). A fragment consisting mostly of the transmembrane and 3 coding regions of 2S was released. The remaining linearized plasmid contained 2S and 1 sequences with restricted regions of homology. Because appropriate crossovers can occur only in a small area delimited by the chosen restriction enzyme or enzymes, we named this method TRCP. Using this method, dozens of chimeric subunits with crossovers in the 5 (extracellular) region were generated in XL1-Blue cells, an endA strain that facilitates plasmid miniprep production. The chimeric open reading frames were subcloned into pGH19 (Liman et al., 1992; Robertson et al., 1996) for expression in oocytes or into pCEP4 (InVitrogen, San Diego, CA) for transient expression in HEK 293 cells. For the TRCP chimeras generated in this study (Fig. 1B), the 2S and 1 amino acids at which the crossovers occur are N40/R28 (40), W82/K70 (82), W107/T95 (107), F113/H101 (113), D161/A149 (161), and L167/K155 (167). Chimeras 40, 82, 107, and 113 were generated by AflII digestion, whereas 161 and 167 used BbsI digestion.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   TRCP. A, Chimeras were generated by placing the 2S coding region (2.0 kb) 5 to and in register with the 1 sequence (1.65 kb) in pBlueScript SK (2.9 kb; Stratagene) to yield pTRCP (6.55 kb). When linearized and introduced into competent coliform cells, the plasmid was recircularized by crossover events in homologous regions within the plasmid construct (see Materials and Methods). By choosing restriction enzymes that cut both 2S and 1 (e.g., AflII), the transmembrane and 3 coding region of 2 was released, and sufficient 1 and 2 5 sequence was left to allow for crossover events. Black, 2S sequence. White, 1 sequence. Gray, crossover areas made available by digestion with AflII. B, TRCP chimeras were screened from four independent trials and contained 5 2S and 3 1 sequence, the amount of which was determined by restriction digest mapping and DNA sequencing. The chimeras () generated by TRCP are named for the amino acid of where the crossover transitions occurs and fell into six major groups (40, 82, 107, 113, 161, and 167). Three additional non-TRCP chimeras were made (see Materials and Methods) and are named for the 2S segments each contains. For example, 40/114-161 contains 2S sequence from Q1 to N40 and from R114 to D161. 114-161 contains only 2S sequence from R114 to D161. Black, 2S sequence. White, 1 sequence. Gray, transmembrane segments M1 through M4.

Transient expression in HEK 293 cells. Rat 1, 2, 2S, and chimeric subunit cDNAs were subcloned into the multiple cloning site of a mammalian expression vector (pCEP4; InVitrogen) for transient transfection of HEK 293 cells (American Type Culture Collection CRL 1573). Cells were grown onto 100-mm tissue culture dishes in minimum essential medium with Earle's salts (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (Hyclone Laboratories, New Brunswick, NJ) in a 37° incubator under a 5% CO₂ atmosphere. Cells were cotransfected at 40-50% confluency with pCEP-1, pCEP-2, pCEP-2, and/or pCEP- using a standard CaHPO4 method (Graham and Eb, 1973). In general, cells were transfected with equal ratios of subunit DNA (5 µg/subunit). Cells were harvested and membrane homogenates prepared 48-72 hr after transfection.

Binding assays. Cells were scraped from the dishes and pelleted by centrifugation (1000 × g, 10 min, 4°). The cells were washed once and resuspended in a HEPES buffer containing 124 mM NaCl, 2.9 mM KCl, 1.3 mM MgSO₄, 1.2 mM KH₂PO₄, 25.0 mM HEPES, 5.2 mM D-glucose, and 2 mM EDTA; pH 7.4 and homogenized using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). The homogenates were centrifuged (30,000 × g, 20 min, 4°), and the resulting pellets were resuspended in HEPES buffer. Protein concentrations were determined using a Bradford assay (BioRad, Hercules, CA) with bovine serum albumin as a standard.

Expression in oocytes. Capped cRNA coding for the wild-type and chimeric subunits was synthesized by in vitro transcription from NheI-linearized cDNA template using the mMessage mMachine T7 kit (Ambion, Austin, TX). Oocytes from Xenopus laevis were prepared by incubating small pieces of ovary in collagenase (2 mg/ml) in ND96/Ca²⁺-free media containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 5 mM HEPES, pH 7.6, for 40 min at room temperature. The digested ovaries were washed several times in ND96, followed by several washes in recording solution (ND96 with 1.8 mM CaCl₂). Individual oocytes were defolliculated manually or en masse by 40-min incubation at room temperature in osmotic shock solution (130 mM K₂HPO₄, 1 mg/ml bovine serum albumin, pH 6.5 with HCl; Pajor, 1995) followed by several washes in recording solution. Within 1 day, they were injected with 5-50 nl of mRNA (10-200 pg/nl/subunit) mixed in a ratio of 1:1 (:, :, or :) or 1:1:10 (:: or ::). These ratios were determined to produce maximal assembly of - or -containing channels (Boileau AJ and Czajkowski C. Improved measurements of GABA-elicited currents and diazepam potentiation in recombinant GABA_A receptor channels expressed in Xenopus oocytes, manuscript in preparation). Oocytes were stored at 17-19° in recording solution supplemented with 100 µg/ml gentamicin and 100 µg/ml bovine serum albumin and were used for electrophysiological experiments 2-14 days after injection. The total amount of cRNA was scaled to yield maximal GABA-induced currents of ~3-8 µA for 122S and 12. The 22S and 2 subunit combinations yielded less current, usually 0.5-3 µA. cRNA concentrations were calculated by UV absorption and corroborated by comparison with RNA standards on 1.5% agarose gels.

Voltage-clamp analysis. Oocytes under two-electrode voltage-clamp (V_hold = 80 mV) were perfused continuously with ND96/Ca²⁺ recording solution at a rate of 5 ml/min. In general, drugs and reagents were dissolved in ND96/Ca²⁺. The stock diazepam solution was made in dimethylsulfoxide. No differences in currents were observed with the vehicle. GABA responses were scaled for run-down or run-up by comparison with a low, nondesensitizing concentration of drug applied just before the drug concentration tested. Diazepam potentiation was recorded at ~EC₇ to EC₂₀ for GABA (1 µM GABA for 122S and 12, 40 µM GABA for 22S). Potentiation is defined as [I_{(GABA + DZ)}/I_GABA)  1], where I_{(GABA + DZ)} is the current response in the presence of diazepam, and I_GABA is the control GABA current. Standard two-electrode voltage-clamp recording was performed using a GeneClamp 500 (Axon Instruments, Burlingame, CA) interfaced to a computer with an IT-16 A/D device (Instrutech, Great Neck, NY). Electrodes were filled with 3 M KCl and had a resistance of 0.5-1.5 M.

	Results

Top Summary Introduction Materials & Methods Results Discussion References

BZD and GABA Binding to Receptors

To create chimeric subunits containing amino-terminal domains of the 2S subunit and carboxyl-terminal domains of the 1 subunit, we modified a published method (Moore and Blakely, 1994) to specifically target crossovers to occur in the extracellular amino-terminal domain before M1 (see Materials and Methods; Fig. 1A). Chimeras () used here, named for the 2S amino acid at which the crossovers occur, are 40, 82, 107, 113, 161, and 167 (Fig. 1B).

To determine whether the chimeric subunits contained appropriate 2S domains for BZD binding, they were individually expressed with wild-type 1 and 2 subunits in HEK 293 cells to form 12 receptors, and the binding of 100 nM [³H]flunitrazepam was measured. Only two chimeras, 161 and 167, which contain the amino-terminal 161 or 167 amino acid residues of the 2S subunit, exhibited significant levels of specific [³H]flunitrazepam binding (Fig. 2). No significant specific [³H]flunitrazepam binding was detected after expression of single subunits of wild-type or chimeric origin; two-subunit combinations using 12, 12S, 22S, or 2; or 12 combinations with 40, 82, 107, or 113.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   161 and 167 receptors bind [3H]flunitrazepam. Chimeric subunits were individually expressed with wild-type 1 and 2 subunits in HEK 293 cells, and the binding of 100 nM [3H]flunitrazepam was measured (see Materials and Methods). Note that only two chimeras, 161 and 167, which contain the amino-terminal 161 and 167 amino acid residues of the 2 sequence respectively, specifically bound [3H]flunitrazepam. Percentages were calculated by normalizing specific [3H]flunitrazepam binding of 122S, 12, or 12 receptors to 122S binding. Results are mean ± standard error. The number of individual experiments are shown in parentheses.

The affinity of 122S, 12161, and 12167 receptors for [³H]flunitrazepam (BZD agonist), Ro15-1788 (BZD antagonist), and Ro15-4513 (BZD inverse agonist) was measured by radioligand saturation and competition experiments to determine whether 161- and 167-containing receptors were altered in their ability to bind different classes of BZDs. Results from saturation binding experiments demonstrated that 12161 and 12167 receptors had B_max values and equilibrium dissociation constants (K_D) for [³H]flunitrazepam similar to those of 122S receptors, with K_D values of 13.3, 11.3, and 9.9 nM, respectively (Fig. 3, Table 1). Competition binding experiments using Ro15-1788 or Ro15-4513 showed no significant differences from 122S in the K_I values for these compounds (Table 1).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Saturation binding of [3H]flunitrazepam to membranes prepared from HEK 293 cells expressing 122 and 12161 receptors. KD and Bmax values for [3H]flunitrazepam were calculated by nonlinear least-squares fit of specifically bound [3H]flunitrazepam (see Materials and Methods). Data shown are from a single experiment repeated multiple times with similar results; points, mean ± standard error of triplicate determinations. Results are summarized in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Binding affinities for three different types of BZDs using wild-type and chimeric receptors The affinity of 122S, 12161, and 12167 receptors for [3H]flunitrazepam (BZD agonist), Ro15-1788 (BZD antagonist), and Ro15-4513 (BZD inverse agonist) was measured by radioligand saturation and competition binding experiments. 12161 and 12167 receptors had an affinity similar to that of 122S for all three types of BZD-site ligands tested. Results shown are mean ± standard error; n is the number of independent experiments.

The dissociation constants for [³H]muscimol binding (a GABA binding site agonist) to 12, 122S, and 12161 receptors also were determined. The affinity and B_max values for [³H]muscimol binding to 161-containing receptors were similar to 122S receptors (12161: K_D = 88.3 ± 5.9 nM, B_max = 1.32 ± 0.19 pmol/mg, 3 experiments; 122S: K_D = 70.0 ± 8.0 nM, B_max = 1.21 ± 0.18 pmol/mg, 20 experiments). 12 receptors bound [³H]muscimol with a ~2-fold higher affinity (K_D = 46.2 ± 9.0 nM, B_max = 1.23 ± 0.14 pmol/mg, 5 experiments).¹ The small but significant difference in [³H]muscimol affinity in 122S and 12161 receptors versus 12 receptors (p < 0.01) may be diagonistic for the presence of 2 domains in the pentameric receptor complex.

Allosteric Coupling of the GABA and BZD Binding Sites

Two-electrode voltage-clamp studies. Because robust BZD binding does not necessarily indicate functional coupling of the BZD and GABA binding sites, the chimeras were tested with two-electrode voltage-clamp for the ability of diazepam to potentiate the GABA-mediated Cl current. 40, 82, 107, and 113 showed no diazepam potentiation of the GABA response when coexpressed with wild-type 1 and 2 cRNA in X. laevis oocytes, whereas 161 and 167 exhibited small but detectable amounts of potentiation. The traces in Fig. 4A show diazepam potentiation of GABA-activated currents from oocytes expressing 122S, 12161, and 12167 GABA_A receptors. Fig. 4B plots the potentiation of GABA-activated currents for 122S, 12161, 12167, and 12 receptors as a function of diazepam concentration. The maximal diazepam potentiation of 12161 and 12167 receptors was dramatically lower (~7-fold) than that for wild-type 122S receptors (Table 2). This result was surprising, considering that 12161 and 12167 receptors bound BZDs with wild-type affinity (Table 1), and indicates an uncoupling of high affinity BZD binding from BZD potentiation. Although the potentiation of 12161 and 12167 receptors was small, it was significant (p < 0.05) at diazepam concentrations above 100 nM compared with 12, 1240, or 12113 (Table 2). On normalization of the data to maximal potentiation, a ~6-fold increase in the EC₅₀ for diazepam potentiation was observed in 161- and 167-containing receptors compared with wild-type receptors (Fig. 4B, inset; Table 2).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Diazepam potentiates 12161 and 12167 receptors. A, Trace recordings from cells injected with chimeric construct 122S (left), 12161 (middle), and 12167 (right). Cells were voltage-clamped at 80 mV and perfused with ND96 recording solution or ND96 with 1 µM GABA or 1 µM GABA plus diazepam (transition to diazepam-containing solutions: white arrowheads). Far left, diazepam concentrations. Cells were washed with ND96 recording solution for 5-20 min between drug applications. Note that wild-type 122S subunits show a large potentiation, whereas chimeras show smaller potentiation even at a high concentration of diazepam (1 µM). B, Oocytes injected with wild-type 122S (1:1:10), 12 (1:1), and 12 (1:1:10) cRNA mixtures were treated with a range of diazepam concentrations in the presence of GABA and further analyzed. A potentiation response ratio was determined by dividing the peak current for 122S (), 12 (), 12161 (), and 12167 () exposed to 1 µM GABA plus diazepam (DZ) by the response to 1 µM GABA alone. Data were fitted to a curve described by the equation Y = Min + (Max  Min)/{1 + 10[(logEC50  X) · n]}, where Max is the maximal potentiation, Min is the potentiation at the lowest drug concentration tested, X is the logarithm of diazepam concentration, EC50 is the half-maximal potentiation response, and n is the Hill coefficient. Data points represent mean potentiation from four or more cells from two or more batches of oocytes. Error bars, standard deviation. The parameters from the curve fits are presented in Table 2. Inset, a plot of the same data after normalizing to the maximum response for 122 (), 12161 (), and 12167 () receptors displays the shift in EC50 value for chimera-containing receptors.

View this table: [in this window] [in a new window]   TABLE 2 Summary of voltage-clamp results Dose-response data for wild-type and chimeric subunit combinations for GABA and diazepam potentiation of GABA-mediated CI current in X. laevis oocytes are tabulated. Two-electrode voltage-clamp and data analysis was performed as described (see Materials and Methods). Mean and standard deviation values for maximum potentiation, EC50 values, and Hill coefficients (nH) were calculated from dose-response data (Figs. 4b and 5) with the use of Prism software.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   GABA dose response for chimeras is similar to that of wild-type 122 receptors. Oocytes were injected with 122 (1:1:10, ), 12 (1:1, ), 22 (1:1, ), and 12161 (1:1:10, ) cRNA to determine whether reduced diazepam potentiation of chimeras was due to any shift in GABA dose-response curves. Data were fitted to a curve described by the equation Y = Min + (Max  Min)/{1 + 10[(logEC50  X) · n]}, where Max is the maximal response, Min is the response at the lowest drug concentration tested, X is the logarithm of GABA concentration, EC50 is the half-maximal response, and n is the Hill coefficient. Dose response for both 12161 and 12167 (not shown, for clarity) are most similar to that of wild-type 122. Data points, mean peak current from four or more cells from two or more batches of oocytes; error bars, standard deviation. Parameters determined from the curve fits are presented in Table 2.

Equilibrium binding studies. To gain further insight into whether the decrease in the allosteric coupling of the GABA and BZD binding sites was due to an intrinsic property of the chimera-containing receptors, the ability of GABA to potentiate [³H]flunitrazepam binding to membrane homogenates prepared from HEK 293 cells expressing 122S, 12161, and 12167 receptors was measured. In this experimental paradigm, only the receptor populations containing a 2S or chimeric subunit were monitored because 12 receptors do not bind BZDs. Fig. 6 plots the potentiation of specific [³H]flunitrazepam binding of 122S and 12161 receptors as a function of GABA concentration. The GABA-mediated potentiation of [³H]flunitrazepam binding in 12161 receptors was nearly abolished at concentrations of GABA up to 100 µM. Similar results were seen for 12167 receptors. In contrast, GABA potentiated [³H]flunitrazepam binding of 122S receptors with an EC₅₀ value of 1.20 ± 0.15 µM and a maximal potentiation of 1.25 ± 0.05 (Fig. 6). These results suggest that the BZD and GABA binding sites are uncoupled in 12161 and 12167 receptors and that the uncoupling is due to a property of the chimera-containing receptors. In addition, these results corroborate the markedly reduced diazepam potentiation observed electrophysiologically.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   GABA potentiation of [3H]flunitrazepam binding on wild-type and chimeric receptors. GABA potentiation of 2.5 nM [3H]flunitrazepam binding was measured in membrane homogenates prepared from HEK 293 cells expressing 122 () and 12161 () receptors (see Materials and Methods). Potentiation was calculated by dividing specific dpm in the presence of GABA by specific dpm in the absence of GABA, and the resulting data were fit to a single-site sigmoidal dose-response curve (see Materials and Methods; Fig. 4). Data points, mean potentiation of binding from eight experiments with 122 and six experiments with 12161. Error bars, standard error.

Further Localization of the BZD Binding Site

By comparing the / crossover positions (Fig. 1B) in chimeras that bound BZDs with high affinity (161, 167) with those that did not (40, 82, 107, and 113), a region of 48 amino acid residues (R114-D161) of the 2S subunit that is essential for BZD binding can be identified. This determination requires that 12 receptor combinations using 40, 82, 107, or 113 subunits were assembled and expressed efficiently. To address this question, the chimeric subunits were individually expressed with 2 subunits in X. laevis oocytes and the ability of GABA to activate a Cl-specific current was tested. Because the dual subunit combinations 12 and 22S form functional GABA-gated receptors when expressed in X. laevis oocytes (Table 2; Sigel et al., 1990) and 2 subunits expressed alone cannot, expression of 2 combinations directly tests the capability of the chimeras to assemble into functional receptors. We observed GABA-mediated Cl currents using all six 2 subunit combinations (data not shown). 240, 282, 2161, and 2167 had maximal GABA current amplitudes similar to 22S (3 µA). The maximal GABA currents of 2107 and 2113 receptors were ~5-fold smaller. Interestingly, although diazepam potentiated the GABA response in 22S receptors (EC₅₀ = 24 ± 2 nM), diazepam did not potentiate the GABA current of any of the 2 receptors (see Discussion). Nevertheless, these results demonstrate that the chimeric subunits can be assembled into functional 2 receptors. If the chimeric subunits assemble into functional 12 receptors in a similar manner, a region of 48 amino acids delimited by 113 to 161 in 2S is required for BZD binding.

To determine whether this region is not only necessary but also sufficient for BZD binding, a chimeric subunit (114-161, Fig. 1B) was constructed that replaced the region from H101 to D148 in the 1 subunit with the homologous 2S region (R114-D161). This chimera, when expressed with wild-type 1 and 2 subunits, did not specifically bind [³H]flunitrazepam, [³H]Ro15-1788, or [³H]Ro15-4513 at concentrations up to 200 nM (data not shown). To determine whether 114-161 could assemble into a functional receptor, it was expressed with wild-type 2 subunits, and the binding of [³H]muscimol was measured. The 114-1612 receptor specifically bound [³H]muscimol with a K_D value of 108 ± 30 nM and a B_max value of 0.6 ± 0.4 pmol/mg (four experiments). Membrane homogenates prepared from HEK 293 cells expressing 2 alone did not specifically bind [³H]muscimol. These data suggest that the lack of BZD binding by 12114-161 receptors cannot be explained by an impairment in the assembly or expression of the 114-161 subunit. Therefore, although the R114-D161 region of 2S may be necessary for BZD binding, it clearly is not sufficient.

Because 114-161 did not bind BZDs, two /// chimeras were constructed (40/114-161 and 82/114-161; Fig. 1B) that replaced in both 40 and 82 the 1 region from H101 to D148 with the homologous 2S region (R114-D161). These chimeras were expressed with wild-type 1 and 2 subunits in HEK 293 cells and the binding of [³H]flunitrazepam was measured. The 1240/114-161 receptors did not specifically bind [³H]flunitrazepam or [³H]Ro15-4513, whereas 1282/114-161 receptors bound [³H]flunitrazepam in a similar fashion to 122S receptors with a K_D of 17.8 ± 5.4 nM and a B_max of 0.36 ± 0.06 pmol/mg (six experiments) (Fig. 7). 1282/114-161 receptors showed no significant differences from 122S receptors in the K_I values for Ro15-1788 (K_I = 12.7 ± 3.1 nM, three experiments) or Ro15-4513 (K_I = 23.0 ± 9.4 nM, four experiments). Thus, only two regions of the 2S subunit, Q1-W82 and R114-D161, are required for high affinity BZD binding. Amino acid sequence comparison of 1240/114-161 receptors, which do not bind BZDs, and 1282/114-161 receptors suggests that high affinity BZD binding requires only the 2S domains K41-W82 and R114-D161.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   82/114-161 receptors bind [3H]flunitrazepam. Chimeric subunits were individually expressed with wild-type 1 and 2 subunits in HEK 293 cells and the binding of 100 nM [3H]flunitrazepam was measured (see Materials and Methods). Percentages were calculated by normalizing specific [3H]flunitrazepam binding of 12 receptors to 122S binding. Results are presented as mean ± standard error. The number of individual experiments is shown in parentheses. , 122S receptors; , 1240/114-161 receptors; , 1282/114-161 receptors.

	Discussion

Top Summary Introduction Materials & Methods Results Discussion References

TRCP. The use of TRCP was successful. By choosing available restriction sites, we specifically targeted DNA sequence crossovers to the amino-terminal regions of the 1 and 2S subunits (see Materials and Methods). Moreover, by engineering a sequence with silent mutations to provide new restriction enzyme sites, one could choose any region to target for crossover events. Thus, TRCP should prove useful for any multisubunit protein.