Abstract
Tracazolate, a pyrazolopyridine, is an anxiolytic known to interact with γ-aminobutyric acid (GABA)A receptors, adenosine receptors, and phosphodiesterases. Its anxiolytic effect is thought to be via its interaction with GABAA receptors. We now report the first detailed pharmacological study examining the effects of tracazolate on a range of recombinant GABAA receptors expressed in Xenopus laevis oocytes. Replacement of the γ2s subunit within the α1β3γ2s receptor with the ε subunit caused a dramatic change in the functional response to tracazolate from potentiation to inhibition. The γ2s subunit was not critical for potentiation because α1β3 receptors were also potentiated by tracazolate. γ2/ε chimeras revealed a critical N-terminal domain between amino acids 206 and 230 of γ2, governing the nature of this response. Replacement of the β3 subunit with the β1 subunit within α1β3γ2s and α1β3ε receptors also revealed selectivity of tracazolate for β3-containing receptors, determined by asparagine at position 265 within transmembrane 2. Replacement of γ2s with γ1 or γ3 revealed a profile intermediate to that of α1β1ε and α1β1γ2s. α1β1δ receptors were also potentiated by tracazolate; however, the maximum potentiation of the EC20 was much greater than on α1β1γ2. Concentration-response curves to GABA in the presence of tracazolate for α1β1ε and α1β1γ2s revealed a concentration-related decrease in maximum current amplitude, but a leftward shift in the EC50 only on α1β1γ2. Like α1β1γ2s, GABA concentration-response curves on α1β1δ receptors were shifted to the left with increased maximum responses. Tracazolate has a unique pharmacological profile on recombinant GABAA receptors: its potency (EC50) is influenced by the nature of the β subunit; but more importantly, its intrinsic efficacy, potentiation, or inhibition is determined by the nature of the third subunit (γ1–3, δ, or ε) within the receptor complex.
The γ-Aminobutyric acid typeA (GABAA) receptor is a major inhibitory neurotransmitter receptor in the vertebrate central nervous system. In most neurons, the binding of the neurotransmitter GABA to a GABAA 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 (α1–6, β1–4, γ1–4, δ, ε, θ, and π) (for reviews, see Barnard et al., 1998; Whiting, 1999). Evidence suggests that in vivo GABAA receptors are pentameric complexes of α, β, and γ subunits with a stoichiometry of 2α:2β:1γ (Chang et al., 1996; Farrar et al., 1999). The stoichiometry of receptors containing δ, ε, and θ is currently unknown, although evidence suggests that δ and ε substitute for a γ subunit (Caruncho and Costa, 1994; Quirk et al., 1995; Whiting et al., 1997), whereas θ replaces a β subunit (Bonnert et al., 1999).
The GABAA 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 GABAA 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 γ2- and ε-containing receptors. In addition the generation of chimeric γ2/ε subunits implicates the region equivalent to γ2 residues 206 to 230 in determining the nature of this modulation.
Materials and Methods
Human GABAA Receptor cDNAs.
The cloning and sequencing of human α1, α6, β1, β3, γ1, γ2s, γ3, δ, and ε and the construction of the single point mutations β1S265N and β3N265S 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 γ2/ε 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. laeviswere 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 GABAA subunit cDNAs engineered into the expression vector pCDM8 or pcDNAI/Amp. The ratio of α:β:γ2s constructs was generally 1:0.5:1, whereas the ratio for α:β was 1:1, for α:β:γ3 was 1:1:1, for α:β:γ1 was 1:1:10, and for αβδ and αβε 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-MΩ 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 EC20concentration of GABA (range EC18–25), 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)nH], where x is the drug concentration, EC50 is the concentration of drug eliciting a half-maximal response, andn H is the Hill coefficient. For some receptor subtypes high concentrations of tracazolate or etazolate (e.g., 100 μM etazolate on α1β3γ2s, and 30 μM tracazolate on α6β3γ2s) 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
Potentiation of α1β1γ2s and α1β3γ2s GABAAReceptors: Selectivity for β3.
GABA EC20responses on α1β1γ2s and α1β3γ2s receptors were potentiated in a concentration-dependent manner by tracazolate and etazolate (Table1; 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 GABAAreceptor [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 α1β3γ2s over α1β1γ2s receptors. Because structurally and functionally tracazolate and etazolate were similar, further investigations were performed with tracazolate only.
β3 Selectivity Is Conferred by Asparagine 265.
Selectivity for β2/3-containing GABAAreceptors over β1 has previously been reported for loreclezole (Wingrove et al., 1994), β-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 β2 and β3 subunit. It was logical therefore to see whether this residue also determined the β3 selectivity of tracazolate. The two mutant β cDNAs (β1Ser265Asn and β3Asn265Ser) were coexpressed with α1 and γ2s and concentration-response curves to tracazolate constructed (Table 1; Fig. 2). Replacement of Ser265 within the β1 subunit with Asn (the β3 counterpart) increased the sensitivity of tracazolate, whereas the opposite mutation (Asnβ3 to Ser) decreased the sensitivity to tracazolate. Hence, the β3 selectivity of tracazolate is determined by asparagine 265 within the β3 subunit.
Interaction Is Not via Benzodiazepine Binding Site.
Concentration-response curves to tracazolate were also constructed on oocytes expressing α1β3 and α6β3γ2s GABAA receptors (Table 1; Fig.3). Similar to α1β3γ2s receptors, control GABA EC20 concentrations on α1β3 and α6β3γ2s receptors were potentiated by tracazolate. Statistical analysis (analysis of variance) revealed no significant differences in the log EC50, Hill coefficient, or maximum potentiation for α1β3, α1β3γ2s, or α6β3γ2s. Unlike compounds that interact at the benzodiazepine site, receptors lacking a γ subunit were also potentiated by tracazolate. Replacement of the α1 subunit with an α6 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 α1β3γ2s receptors (198 ± 42%, n = 5 in the absence versus 223 ± 31%, n = 4 in the presence).
Tracazolate Inhibits α1β1ε and α1β3ε GABAAReceptors.
In addition to replacing the α and β subunits, we studied the effects of replacing the γ2 subunit. ε-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 α1β1ε and α1β3ε 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., β3 homomeric receptors (Wooltorton et al., 1997); α1β2L259Sγ2s (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 α1β3ε receptors was undertaken in separate experiments. To enable direct comparison of the modulatory effect of tracazolate on γ2- and ε-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 α1β3γ2s and α1β1γ2s 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 α1β3γ2, tracazolate caused inhibition of the control GABA EC20response (Fig. 4; Table 2). The GABA response could be almost completely inhibited, and the IC50 values for inhibition by tracazolate on α1βε receptors were similar to the EC50values obtained on α1βγ2s receptors.
Outward Current to Tracazolate on α1β3ε Receptors Is Carried by Cl− Ions.
Concentration-response curves to the direct effects of tracazolate were constructed on α1β3ε 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 α1β3ε receptors (Neelands et al., 1999). The holding current of α1β1ε and α1β3ε 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 α1β3ε 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).
The current-voltage relationship was determined for the direct effect of 3 μM tracazolate on α1β3ε receptors. The data were best fitted to a linear regression (r 2 = 0.94 ± 0.02, n = 4) and revealed a reversal potential of −25.7 ± 1.3 mV, n = 4, which was similar to the predicted reversal potential for Cl− ions of −25.4 mV in X. laevisoocytes with an external Cl− concentration of 89.91 mM (MBS used in this study) and an internal Cl− concentration of 33.4 mM (Barish, 1983), indicating that the carrier of the direct effect is Cl− ions.
Modulation of α1β1γ1, α1β1γ3, and α1β1δ Receptors.
The opposing effects observed with tracazolate on γ2s- and ε-containing receptors prompted studies on γ1-, γ3-, and δ-containing receptors. These subunits were coexpressed with α1 and β1 subunits and concentration-response curves to tracazolate constructed. As can be seen in Fig. 6A tracazolate behaved differently on α1β1γ1 and α1β1γ3 receptors compared with α1β1γ2s and α1β1ε. Concentrations up to 10 μM produced a small degree of potentiation of the GABA EC20 response (19 and 30% for 10 μM tracazolate on α1β1γ1 and α1β1γ3, respectively), whereas higher concentrations inhibited the GABA EC20response. The potentiating portion of the concentration-response curve revealed similar EC50 values, Hill coefficients, and maximum responses for α1β1γ1 and α1β1γ3 receptors (Table 1). The data obtained for α1β1γ1 and α1β1γ3 receptors were not significantly different from one another (P > 0.05, unpaired Student's t test); however, comparison with α1β1γ2s revealed a 10-fold decrease in EC50.
GABA EC20 responses for α1β1δ receptors were potentiated by tracazolate to levels substantially greater than that produced by a maximum GABA concentration (Fig. 6B). Potentiation of a GABA EC20 concentration by tracazolate above and beyond the maximum current elicited by GABA was not observed with any other subunit combination examined. The log EC50 values and Hill coefficients, however, were not significantly different between α1β1δ and α1β1γ2s.
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 α1β3ε, α1β3γ2s, and α1β1δ receptors.
On α1β3γ2s receptors, 1 μM tracazolate produced a significant (P < 0.05) 2.5 ± 0.3-fold shift to the left of the GABA EC50 with no significant effect on the maximum response or Hill coefficient (Fig.7A). Higher concentrations of tracazolate (10 and 30 μM) further increased this leftward shift of the GABA concentration-response curve (21.6 ± 5.5- and 39.3 ± 15.8-fold, respectively). In addition the maximum response of GABA in the presence of 10 and 30 μM tracazolate was significantly reduced compared with the maximum obtained for the control GABA concentration-response curve (67.6 ± 4 and 40.0 ± 2.9%, respectively). Tracazolate (30 μM) also significantly reduced the Hill coefficient of the GABA concentration-response curve compared with the control (1.54 ± 0.03 versus 0.98 ± 0.14,P < 0.05).
For α1β3ε receptors, the inward current alone to GABA and tracazolate was measured omitting any direct effect. The log EC50 values and Hill coefficients for the control GABA concentration-response curves compared with those in the presence of 1 and 3 μM tracazolate were not significantly different, whereas the maximum response obtainable to GABA in the presence of 1 and 3 μM tracazolate were significantly lower (P < 0.05) (Fig.7B).
Similar to α1β3γ2s receptors, concentration-response curves to GABA on α1β1δ receptors were shifted to the left by 10 and 30 μM tracazolate (7.6 ± 1.8- and 15.8 ± 1.9-fold, respectively). However, unlike α1β1γ2s the maximum response to GABA in the presence of 10 and 30 μM tracazolate was significantly larger (202.9 ± 27.7 and 305.1 ± 34.2%, respectively) (Fig. 7C).
Domain 206 to 230 Determines Functional Response to Tracazolate.
As described above, tracazolate is a positive modulator at receptors containing a γ subunit but a negative modulator when substituted by ε. To investigate the amino acid determinants of this effect, a series of chimeric γ2/ε subunits were constructed, each having an N-terminal γ2 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 α1β3 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 α1β3γ2s receptors and inhibition on α1β3ε receptors. Similar to α1β3ε, chimera C-A and C-B were negatively modulated by tracazolate (α1β3ε: −79.1 ± 3.4%, n = 6; α1β3C-A: −46.4 ± 8.1%, n = 4; and α1β3C-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 α1β3γ2s (α1β3C-C: 113 ± 15%, n = 4; α1β3C-D: 88 ± 6%,n = 3; and α1β3γ2s: 198 ± 42%,n = 5; Fig. 9). These results implicate a residue or residues within the γ2 domain 206 to 230 that confer the functional response observed with tracazolate. However, replacement of this whole region in γ2 with the homologous portion of ε (chimera C-E) did not alter the functional response to tracazolate [i.e., positive modulation similar to that of α1β3γ2 receptors (α1β3C-E: 210 ± 51%, n = 4; Fig.9)], suggesting that although this region may be necessary, other residues are also required to confer inhibition.
Discussion
Although tracazolate was first synthesized nearly 30 years ago, this is the first detailed electrophysiological study of its effects on recombinant GABAA 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 β 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 (γ1–3, δ, or ε) 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 α1β3. This result is in contrast to benzodiazepine compounds, which, at relevant concentrations, do not modulate αβ receptors (Levitan et al., 1988; Pritchett et al., 1988). In addition, tracazolate did not displace [3H]flumazenil from α3β3γ2s GABAA receptors stably expressed in Ltk− cells (data not shown) nor was the functional response on α1β3γ2s receptors inhibited by flumazenil.
The type of β subunit present within the receptor complex has previously been shown not to effect the modulation obtained with benzodiazepine site ligands (Hadingham et al., 1993). Tracazolate, however, was significantly more potent on α1β3γ2s receptors compared with α1β1γ2s receptors. Recently, an increasing number of structurally unrelated compounds have been identified that also show this selectivity (e.g., loreclezole, β-carbolines, etomidate, furosemide, and mefenamic acid). For each compound, this selectivity has been shown to be due to the presence of an asparagine residue at the homologous position 264 and 265 within TM2 of the β2 and β3 subunit, respectively. Similarly, this asparagine residue was shown to be responsible for the β3 selectivity observed with tracazolate. Collectively, these results demonstrate that tracazolate does not interact with the benzodiazepine site and are in agreement with the previous biochemical and electrophysiological data obtained for the pyrazolopyridines (Williams and Risley, 1979; Barnes et al., 1983).
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 α1β1/3γ2s receptors tracazolate produced concentration-related potentiation of control GABA EC20 responses; however, for α1β1/3ε receptors GABA EC20 responses were inhibited by tracazolate. Receptors containing a γ1 or a γ3 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 β subunit selectivity.
The largest degree of potentiation was observed with α1β1δ receptors. On this receptor subtype, tracazolate potentiated the GABA EC20 response by 1368 ± 377%. This current was approximately 3 times that elicited by a maximum concentration of GABA. Potentiation of a GABA EC10–25 response beyond that of the maximum GABA response has previously been demonstrated for isoflurane on α1β1δ receptors (Lees and Edwards, 1998). One possible explanation of these results is that on α1β1δ receptors GABA behaves as a partial agonist with a low probability of opening. This probability of opening is increased in the presence of tracazolate or isoflurane, giving rise to a supramaximal response. Further evidence for GABA behaving as a partial agonist has been demonstrated using an Ltk− cell line stably expressing α4β3δ receptors in which concentration-response curves to 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol elicit significantly larger responses than GABA (Adkins et al., 2001; Brown et al., 2001).
Outward Current to Tracazolate on α1β3ε Receptors.
Spontaneous channel openings in the absence of GABA have been demonstrated in outside-out patches pulled from fibroblasts transiently transfected with α1β3ε (Neelands et al., 1999). In addition to picrotoxin and tracazolate, Zn2+ ions and bicuculline have been shown to elicit outward currents on α1β3ε 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 β2 subunit and coexpression with α1γ2s produced a receptor with a high degree of constitutive activity, which, like α1β3ε 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 α1β3ε 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 α1β3γ2s, α1β3ε, and α1β1δ receptors revealed further insights into the mechanism of action of tracazolate. GABA concentration-response curves on both α1β3γ2s and α1β1δ 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 α1β3γ2s tracazolate reduced the maximum response to GABA, whereas on α1β1δ this was increased. The leftward shift in the GABA concentration-response curve with a reduction in the maximum response for α1β3γ2s 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 β2/3-containing receptors over β1-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).
On α1β3ε receptors tracazolate behaved as a noncompetitive antagonist, reducing the maximum response to GABA with no change in the log EC50 value or Hill coefficients.
Identification of Region Critical for Functional Efficacy of Tracazolate.
Chimeras C-A and C-B when coexpressed with α1β3 subunits revealed similar characteristics to α1β3ε 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 α1β3γ2s 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 γ2 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 γ2 was replaced by that of ε, suggests a role for an additional C-terminal element in the transduction process, indicating that this domain is necessary but not sufficient to confer ε-like inhibition.
This region just before TM1 is in the vicinity of the loop C domain, which includes several amino acid positions that influence ligand binding in subunits of the Cys-loop receptor family (Vafa and Schofield, 1998). The loop C ligand-binding domain of the α subunit has several amino acid positions that have been suggested to have a role in the function of benzodiazepine site ligands [e.g., positions 201 (Pritchett and Seeburg, 1991), 205 (Renard et al., 1999), and 207 and 210 (Amin et al., 1997; Buhr et al., 1997)]. Boileau et al. (1998)and Boileau and Czajkowski (1999) have investigated the mechanism of benzodiazepine action using chimeric γ2/α1 subunits. These studies are not easily comparable with those carried out herein because the chimeras used were constructed from subunits of nonequivalent classes. Nevertheless, it is interesting to note that the region of the subunit that was identified as being important for benzodiazepine potentiation is overlapping with that which we have identified in this study. From these data, it is clear that the region just before TM1 is important for allosteric effects, perhaps not surprising given its vicinity to the channel.
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 β subunit is switched. We observed that under conditions where the receptor rapidly entered the desensitized state (e.g., α1β1/3ε, or high concentrations of GABA on α1β3γ2s) the functional response to tracazolate was inhibition, whereas in conditions with little desensitization (e.g., α1β1δ or low GABA concentrations on α1β3γ2s) the functional response was potentiation. The GABAA receptor subtypes compared in this study differ markedly in their rate of desensitization, with αβε receptors desensitizing faster than αβγ2s, which in turn desensitize faster than αβδ 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.
In conclusion, this study represents the first functional characterization of the modulatory effects of tracazolate on recombinant GABAA receptors and reveals that tracazolate has a unique profile unlike any other allosteric modulator characterized to date. Mutagenesis studies, aimed at identifying the molecular determinants responsible for the opposing functional effects of tracazolate on γ- and ε-containing receptors have highlighted region 206 to 230 in γ2 as being necessary but not sufficient in determining the different functional effects.
Footnotes
- Received September 26, 2001.
- Accepted December 21, 2001.
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S.-A.T. and P.B.W. contributed equally to this work.
Abbreviations
- GABA
- γ-aminobutyric acid
- MBS
- modified Barth's solution
- TM
- transmembrane
- SB-205384
- 4-amino-7-hydroxy-2-methyl-5,6,7,8-tetrahydrobenzo [b]-thieno[2,3-b]pyridine-3-carboxylic acid but-2-ynyl ester
- The American Society for Pharmacology and Experimental Therapeutics