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The Journal of Neuroscience, December 1, 1999, 19(23):10213-10220

Identification of Transduction Elements for Benzodiazepine Modulation of the GABA_A Receptor: Three Residues Are Required for Allosteric Coupling

Andrew J. Boileau and Cynthia Czajkowski

Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53706

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Modulation of GABA_A receptors by benzodiazepines (BZDs) is believed to involve two distinct steps: a recognition step in which BZDs bind and a conformational transition step in which the affinity of the receptor for GABA changes. Previously, using ₂/₁ chimeric subunits (), we demonstrated that although the N-terminal 167 ₂ amino acid residues confer high-affinity BZD binding, other ₂ domains couple BZD binding to potentiation of the GABA-mediated Cl current (I_GABA). To determine which ₂ regions couple binding to potentiation, we generated s with longer N-terminal ₂ segments for voltage-clamp experiments in Xenopus oocytes. Chimeras containing greater than the N-terminal 167 ₂ residues showed incremental gains in maximal potentiation for diazepam enhancement of I_GABA. Residues in ₂199-236, ₂224-236 (pre-M1), and particularly ₂257-297 (M2 and surrounding loops) are important for BZD potentiation. For several positive BZD modulators tested, the same regions restored potentiation of I_GABA. In contrast, -carboline inverse-agonism was unaltered in chimeric receptors, suggesting that structural determinants for positive and negative BZD allosteric modulation are different. Dissection of the ₂257-297 domain revealed that three residues in concert, ₂T281, ₂I282 (M2 channel vestibule), and ₂S291 (M2-M3 loop) are necessary to impart full BZD potentiation to chimeric receptors. Thus, these residues participate in coupling distant BZD-binding events to conformational changes in the GABA_A receptor. The location of these novel residues provides insight into the mechanisms underlying allosteric coupling for other members of the ligand-gated ion channel superfamily.

Key words: GABA; GABA_A receptor; benzodiazepines; benzodiazepine-binding site; allosteric coupling; chimeric subunits; mutagenesis; inverse agonist; positive modulation; subunit; subunit; M2 domain; M2-M3 loop; Xenopus oocytes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GABA_A receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and are members of a ligand-gated ion channel (LGIC) superfamily (Ortells and Lunt, 1995), which also includes receptors for acetylcholine, glycine, and serotonin. The GABA_A receptor gene family comprises several different classes and subtypes of receptor subunits including 6, 4, 3, 1, 1, and 1 (Barnard et al., 1998). GABA_A receptors are pentameric proteins containing an integral chloride-selective channel with specific binding sites for GABA, benzodiazepines (BZDs), barbiturates, and steroids (Smith and Olsen, 1995). BZDs, clinically used for their anxiolytic and antiepileptic actions, exert their therapeutic effects by allosteric modulation of GABA_A receptors (Sieghart, 1995). Positive BZD modulators increase the opening frequency of the Cl channel in the presence of GABA, whereas negative BZD modulators (e.g., -carbolines) decrease the opening frequency (Rogers et al., 1994). Because the therapeutic clinical value of these drugs depends on their ability to exert positive modulation on I_GABA, we were interested in identifying the structural determinants underlying BZD efficacy in potentiating GABA-gated currents.

Evidence suggests that both the  and  subunits play critical roles in BZD binding and potentiation. Using the agonist-binding site of the nicotinic ACh receptor as an archetype for pentameric LGIC receptors (Czajkowski et al., 1993), the BZD-binding site of the GABA_A receptor has been modeled with the  subunit apposed to an  subunit, and both subunits contributing to the binding site at the interface (Galzi and Changeux, 1994; Smith and Olsen, 1995). Although several studies have begun to identify amino acids in both  and  subunits involved in BZD binding (for review, see Sigel and Buhr, 1997), little is known about the structural components involved in coupling BZD binding to BZD potentiation of GABA-gated current (I_GABA).

Our previous studies using ₂/₁ chimeric subunits demonstrated that chimeras () containing the N-terminal 161 amino acid residues of ₂ exhibit wild-type binding but drastically impaired potentiation of I_GABA by BZDs (Boileau et al., 1998). To further delineate the regions unique to the ₂ subunit that confer BZD potentiation, we generated additional ₂/₁ chimeras, expanding the length of the N-terminal ₂ portion and reducing the ₁ C-terminal portion (Fig. 1). Two main regions of the  subunit, ₂224-236 and ₂257-297, improve the allosteric coupling of BZD binding to potentiation of I_GABA. The largest gain in potentiation is conferred by the ₂257-297 region, which surrounds and includes the M2 transmembrane segment. Further investigation revealed that a triplet set of residues, ₂T281, I282, and S291 underlie this function of the ₂ subunit. Unlike positive modulatory BZD compounds, the negative modulator 3-carbomethoxy-4-ethyl-6,7-dimethoxy--carboline (DMCM) exhibits full wild-type inhibition for all chimeric receptors.

View larger version (19K): [in this window] [in a new window]   Figure 1.   Chimeric 2/1 subunits and mutants. A, Chimeras () used in this study contain 5' 2 and 3' 1 sequences and are named for the 2 amino acid residue where the crossover with 1 occurs (arrows) in the mature rat protein sequence. For example, 161 contains 2 residues from position 1 to 161 and 1 residues in the remainder of the subunit. Chimeric crossovers depicted are for 161, 167, 198, 223, 236, 256, and 297. B, Shown are aligned 1 and 2 protein sequence segments containing the putative transmembrane domains M1, M2, and the beginning of M3, with the relative crossover positions of 236, 256, and 297 indicated (arrows). Mutants were constructed in the background of 236. Numbering for  and 2 subunits is identical. Boxes indicate blocks of mutations constructed simultaneously: box a represents the substitution of 1 RES residues to the aligned 2 residues KDA (RESKDA); box b corresponds to VFGVSLGI; box c corresponds to ISTI; box d corresponds to NAAKSV; box e corresponds to a subset of box d, AASV. Residues highlighted in black are important for positive BZD modulation of IGABA, and their positions in 2 are indicated below them.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Molecular cloning. Chimeras are named for the last ₂ amino acid residue before the crossover with ₁ in the mature rat protein sequence. Thus, numbering for  and ₂ subunits is identical. For chimeras used in this study (Fig. 1), the ₂ and ₁ amino acid residues at the crossovers are "D161/A149"(161), "L167/K155" (167), "L198/N188" (198), "M223/T213" (223), "F236/V226" (236), "W256/L246" (256), and "D297/W287" (297). 161 and 167 were generated by a targeted random chimera production method (Boileau et al., 1998). All others (Fig. 1A) were produced by recombinant PCR using overlapping complementary oligonucleotides designed to create a / crossover at the desired amino acid junction. Using a ₂ sense oligonucleotide and an antisense / junction oligonucleotide with ₂ cDNA as a template, upstream PCR fragments with ₂ 5' and an ₁ 3' overhang were generated. Simultaneously, using a sense junction oligonucleotide and an ₁ antisense oligonucleotide with ₁ cDNA as template, downstream PCR fragments with ₂ 5' overhang and ₁ 3' sequences were also prepared in separate PCR reactions. Upstream and downstream PCR fragments were then combined and amplified to create / DNA cassette fragments that were subcloned into 167 cDNA using AflII and NcoI, thus replacing the cassette region of 167 with the new chimeric junction sequences.

Mutant subunit fragments were generated using the same recombinant PCR method and subcloned into the background of the 236 subunit to generate different combinations of mutants (Fig. 1B). Single and multiple mutants were named according to the ₁ to ₂ substitutions made in 236. For example, the "236-RESKDA" mutant is 236 with ₁ Arg-Glu-Ser (RES) residues replaced by the aligned ₂ sequence at positions 259-261, Lys-Asp-Ala (KDA). The "236-A291S" mutant replaces ₁ Ala with ₂ Ser291 in 236; the "236-I281T + A291S" mutant is 236 with an I281T and an A291S mutation. The chimeric and mutant subunits were subcloned in pGH19 vector (Liman et al., 1992; Robertson et al., 1996) for expression in Xenopus oocytes. All chimeras were verified by restriction digest and double-stranded DNA sequencing using standard techniques (Sambrook et al., 1989).

Expression of rat GABA_A receptors in Xenopus oocytes. Capped cRNA coding for the wild-type and chimeric subunits was synthesized by in vitro transcription from NheI-linearized cDNA template in pGH19 using the mMessage mMachine T7 kit (Ambion). Oocytes from Xenopus laevis were prepared as previously described (Boileau et al., 1998) and injected with 28 nl of mRNA (10-200 pg/nl/subunit) mixed in a ratio of 1:1 (:), or 1:1:20 (:: or ::). Excess molar ratios of  or  cRNA were injected to ensure expression of these subunits in the receptor complex (Boileau et al., 1998). Oocytes were stored at 17-19°C in recording solution supplemented with 100 µg/ml gentamycin and 100 µg/ml BSA, and were used for electrophysiological experiments 2-14 d after injection. Total amount of cRNA was scaled to yield maximal GABA-induced currents of ~3-8 µA for ₁₂₂ and ₁₂. cRNA concentrations were calculated by UV absorption and corroborated by comparison to 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 recording solution containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, and 5 HEPES, pH 7.4 at a rate of 5 ml/min. In general, drugs and reagents were dissolved in ND96. Stock drug solutions (1000-10,000×) were made in dimethylsulfoxide. No differences in currents were observed with the vehicle. Maximal current (~3-8 µA) for all receptors was achieved with 10 mM GABA. For some chimeras and mutants, GABA EC₅₀ was estimated by examining the ratio of response to 1 µM versus 10 mM GABA. GABA (1 µM) elicits currents corresponding to EC₅ for both ₁₂ (range, EC_2-7) and ₁₂₂ (EC_4.8 ± 1.7) receptors. Using a standard Hill equation to model the GABA dose responses and the determined 1 µM/10 mM GABA current ratio, we calculate that the chimeric and mutant receptors show no more than a twofold shift in GABA EC₅₀ from wild-type receptors (EC₅₀ = 12 µM). Greater than twofold shifts are detectable in our assay.

BZD potentiation or -carboline inhibition I_GABA were recorded at 1 µM GABA (EC₅). Potentiation is defined as (I_{GABA + DRUG}/I_GABA)  1), where I_{GABA + DRUG} is the current response in the presence of the drug tested, and I_GABA is the control GABA-induced current. For single concentration experiments, BZD site ligands were tested at concentrations eliciting maximal effects on I_GABA in wild-type ₁₂₂ receptors. If differences in potentiation for mutant receptors were caused entirely by a shift in GABA affinity, the Hill equation predicts that a sixfold shift in GABA affinity, and only to the left, would be required to account for a 50% reduction in maximal potentiation. This would result in a 1 µM/10 mM GABA ratio corresponding to EC_27-32 (Hill coefficient varied from 1 to 2), well out of our observed range. Standard two-electrode voltage-clamp recording was performed using a GeneClamp 500 (Axon Instruments, Foster City, CA) interfaced to a computer with an IT-16 A/D device (Instrutech). Electrodes were filled with 3 M KCl and had a resistance of 0.5-1.5 M.

Data acquisition and analysis were performed using AxoData, AxoGraph (Axon Instruments), and Prism software (Graphpad). Statistical comparisons of potentiation data employed one-way ANOVA with Dunnet and Tukey post-tests for multiple independent samples using Prism software.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Regions of the ₂ subunit responsible for BZD binding can be separated from domains required for coupling BZD binding to potentiation of I_GABA (Boileau et al., 1998). This "uncoupling" of high-affinity BZD binding and potentiation is apparent in the / chimera 161, which binds BZDs with wild-type affinity when expressed with wild-type ₁ and ₂ subunits, but displays drastically impaired diazepam modulation of I_GABA. In an effort to identify regions of the ₂ subunit required for full BZD potentiation, several additional chimeras (Fig. 1A) were constructed with longer ₂ N-terminal domains. Chimeras used here, named for the ₂ amino acid where the crossovers occur, are 161, 167, 198, 223, 236, 256, and 297 (see Materials and Methods). These chimeras were expressed in Xenopus oocytes and screened by two-electrode voltage clamp to test for restoration of allosteric modulation of I_GABA with several structurally diverse BZD-binding site ligands. The expression, BZD radioligand binding, and diazepam modulation of GABA-gated currents for ₁₂161 and ₁₂167 receptors have been described previously (Boileau et al., 1998).

Different domains alter BZD EC₅₀ and potentiation

Chimeric cRNA was coinjected with wild-type ₁ and ₂ subunit cRNA into oocytes and tested for diazepam potentiation of I_GABA with 1 µM GABA. This concentration corresponded to EC₅ for GABA for both wild-type and chimeric receptors. Both maximal potentiation and EC₅₀ for diazepam modulation of I_GABA were measured. Dose-response curves and traces for diazepam potentiation of the GABA response for selected chimera-containing and wild-type receptors are depicted in Figure 2. At concentrations >1 µM diazepam, potentiation of wild-type receptors begins to depress (Fig. 2, dashed line; Amin et al., 1997). The decrease may be attributable to channel block and/or BZD blockage of the GABA-binding site. Receptors containing 161 and 167 exhibited low potentiation, whereas chimeras with increasingly longer N-terminal ₂ segments exhibited incremental gains in maximal potentiation (Table 1, Fig. 2). Full potentiation was restored with 297, which contains ₂ residues up to the beginning of the M3 transmembrane domain. For maximal potentiation, data from chimeric and wild-type receptors yielded the series 161  167 < 198  223 < 236  256 297  .

View larger version (32K): [in this window] [in a new window]   Figure 2.   Diazepam potentiation of IGABA for 12 receptors. A, Trace recordings from cells injected with 122 (leftmost), and chimeric constructs. Cells were voltage-clamped at 80 mV and perfused with ND96 recording solution or ND96 with 1 µM GABA or 1 µM GABA plus 1 µM diazepam (transition to diazepam-containing solutions indicated by white arrowheads). Cells were washed with ND96 recording solution for 5-20 min between drug applications. 12198 exhibits unusually fast desensitization properties. Note that chimeras show incrementally larger potentiation up to 12297, which is similar to wild-type 122. B, Oocytes injected with wild-type 122 (1:1:20) and 12 (1:1:20) 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 122 (), 12167 (), 12198 (), 12223 (), 12236 (), 12256 (), and 12297 () 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((LogEC50X)·nH)), 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 nH is the Hill coefficient. Data points represent mean potentiation from four or more cells from two or more batches of oocytes. Error bars indicate SD. The parameters from the curve fits are presented in Table 1. Inset, A plot of the same data after normalizing to the maximum diazepam potentiation for each receptor.

View this table: [in this window] [in a new window]   Table 1.   Summary of dose-response data for diazepam potentiation of chimeric and wild-type receptors

Differences in EC₅₀ for diazepam potentiation of I_GABA between chimeric and wild-type receptors were also measured (Fig. 2, inset; Table 1). Diazepam EC₅₀ values for receptors containing 223 (120 ± 43 nM), 236 (164 ± 29 nM), 256 (73 ± 26 nM), and 297 (98 ± 5 nM) were not significantly different from those for wild-type ₁₂₂ receptors (64 ± 15 nM). However, the EC₅₀ values for diazepam were significantly higher (p < 0.01) for receptors containing 161, 167, and 198 (310 ± 45, 437 ± 87, and 340 ± 118, respectively). A significant decrease in diazepam EC₅₀ occurred between 198 and 223 (Fig. 2, inset). It was also noted that 198 displays unusually fast desensitization to application of either GABA or GABA plus diazepam. The reason for this is unclear, and was not pursued. Interestingly, although the diazepam EC₅₀ for ₁₂256 receptors was similar to wild-type ₁₂₂ receptors, the maximal potentiation was reduced by ~60% compared to wild-type receptors (Fig. 2; see Fig. 4). The most significant gain of potentiation occurred between 256 and 297 (Fig. 1); ₁₂297 receptors exhibit potentiation and EC₅₀ for diazepam indistinguishable from wild-type receptors.

Positive BZD modulators use the 257-297 domain

To explore whether potentiation or inhibition by other drugs acting at the BZD site would require similar regions of the ₂ subunit, the chimeras were tested with several different BZD-binding site ligands. Surprisingly, the -carboline DMCM, an inverse agonist at the BZD site, inhibited I_GABA in all chimeric receptors to the same extent as wild-type ₁₂₂ receptors (Fig. 3). In contrast, the positive modulatory BZD site ligands tested showed significant differences in potentiation between chimeric receptors (Fig. 4, Table 2). The differences between chimeric receptors for positive modulation could be caused by inefficient expression of the chimera in a receptor complex, yielding a mixture of ₁₂ and ₁₂ receptors that would appear to have reduced potentiation. However, the observation that each chimera responds to DMCM akin to ₁₂₂ receptors, and not ₁₂ receptors, allays this concern.

View larger version (42K): [in this window] [in a new window]   Figure 3.   Chimeric receptors are indistinguishable from wild-type receptors for negative modulation by DMCM. Negative modulation of 1 µM GABA responses by the -carboline DMCM (1 µM) is graphed for wild-type 122, 12161, 12167, 12223, 12236, 12256, and 12297 chimeric receptors. No significant difference in the inhibition of the GABA current was measured between wild-type and chimeric receptors.

View larger version (32K): [in this window] [in a new window]   Figure 4.   Positive BZD-site modulators potentiate the GABA responses of chimeric receptors to different extents depending on the amount of 2 subunit in each receptor. Potentiation of 1 µM GABA responses is graphed for wild-type 122, 12161, 12167, 12223, 12236, 12256, 12297, and "12236-ISTI + A291S" chimeric receptors using 1 µM diazepam, 1-3 µM flurazepam (A), 1-3 µM midazolam (B), 10 µM zolpidem (C), and 1 µM CL218,872 (D). Also depicted in B is inhibition of the midazolam (MZ) potentiation by 1 µM Ro15-1788 (hatched bars) for chimeric and wild-type receptors. Open bars indicate the potentiation of IGABA by 1 µM Ro15-1788. Asterisks indicate level of significance (*p < 0.01; **p < 0.001) comparing mean potentiation for that chimera with the preceding chimera in the series (left to right). Error bars indicate SD.

View this table: [in this window] [in a new window]   Table 2.   Recovery of BZD potentiation of IGABA in chimeric receptors

Classical 1,4-benzodiazepines such as diazepam and water-soluble flurazepam exhibited similar increments in potentiation of I_GABA in chimeras with increasingly larger N-terminal segments (Fig. 4A). The strong positive modulators midazolam (a triazolobenzodiazepine, Fig. 4B) and zolpidem (an imidazopyridine, Fig. 4C), and the partial agonist CL218,872 (a triazolopyridazine, Fig. 4D) also showed increments in potentiation from 167 to 297, indicating a common pattern of residues required for positive BZD allosteric modulation.

Table 2 summarizes the effects of various BZD site ligands on the potentiation of I_GABA for chimeric receptors compared to wild-type ₁₂₂ receptors. For all positive modulators tested, a correlation between recovery of potentiation and length of the chimeric ₂ sequence emerges. Small increases in potentiation are observed between 167 and 223 (~10-15%), and between 223 and 236 (~10-25%) for all of the positive modulators tested. Whether a significant recovery of potentiation occurred between 167 and 198 or between 198 and 223 depended on the drug tested. The largest increases in potentiation (50-60%) for all of the positive modulatory BZDs tested occurred between 256 and 297, indicating that amino acid residues surrounding and/or including M2 are required for full potentiation of I_GABA by these drugs.

In addition, we tested the activity of the imidazobenzodiazepine antagonist Ro15-1788 (flumazenil; Fig. 4B) in wild-type and chimera-containing receptors. Ro15-1788 (1 µM) was effective at blocking the midazolam-induced (1 µM) potentiation of I_GABA in all chimeric receptors (Fig. 4B, hatched bars). At micromolar concentrations, Ro15-1788 acts as a weak positive modulator of GABA activation (Fig. 4B, open bars; Mihic et al., 1997). The pattern of potentiation in wild-type and chimeric receptors was similar to that seen for other positive modulators. After subtracting out the minor background Ro15-1788-induced potentiation, the midazolam-induced potentiation is inhibited by Ro15-1788 to the same extent for wild-type and all chimeric receptors (91.5 ± 3.7%).

Mutations conferring positive modulation of I_GABA

Because the largest gain in BZD potentiation is mediated by the ₂I257-D297 domain, we focused on identifying the amino acid residue or residues in this region that contribute to BZD modulation of I_GABA. To determine whether ₁₂236 receptors are altered in their ability to bind BZDs, we performed radioligand-binding assays. The K_D for [³H]flunitrazepam binding in ₁₂236 receptors (3 ± 1 nM; n = 2) was similar to the values obtained for ₁₂161 (11.3 ± 1.7 nM) and ₁₂₂ (9.9 ± 0.8 nM; Boileau et al., 1998). Several mutants were constructed in a 236 background, corresponding to nonidentical residues between ₂ and ₁ in the M1-M2 loop, the M2 transmembrane domain, and the M2-M3 loop (Fig. 1B). The first set of such mutants with ₁ amino acid residues substituted to the homologous ₂ residues were 236-RESKDA (Fig. 1B, box a), 236-RESKDA + NAAKSV (box a + box d), 236-VFGVSLGI (box b), 236-VFGVSLGI + NAAKSV (box b + box d), 236-ISTI (box c), and 236-ISTI + AASV (box c + box e). Of these, only 236-ISTI + AASV (box c + box e) exhibited full potentiation of I_GABA by diazepam. All single-, double-, and triple-mutant combinations of the substitutions present in 236-ISTI + AASV (box c + box e) were generated and tested with 1 µM diazepam for potentiation of I_GABA (Fig. 5). Of these, only the triple mutant combination 236-ISTI + A291S (Fig. 1B, black outlined residues) restored full potentiation to 236; all values for diazepam potentiation of I_GABA by mutant receptors depicted in Figure 5 are significantly different (p < 0.01) from those for 297 except for the quadruple mutant 236-ISTI + AASV (box c + box e) and the triple mutant 236-ISTI + A291S.

View larger version (54K): [in this window] [in a new window]   Figure 5.   Diazepam potentiation of chimeric and chimeric mutant receptors. Potentiation of 1 µM GABA current using 1 µM diazepam (DZ) is graphed for 12236 (white), mutants, and mutant combinations made in the background of 12236 receptors (gray) and 12297 (black) chimeric receptors. Potentiation is defined as (IGABA + DZ/IGABA)  1), where IGABA + DZ is the current response in the presence of diazepam, and IGABA is the control GABA-induced current. Dashed lines at 1.0 and 2.0 on the ordinate axis are shown for ease of comparison. Only the 12236-ISTI + A291S and 12236-ISTI + AASV chimeric receptors show no significant difference from 12297 receptors.

Diazepam dose-response curves for the components of 236-ISTI + A291S, namely 236-ISTI, 236-A291S and the other two double mutants 236-I281T + A291S and 236-S282I + A291S demonstrate that all three mutations are necessary for full potentiation for diazepam (Fig. 6). Summary data for maximal potentiation and EC₅₀ values are shown in Table 1. Although 236-A291S alone did restore some of the potentiation to 236, it is still significantly less than for 297 (p < 0.01). The triple mutant also exhibited wild-type maximal potentiation for other BZD-positive modulators (Fig. 4, Table 2).

View larger version (28K): [in this window] [in a new window]   Figure 6.   Diazepam potentiation of IGABA for mutant 12236 receptors. A, Trace recordings from cells injected with wild-type 1, 2, and either 236 (left), mutant 236 (middle), or 297 (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 1 µM diazepam (transition to diazepam-containing solutions indicated by white arrowheads). Cells were washed with ND96 recording solution for 5-20 min between drug applications. Note that only the 12236-ISTI + A291S mutant chimeric receptors show potentiation as large as 12297 receptors. B, Oocytes injected with chimeric and chimeric mutant 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 12297 (), 12236-ISTI + A291S (), 12236-A291S (), 12236-ISTI (), 12236-I281T + A291S (), 12236-S282I + A291S (), and 12236 () 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)·nH)), 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 nH is the Hill coefficient. Data points represent mean potentiation from six or more cells from two or more batches of oocytes. Error bars indicate SD. The parameters from the curve fits are presented in Table 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We previously reported that chimeric subunits with ₂ sequence in the first 161 amino acid residues and ₁ sequence in the remainder exhibit high-affinity BZD binding, but impaired BZD modulation of I_GABA (Boileau et al., 1998). Efficient coupling of BZD binding to potentiation of I_GABA is therefore mediated, at least in part, by ₂ amino acid residues that are not directly involved in the binding of BZDs. In this study, we identified two novel ₂ regions, ₂224-236 (pre-M1) and ₂257-297 (M2 and surrounding loops) that are necessary for full restoration of coupling between high-affinity BZD binding and full BZD potentiation of I_GABA by several disparate BZD positive allosteric modulators (Figs. 2, 4). In addition, a threefold shift in EC₅₀ is observed between ₁₂198 and ₁₂223 receptors (Table 1). This change in diazepam EC₅₀ may account for some or all of the increased potentiation observed in ₁₂223 receptors as compared to ₁₂167 receptors (Figs. 2, 4).

For all the BZD positive allosteric modulators tested, the largest recovery in BZD potentiation of I_GABA (50-60%) is conferred by one or more ₂ residues in and/or surrounding the M2 region between 256 and 297. For this region, a change in diazepam EC₅₀ cannot explain the complete restoration in the ability of diazepam to potentiate I_GABA in ₁₂297 receptors as compared to ₁₂256 receptors because the diazepam EC₅₀ values for ₁₂297 and ₁₂256 receptors are not significantly different from each other or wild-type receptors (Table 1). The differences in levels of potentiation for chimeric receptors are also not caused by inefficient expression of the chimeric subunit, as evidenced by the equivalence of DMCM inhibition of I_GABA for each chimeric receptor and ₁₂₂ receptors (Fig. 3). Thus, DMCM inhibition of I_GABA serves as a useful benchmark and control for chimeric insertion into the receptor complex. Finally, the differences in potentiation are not attributable to differences in GABA dose responses. Previously, we established that ₁₂161 and ₁₂167 receptors have EC₅₀ values for GABA similar to ₁₂₂ and ₁₂ receptors (Boileau et al., 1998). In addition, we have calculated that the EC₅₀ values for all of the chimeric receptors are shifted by less than twofold compared to wild-type receptors (see Materials and Methods), which could not alone account for the decrease in maximal potentiation observed. Thus, we conclude that the ₂257-297 region contains structural determinants required for coupling BZD binding to BZD potentiation of I_GABA.

Because DMCM inhibits ₁₂167 receptors to a similar extent as wild-type ₁₂₂ receptors (Fig. 3), negative modulation of the GABA-gated current by -carboline-binding to the BZD site must be transduced through different structural elements, i.e., using ₂ residues located within the first 167 amino acid residues, with or without downstream ₂ residues that are conserved in the ₁ and ₂ subunits. Because we are comparing  to  subunits for differences in BZD function, we cannot detect amino acid residues important for processes that are conserved between the two subunit subtypes.

The specific residues in the ₂257-297 region that underlie BZD potentiation of I_GABA were identified. Of the twelve nonidentical residues between 256 and 297, the residues ₂T281, I282, and S291 are, in combination, necessary to confer wild-type potentiation of I_GABA by positive BZD modulators (Figs. 4-6). Although we cannot rule out the possibility that these mutations somehow serve to relieve a conformational dysfunction in the structure of the 236 subunit and do not play a direct role in BZD actions, we think this unlikely. Wild-type responses to DMCM and equivalent values for competitive displacement by Ro15-1788 for all the chimeric receptors suggest that the overall structures of the chimeras are not severely disrupted. Equilibrium binding values for [³H]flunitrazepam to ₁₂161, ₁₂167, and ₁₂236 receptors were similar to wild-type receptors, indicating that the BZD-binding site is intact. Further support for our conclusion that ₂T281, I282 and S291 are compulsory ₂ elements for BZD activity derives from the fact that all three residues are conserved in all known  subunits cloned from various species but vary in other subunit subtypes. These identified residues, however, are not the sole ₂ determinants controlling BZD potentiation. Certainly other ₂ amino acid residues also play a role, particularly residues in the pre-M1 regions (e.g., ₂224-236), which have yet to be identified, and/or amino acid residues that are conserved between the  and  subunits.

Alterations in the ability of positive BZD modulators to potentiate I_GABA can arise from several possible sources, including alterations at the BZD-binding site (Colquhoun, 1998), disruptions of the coupling (transduction of BZD binding to potentiation) machinery, and/or modifications of the ion channel pore itself. An example of a mutation that alters BZD "efficacy" of several BZD ligands is the ₂ subunit mutation T142S (Mihic et al., 1994); both a competitive antagonist at the BZD-binding site (Ro15-1788) and a weak negative modulator (Ro15-4513) take on the character of weak positive modulators. Because we previously demonstrated that high-affinity BZD binding is localized to the N-terminal 161 ₂ amino acid residues (Boileau et al., 1998), the novel regions and residues identified in this study control BZD potentiation by influencing the coupling machinery and/or the ion channel pore itself rather than the BZD-binding site. For the chimeric receptors described in this study, strong positive BZD modulators behave like weak modulators or BZD antagonists. Additionally, we observe that ₁₂161, ₁₂167, and ₁₂236 receptors exhibit wild-type, high-affinity radioligand binding. Together, these observations suggest that we are measuring disruptions in coupling rather than binding. Because ₂T281 and I282 are likely to line the water-accessible surface of the Cl channel based on homology with the ₁ subunit (Xu and Akabas, 1996), it is tempting to speculate that this region controls BZD potentiation by affecting the ion channel. These ₂ residues may influence ion channel gating, possibly facilitating channel opening by GABA when BZDs are present.

Structurally, allosteric coupling between GABA and BZD-binding sites could occur exclusively in the N-terminal extracellular domains of the receptor, from one binding site to the other. The Monod-Wyman-Changeux allosteric model predicts that ligand-binding events cause allosteric transitions that change the state of the receptor, which result in changes in the binding sites (Monod et al., 1965). Our chimeric receptors have either weakened or deleted allosteric transitions that are restored by ₂ segments distant from the presumed binding sites, and in particular by the combination of the channel-lining residues ₂T281 and I282 coupled with the M2-M3 loop residue ₂S291. Thus, allosteric coupling between the GABA and BZD-binding sites requires transduction through transmembrane or intracellular regions and then back out to the extracellular binding regions, subsequently affecting the kinetics of GABA binding at one or both cooperative GABA-binding sites.

Our results demonstrate that two M2 residues (₂ T281, I282) and a M2-M3 extracellular loop residue (₂ S291) are required for full, wild-type BZD potentiation of I_GABA by a variety of BZD ligands. It is interesting that these ₂ residues map to corresponding regions as residues in other GABA_A receptor subunits and the homologous glycine receptor believed to be involved in "direct" channel gating by agonists. In the GABA_A receptor, when the M2 central leucine is substituted with threonine in the human ₁ subunit, GABA activation is abolished, even though binding with the GABA agonist [³H]muscimol is unaltered (Tierney et al., 1996). In the glycine receptor, mutation of ₁ R271 (in the M2-M3 loop) to leucine or glutamine converts glycinergic agonists -alanine and taurine from agonists to competitive antagonists for glycine (Rajendra et al., 1995). Our results that residues in M2 and the M2-M3 loop are necessary for coupling BZD binding to BZD potentiation suggests that not only are the BZD and GABA-binding sites structurally conserved (Olsen et al., 1996), but the regions of the receptor involved in the coupling of binding to their functional effects are also conserved. We speculate that the BZD-binding site may in reality be a very low-affinity GABA-binding site that over evolutionary time has acquired the ability to bind BZDs and when the ₂ M2 region is present, BZDs may act as coagonists and increase channel opening frequency.

Mutations within the M2 region and surrounding loops in the GABA_A receptor also affect the actions of other GABA_A receptor modulators. Mutations in two specific M2 amino acid residues, ₁S267 or the aligned