Introduction

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The -Aminobutyric acid type_A (GABA_A) receptor is a major inhibitory neurotransmitter receptor in the vertebrate central nervous system. In most neurons, the binding of the neurotransmitter GABA to a GABA_A receptor induces an inward Cl current, which results in membrane hyperpolarization and reduced neuronal excitability. This ligand-gated ion channel is a heteromeric complex assembled from a number of different subunits (1-6, 1-4, 1-4, , , , and ) (for reviews, see Barnard et al., 1998; Whiting, 1999). Evidence suggests that in vivo GABA_A receptors are pentameric complexes of , , and  subunits with a stoichiometry of 2:2:1 (Chang et al., 1996; Farrar et al., 1999). The stoichiometry of receptors containing , , and  is currently unknown, although evidence suggests that  and  substitute for a  subunit (Caruncho and Costa, 1994; Quirk et al., 1995; Whiting et al., 1997), whereas  replaces a  subunit (Bonnert et al., 1999).

The GABA_A receptor is allosterically modulated by a large number of compounds, including benzodiazepines; general anesthetic agents, such as halothane, barbiturates, and etomidate; and neuroactive steroids (Lambert et al., 1995; Sieghart, 1995; Whiting et al., 1995). For a number of these compounds, studies with recombinant receptors have focused on defining receptor subtype selectivity, the amino acids involved in binding, and the mechanism of action. One chemical class of compounds, which is known to modulate GABA_A receptors, that has received little attention in recent years is the pyrazolopyridines, which include tracazolate, etazolate, and cartazolate (Barnes et al., 1983). Behavioral studies have shown that tracazolate and etazolate possess anxiolytic and anticonvulsant activity (Patel et al., 1985; Young et al., 1987). Compared with the standard benzodiazepine chlordiazepoxide, tracazolate was 2 to 20 times less potent as an anxiolytic, but interestingly displayed a much larger window of separation between the anxiolytic effect and potential side effects (sedation, motor incoordination, and its interaction with ethanol and barbital) (Patel et al., 1985).

Herein, we demonstrate that these compounds, particularly tracazolate, possess unique features, modulating these receptors in an allosteric manner previously undescribed, with dimetrically opposite actions on 2- and -containing receptors. In addition the generation of chimeric 2/ subunits implicates the region equivalent to 2 residues 206 to 230 in determining the nature of this modulation.

	Materials and Methods

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Human GABA_A Receptor cDNAs. The cloning and sequencing of human 1, 6, 1, 3, 1, 2s, 3, , and  and the construction of the single point mutations 1S265N and 3N265S have been reported previously (Wingrove et al., 1994, 1997; Thompson et al., 1997, 1999a; Whiting et al., 1997, and references therein).

Construction of Chimeric Subunits. Seventeen chimeric 2/ subunits were constructed of which only the five most informative are described herein (Fig. 8). Unique restriction endonuclease sites were introduced into the wild-type sequences by site-directed mutagenesis as described previously (Wingrove et al., 1994). Restriction fragments were gel-purified and ligated using standard techniques. The integrity of chimeric subunits was confirmed by DNA sequencing using an ABI 373 automated sequencer (Applied Biosystems, Foster City, CA).

Expression in Xenopus laevis Oocytes and Electrophysiological Recordings. Adult female X. laevis were anesthetized by immersion in a 0.1% solution of 3-aminobenzoic acid ethylester (pH adjusted to toad housing water with 1 M NaHCO₃, pH 7.2-8.0) for 30 to 45 min. Ovary tissue was removed via a small abdominal incision and stage V and VI oocytes were isolated with fine forceps. After mild collagenase treatment to remove follicle cells (type IA, 0.5 mg/ml, for 6 min), the oocyte nuclei were directly injected with 10 to 20 nl of injection buffer (88 mM NaCl, 1 mM KCl, 15 mM HEPES, at pH 7, filtered through nitrocellulose) containing different combinations of human GABA_A subunit cDNAs engineered into the expression vector pCDM8 or pcDNAI/Amp. The ratio of ::2s constructs was generally 1:0.5:1, whereas the ratio for : was 1:1, for ::3 was 1:1:1, for ::1 was 1:1:10, and for and was 1:0.5:3 with 1 corresponding to 6 ng/µl of cDNA. Confirmation that all the subunits injected were being expressed was routinely checked using Zn²⁺, flunitrazepam, or picrotoxin. Oocytes were maintained at 19-20°C in modified Barth's solution (MBS) consisting of 88 mM NaCl, 1 mM KCl, 10 mM HEPES, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂, 0.91 mM CaCl₂, 2.4 mM NaHCO₃, at pH 7.5 supplemented with 50 µg/ml gentamicin, 10 µg/ml streptomycin, 10 units/ml penicillin, and 2 mM sodium pyruvate, for up to 6 days. For electrophysiological recordings, oocytes were placed in a 50-µl bath and continually perfused at 4 to 6 ml/min with MBS. Cells were impaled with two 1- to 3-M electrodes containing 2 M KCl and voltage-clamped at 70 mV. In all experiments, drugs were applied in the perfusate until the peak of the response was observed. The magnitude of modulation of GABA-evoked currents by allosteric modulators is critically dependent upon the concentration of GABA used (Parker et al., 1986; Wafford et al., 1994). For this reason the modulatory effect of tracazolate was examined against an EC₂₀ concentration of GABA (range EC_18-25), which was determined for every individual oocyte. In all experiments, except those designed to investigate the direct effect of tracazolate, tracazolate was preapplied for 30 s before being coapplied with the appropriate concentration of GABA. To minimize the effect of receptor desensitization, agonist applications were separated by a period of at least 3 min upon recovery back to baseline. All data were expressed as either a percentage modulation of the GABA EC₂₀ value, or as a percentage of the maximal response to GABA. Curves were fitted using a nonlinear square-fitting program to the equation f(x) = B_max/[1 + (EC₅₀/x) ^n_H], where x is the drug concentration, EC₅₀ is the concentration of drug eliciting a half-maximal response, and n_H is the Hill coefficient. For some receptor subtypes high concentrations of tracazolate or etazolate (e.g., 100 µM etazolate on 132s, and 30 µM tracazolate on 632s) produced responses substantially smaller than the previous response. In these instances curves were fitted to the concentration before this point. Data are presented as the arithmetic mean ± S.E.M. or geometric mean (S.E.M., +S.E.M.) from a number (n) of different cells. Differences between means were evaluated by analysis of variance and Student's t test and considered significant if P < 0.05.

Solutions and Solvents. GABA 1 M stock was dissolved in MBS, ZnCl₂ 1 M stock in 0.2 M HCl, and picrotoxin and tracazolate 100 mM stocks and flumazenil 10 mM stock in 100% dimethyl sulfoxide. The limit of solubility of tracazolate in MBS was 100 µM. The maximum concentration of vehicle (0.1% dimethyl sulfoxide) was without effect. GABA, ZnCl₂, picrotoxin, and tracazolate were obtained from Sigma Chemical (St. Louis, MO), whereas the Chemistry Department at Merck Sharp and Dohme (Harlow, Essex, UK) synthesized flumazenil.

	Results

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Potentiation of 112s and 132s GABA_A Receptors: Selectivity for 3. GABA EC₂₀ responses on 112s and 132s receptors were potentiated in a concentration-dependent manner by tracazolate and etazolate (Table 1; Fig. 1). As stated above, on some receptor subtypes, high concentrations of tracazolate or etazolate produced responses substantially smaller than the previous response. These data points were not included in the curve fitting and to aid visualization of the data were omitted from the graphs. This decrease in apparent efficacy at high concentrations may be an artifact of the slow application time, inherent with the X. laevis oocyte system, which may allow receptor desensitization to occur during the rising phase of the inward current to GABA, resulting in a truncated response. Other explanations such as channel blockade, however, cannot be eliminated from the present data. The maximum potentiation (fitted to the ascending portion) ranged from between 168 and 315% and is comparable with that seen with both full benzodiazepine agonists (Wafford et al., 1993) and many other nonbenzodiazepine modulators of the GABA_A receptor [e.g., loreclezole (Wafford et al., 1994), pentobarbital (Thompson et al., 1996), and neurosteroids (Lambert et al., 1995)]. No significant direct activation by either tracazolate or etazolate was observed over the concentration range examined (30 nM-100 µM). Interestingly, both compounds displayed a significant 6- to 9-fold selectivity for 132s over 112s receptors. Because structurally and functionally tracazolate and etazolate were similar, further investigations were performed with tracazolate only.

View this table: [in this window] [in a new window]   TABLE 1 Summary of concentration-response data for tracazolate and etazolate potentiation of control GABA EC20 responses for wild type and mutant GABAA receptors Data for the EC50 are the geometric mean (S.E.M., + S.E.M.) and for the maximum potentiation and Hill coefficient are the arithmetic mean ± S.E.M.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Tracazolate and etazolate potentiate control GABA EC20 responses on 112s and 132s receptors with selectivity for 132s receptors. A, representative trace showing a concentration-response curve to tracazolate on an oocyte expressing 132s receptors. The oocyte was voltage-clamped at 70 mV. The horizontal lines indicate application of tracazolate and GABA. To observe any direct effect and to ensure complete binding, increasing concentrations of tracazolate were applied for 30 s before the coapplication of the GABA EC20 concentration and the tracazolate concentration. Note the reduction in response to 30 µM tracazolate compared with 10 µM tracazolate, which was observed in three of the five cells examined. B, modulation of the control GABA EC20 response by increasing concentrations of tracazolate on oocytes expressing 112s () and 132s () GABAA receptors. C, modulation of the control GABA EC20 response by increasing concentrations of etazolate on oocytes expressing 112s () and 132s () GABAA receptors. Data represent the mean ± S.E.M. from the number of cells indicated from two or more batches of oocytes.

3 Selectivity Is Conferred by Asparagine 265. Selectivity for 2/3-containing GABA_A receptors over 1 has previously been reported for loreclezole (Wingrove et al., 1994), -carbolines (Stevenson et al., 1995), etomidate (Hill-Venning et al., 1997), furosemide (Thompson et al., 1999a), and mefenamic acid (Halliwell et al., 1999). For all these compounds this selectivity has been shown to be due to a critical asparagine residue at position 264 and 265 (numbering according to mature polypeptide sequence) within TM2 of the 2 and 3 subunit. It was logical therefore to see whether this residue also determined the 3 selectivity of tracazolate. The two mutant  cDNAs (1Ser265Asn and 3Asn265Ser) were coexpressed with 1 and 2s and concentration-response curves to tracazolate constructed (Table 1; Fig. 2). Replacement of Ser265 within the 1 subunit with Asn (the 3 counterpart) increased the sensitivity of tracazolate, whereas the opposite mutation (Asn3 to Ser) decreased the sensitivity to tracazolate. Hence, the 3 selectivity of tracazolate is determined by asparagine 265 within the 3 subunit.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   3 selectivity of tracazolate is conferred by asparagine 265. Modulation of the control GABA EC20 response by increasing concentrations of tracazolate on oocytes expressing 112s (), 132s (), 11Ser265Asn2s (), and 13Asn265Ser2s () GABAA receptors. Data represent the mean ± S.E.M. from the number of cells indicated from two or more batches of oocytes.

Interaction Is Not via Benzodiazepine Binding Site. Concentration-response curves to tracazolate were also constructed on oocytes expressing 13 and 632s GABA_A receptors (Table 1; Fig. 3). Similar to 132s receptors, control GABA EC₂₀ concentrations on 13 and 632s receptors were potentiated by tracazolate. Statistical analysis (analysis of variance) revealed no significant differences in the log EC₅₀, Hill coefficient, or maximum potentiation for 13, 132s, or 632s. Unlike compounds that interact at the benzodiazepine site, receptors lacking a  subunit were also potentiated by tracazolate. Replacement of the 1 subunit with an 6 subunit did not alter the concentration-response curve to tracazolate. Finally, 300 nM flumazenil, a benzodiazepine site antagonist, did not affect the degree of potentiation elicited by 10 µM tracazolate on 132s receptors (198 ± 42%, n = 5 in the absence versus 223 ± 31%, n = 4 in the presence).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Tracazolate potentiates control GABA EC20 responses on 13 and 632s; interaction is not via the benzodiazepine binding site. Modulation of the control GABA EC20 response by increasing concentrations of tracazolate on oocytes expressing 13 (), 132s (), and 632s () GABAA receptors. Data represent the mean S.E.M. from the number of cells indicated from two or more batches of oocytes.

Tracazolate Inhibits 11 and 13 GABA_A Receptors. In addition to replacing the  and  subunits, we studied the effects of replacing the 2 subunit. -Containing receptors reveal some unusual properties, including a proportion of constitutively active channels, fast desensitization kinetics, and a transient rebound current on GABA washout (Whiting et al., 1997; Neelands et al., 1999). Unlike the receptor combinations mentioned above, on 11 and 13 receptors, tracazolate showed a significant direct effect, with low concentrations producing an inward current, whereas higher concentrations produced an inward current followed by an outward current (Fig. 4, A and B). It has been shown previously that constitutively open channels can be modulated by certain allosteric modulators [e.g., 3 homomeric receptors (Wooltorton et al., 1997); 12L259S2s (Thompson et al., 1999b)] and hence the effect of an allosteric modulator on the constitutively open channels and the GABA-activated channels can be difficult to separate. Further characterization of the direct effect of tracazolate on 13 receptors was undertaken in separate experiments. To enable direct comparison of the modulatory effect of tracazolate on 2- and -containing receptors, the application time of tracazolate before coapplication of tracazolate and GABA was kept at 30 s. To further facilitate comparison with the results obtained with 132s and 112s receptors, the inward current to GABA in the presence of tracazolate was normalized with respect to the response evoked by the control GABA EC₂₀ response (i.e., the effect of tracazolate on the constitutively open channels was omitted). As illustrated in Fig. 5A the direct effect of tracazolate could take up to 240 s to reach steady state, hence one caveat with the experimental design described above is the introduction of a small degree of error in the measurement of the inward current to GABA. Unlike its effects on 132, tracazolate caused inhibition of the control GABA EC₂₀ response (Fig. 4; Table 2). The GABA response could be almost completely inhibited, and the IC₅₀ values for inhibition by tracazolate on 1 receptors were similar to the EC₅₀ values obtained on 12s receptors.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Tracazolate inhibits control GABA EC20 responses on 11 and 13 receptors. A, representative trace showing a concentration-response curve to tracazolate on an oocyte expressing 13 receptors. The oocyte was voltage-clamped at 70 mV. The horizontal lines indicate application of tracazolate and GABA. Preapplication (30 s) of low concentrations of tracazolate (10 nM-1 µM) produced a small inward current, whereas concentrations above 1 µM produced an inward current followed by an outward current. B, expanded response to 3 µM tracazolate illustrating the inward current to GABA in the presence of tracazolate, which was normalized to the control GABA EC20 response. C, modulation of the control GABA EC20 response by increasing concentrations of tracazolate on oocytes expressing 11 () and 13 () GABAA receptors. Data represent the mean ± S.E.M. from the number of cells indicated from two or more batches of oocytes.

View larger version (9K): [in this window] [in a new window]   Fig. 5.   Outward current to tracazolate on 13 receptors. A, representative trace showing the direct effect of tracazolate on an oocyte expressing 13 receptors. The oocyte was voltage-clamped at 70 mV. The horizontal lines indicate application of tracazolate. B, concentration-response curve for the outward current to tracazolate on 13 receptors () normalized to 100 µM picrotoxin. Data represent the mean ± S.E.M. from the number of cells indicated from two or more batches of oocytes.

View this table: [in this window] [in a new window]   TABLE 2 Summary of concentration-response data for tracazolate inhibition of control GABA EC20 responses for 11 and 13 GABAA receptors Data for the EC50 are the geometric mean (S.E.M., + S.E.M.) and for the maximum potentiation and Hill coefficient are the arithmetic mean ± S.E.M..

Outward Current to Tracazolate on 13 Receptors Is Carried by Cl Ions. Concentration-response curves to the direct effects of tracazolate were constructed on 13 receptors. As can be observed in Fig. 5A, a small inward current was observed followed by a larger outward current, which increased with increasing concentration up to a maximum response at 10 µM. At high concentrations, the outward current took up to 240 s to reach a plateau followed by a washout period of up to 10 min to reestablish the baseline value; this was hypothesized to be due to block of constitutive activity. The outward current was measured using the peak of the inward current as the start value and normalized to the outward current induced by 100 µM picrotoxin. Picrotoxin (10 µM) has previously been shown to cause an 80 to 90% reduction in the holding current of oocytes expressing 13 receptors (Neelands et al., 1999). The holding current of 11 and 13 receptors in the presence of 100 µM picrotoxin is similar to that observed in uninjected oocytes (S. A. Thompson and K. A. Wafford, unpublished observations), suggesting that 100 µM picrotoxin blocks the majority of the constitutively active channels. In addition the baseline (holding current) upon washout of tracazolate was not completely reestablished. A gradual reduction of the holding current was also observed for oocytes expressing 13 receptors, which were voltage-clamped at 70 mV and left for 1 to 2 h (data not shown), suggesting a long-term shift in the leak current possibly due to chloride redistribution through the constitutively active channel. Interestingly, the IC₅₀ of the direct effect of tracazolate (i.e., inhibition of the constitutive activity) [1.4 (1.1, 1.6) µM, n = 4] was not significantly different from the IC₅₀ value for inhibition of a GABA-activated EC₂₀ response [1.2 (0.9, 1.5) µM, n = 4] (Fig. 5B).

Modulation of 111, 113, and 11 Receptors. The opposing effects observed with tracazolate on 2s- and -containing receptors prompted studies on 1-, 3-, and -containing receptors. These subunits were coexpressed with 1 and 1 subunits and concentration-response curves to tracazolate constructed. As can be seen in Fig. 6A tracazolate behaved differently on 111 and 113 receptors compared with 112s and 11. Concentrations up to 10 µM produced a small degree of potentiation of the GABA EC₂₀ response (19 and 30% for 10 µM tracazolate on 111 and 113, respectively), whereas higher concentrations inhibited the GABA EC₂₀ response. The potentiating portion of the concentration-response curve revealed similar EC₅₀ values, Hill coefficients, and maximum responses for 111 and 113 receptors (Table 1). The data obtained for 111 and 113 receptors were not significantly different from one another (P > 0.05, unpaired Student's t test); however, comparison with 112s revealed a 10-fold decrease in EC₅₀.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Concentration-response curves to tracazolate on oocytes expressing 111, 113s, and 11 receptors. A, modulation of the control GABA EC20 response by increasing concentrations of tracazolate on oocytes expressing 111 (), 112s (), 113 (), and 11 () GABAA receptors. B, modulation of the control GABA EC20 response by increasing concentrations of tracazolate on oocytes expressing 111 (), 112s (), 113 (), 11 (), and 11 () GABAA receptors. Data represent the mean ± S.E.M. from the number of cells indicated from two or more batches of oocytes.

Effect of Tracazolate on GABA Concentration-Response Curves. The studies mentioned above only examined the effect that various concentrations of tracazolate have on a single, low concentration of GABA (EC₂₀). To further understand the mechanism of action of tracazolate, its effect on a range of GABA concentrations was examined. GABA concentration-response curves were constructed in the absence and then the presence of a single concentration of tracazolate on oocytes expressing 13, 132s, and 11 receptors.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Concentration-response curves for GABA in the absence and presence of tracazolate on 132s, 13, and 11. A, concentration-response curves for GABA (), GABA + 1 µM tracazolate (), GABA + 10 µM tracazolate (), and GABA + 30 µM tracazolate () on 132s receptors. B, concentration-response curves for GABA (), GABA + 1 µM tracazolate (), and GABA + 3 µM tracazolate () on 13 receptors. C, concentration-response curves for GABA (), GABA + 10 µM tracazolate (), and GABA + 30 µM tracazolate () on 11 receptors. Data represent the mean ± S.E.M. from the number of cells indicated from two or more batches of oocytes.

Domain 206 to 230 Determines Functional Response to Tracazolate. As described above, tracazolate is a positive modulator at receptors containing a  subunit but a negative modulator when substituted by . To investigate the amino acid determinants of this effect, a series of chimeric 2/ subunits were constructed, each having an N-terminal 2 domain. The junction between these two subunits was moved incrementally through to the start of TM2 from chimera C-A to C-D. The results from only the five most informative constructs are described previously (Fig. 8A). Chimeric subunits were coexpressed with 13 and tested for modulation of a GABA EC₂₀ response by 10 µM tracazolate. Tracazolate (10 µM) was chosen because this concentration produced the greatest window of separation between the potentiation on 132s receptors and inhibition on 13 receptors. Similar to 13, chimera C-A and C-B were negatively modulated by tracazolate (13: 79.1 ± 3.4%, n = 6; 13C-A: 46.4 ± 8.1%, n = 4; and 13C-B: 80.4 ± 6.6%, n = 4). Conversely, chimeras C-C and C-D were positively modulated by tracazolate to levels not significantly different from 132s (13C-C: 113 ± 15%, n = 4; 13C-D: 88 ± 6%, n = 3; and 132s: 198 ± 42%, n = 5; Fig. 9). These results implicate a residue or residues within the 2 domain 206 to 230 that confer the functional response observed with tracazolate. However, replacement of this whole region in 2 with the homologous portion of  (chimera C-E) did not alter the functional response to tracazolate [i.e., positive modulation similar to that of 132 receptors (13C-E: 210 ± 51%, n = 4; Fig. 9)], suggesting that although this region may be necessary, other residues are also required to confer inhibition.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   A, diagrammatic representation of 2/ chimeric subunits. The subunits are represented as rectangles with the putative signal peptide and transmembrane domains shown as black boxes. The 2 subunit and portions created from it are in white and similarly the  subunit is in gray. The five most informative chimeric subunits of those synthesized, C-A to C-E, are illustrated. The 2 portion of each chimeric subunit is as follows: C-A, 1 to 147; C-B, 1 to 205; C-C, 1 to 230; C-D, 1 to 265; and C-E, 1 to 205 and 231 to 428 (mature peptide numbering). B, partial amino acid sequence alignment of the N termini of 2 and  subunits. The aligned amino acid sequences of the 2 and  subunits is shown for the region immediately N-terminal to, and including, the TM1, which is overlined. The domain identified as important for tracazolate function (2 residues 206-230) is boxed and within this region the nonconserved amino acids are in lowercase.

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Tracazolate modulation of receptors containing chimeric 2s/ subunits. Wild-type and chimeric subunits were coexpressed with 13 in X. laevis oocytes and assayed for modulation of a GABA EC20 response by 10 µM tracazolate. Data represent the mean ± S.E.M. from the number of cells indicated from two or more batches of oocytes.

	Discussion

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Although tracazolate was first synthesized nearly 30 years ago, this is the first detailed electrophysiological study of its effects on recombinant GABA_A receptors. Tracazolate binds to neither the benzodiazepine nor the GABA binding site but to another as-yet-unidentified site. Its potency (EC₅₀) is influenced by the nature of the  subunit; more importantly, however, its intrinsic efficacy (i.e., whether it potentiates or inhibits GABA) is critically determined by the nature of the third subunit (1-3, , or ) within the receptor complex. Tracazolate may prove to be a useful tool to aid identification of receptor subtypes within neuronal preparations.

Tracazolate Does Not Act via Benzodiazepine Binding Site. Tracazolate produced concentration-related potentiation of control GABA EC₂₀ responses on oocytes expressing the binary receptor 13. This result is in contrast to benzodiazepine compounds, which, at relevant concentrations, do not modulate receptors (Levitan et al., 1988; Pritchett et al., 1988). In addition, tracazolate did not displace [³H]flumazenil from 332s GABA_A receptors stably expressed in Ltk cells (data not shown) nor was the functional response on 132s receptors inhibited by flumazenil.

Importance of Third Subunit within Receptor Complex. The nature of the third subunit within the receptor complex was critical in determining the functional response to tracazolate. For 11/32s receptors tracazolate produced concentration-related potentiation of control GABA EC₂₀ responses; however, for 11/3 receptors GABA EC₂₀ responses were inhibited by tracazolate. Receptors containing a 1 or a 3 subunit produced an intermediate profile with low concentrations of tracazolate potentiating to a small degree the GABA EC₂₀, whereas higher concentrations caused inhibition. These differing functional effects are in contrast to the general anesthetic agents such as pentobarbitone and propofol and the neuroactive steroids, which potentiate all the receptor subtypes examined herein (Whiting et al., 1997; Thompson et al., 1998; Maitra and Reynolds, 1999). In addition, these agents are also dissimilar to tracazolate because they do not display  subunit selectivity.

Outward Current to Tracazolate on 13 Receptors. Spontaneous channel openings in the absence of GABA have been demonstrated in outside-out patches pulled from fibroblasts transiently transfected with 13 (Neelands et al., 1999). In addition to picrotoxin and tracazolate, Zn²⁺ ions and bicuculline have been shown to elicit outward currents on 13 receptors (Neelands et al., 1999). One explanation for the outward current observed with these agents is that they inhibit the constitutively active currents. Mutation of the 9' leucine residue within the 2 subunit and coexpression with 12s produced a receptor with a high degree of constitutive activity, which, like 13 receptors, was reduced in the presence of picrotoxin, bicuculline, gabazine, and Zn²⁺ (Thompson et al., 1999b). Our results demonstrate that the outward current to tracazolate on 13 receptors is carried by chloride ions, suggesting that like other agents it inhibits constitutive activity. The small inward current to low concentrations of tracazolate may represent initial potentiation of the constitutive activity, which is then superseded by the outward current.

Differing Effects on GABA Concentration-Response Curves. Concentration-response curves to GABA in the absence and presence of tracazolate on 132s, 13, and 11 receptors revealed further insights into the mechanism of action of tracazolate. GABA concentration-response curves on both 132s and 11 receptors were shifted to the left with significantly lower EC₅₀ values. The maximum response to GABA, however, in the presence of increasing concentrations of tracazolate, were shifted in opposing directions; on 132s tracazolate reduced the maximum response to GABA, whereas on 11 this was increased. The leftward shift in the GABA concentration-response curve with a reduction in the maximum response for 132s receptors is similar to that reported for loreclezole (Wafford et al., 1994) and SB-205384 (Meadows et al., 1997) and may indicate a common mechanism of action of these compounds. The similarities of these three compounds also extend to the selectivity for 2/3-containing receptors over 1-containing receptors. Benzodiazepine site ligands also produce a leftward shift in the GABA concentration-response curve; however, they cause no reduction in the maximum response (Sigel and Baur, 1988; Maksay et al., 2000).

Identification of Region Critical for Functional Efficacy of Tracazolate. Chimeras C-A and C-B when coexpressed with 13 subunits revealed similar characteristics to 13 receptors (i.e., inhibition of the GABA EC₂₀ response by tracazolate), a direct effect to tracazolate, and the presence of a rebound current upon washout of GABA. Chimeras C-C and C-D, however, were similar to 132s receptors; i.e., they were potentiated by tracazolate and showed no direct effect to tracazolate or rebound currents upon washout of GABA. The switch from negative to positive modulation occurred between chimera C-B to C-C, implicating the region equivalent to 2 residues 206 to 230 in determining the direction of modulation by tracazolate. However, the potentiation observed with chimera C-E, in which only this domain of 2 was replaced by that of , suggests a role for an additional C-terminal element in the transduction process, indicating that this domain is necessary but not sufficient to confer -like inhibition.

Mechanism of Action. The results obtained in this study lead us to speculate on the possible mechanism of action of tracazolate. We hypothesize that a single common site is present, and the observed inhibition or potentiation relates to the nature of the GABA subtype. This is supported by the same apparent EC₅₀ for inhibition or potentiation and an identical shift of EC₅₀/IC₅₀ when the  subunit is switched. We observed that under conditions where the receptor rapidly entered the desensitized state (e.g., 11/3, or high concentrations of GABA on 132s) the functional response to tracazolate was inhibition, whereas in conditions with little desensitization (e.g., 11 or low GABA concentrations on 132s) the functional response was potentiation. The GABA_A receptor subtypes compared in this study differ markedly in their rate of desensitization, with receptors desensitizing faster than 2s, which in turn desensitize faster than receptors (Saxena and Macdonald, 1994; Whiting et al., 1997; Brown et al., 2001). One possible interpretation of the data is that tracazolate displays higher affinity for the desensitized state than for the agonist bound state of the receptor. A similar mechanism has been proposed for the action of ifenprodil on N-methyl-D-aspartate receptors (Kew et al., 1996). Experiments to investigate the effects of tracazolate on the kinetics of GABA will be required to validate this hypothesis.