Tracazolate Reveals a Novel Type of Allosteric Interaction with Recombinant -Aminobutyric AcidA Receptors

Tracazolate, a pyrazolopyridine, is an anxiolytic known to interact with -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 2s subunit within the 1 3 2s receptor with the subunit caused a dramatic change in the functional response to tracazolate from potentiation to inhibition. The 2s subunit was not critical for potentiation because 1 3 receptors were also potentiated by tracazolate. 2/ chimeras revealed a critical Nterminal domain between amino acids 206 and 230 of 2, governing the nature of this response. Replacement of the 3 subunit with the 1 subunit within 1 3 2s and 1 3 receptors also revealed selectivity of tracazolate for 3-containing receptors, determined by asparagine at position 265 within transmembrane 2. Replacement of 2s with 1 or 3 revealed a profile intermediate to that of 1 1 and 1 1 2s. 1 1 receptors were also potentiated by tracazolate; however, the maximum potentiation of the EC20 was much greater than on 1 1 2. Concentration-response curves to GABA in the presence of tracazolate for 1 1 and 1 1 2s revealed a concentration-related decrease in maximum current amplitude, but a leftward shift in the EC50 only on 1 1 2. Like 1 1 2s, GABA concentration-response curves on 1 1 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 subunit; but more importantly, its intrinsic efficacy, potentiation, or inhibition is determined by the nature of the third subunit ( 1–3, , or ) within the receptor complex. The -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 ( 1–6, 1–4, 1–4, , , , and ) (for reviews, see Barnard et al., 1998; Whiting, 1999). Evidence suggests that in vivo GABAA 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 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, S.-A.T. and P.B.W. contributed equally to this work. ABBREVIATIONS: GABA, -aminobutyric acid; MBS, modified Barth’s solution; TM, transmembrane; SB-205384, 4-amino-7-hydroxy-2-methyl5,6,7,8-tetrahydrobenzo [b]-thieno[2,3-b]pyridine-3-carboxylic acid but-2-ynyl ester. 0026-895X/02/6104-861–869$7.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 4 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1378/972492 Mol Pharmacol 61:861–869, 2002 Printed in U.S.A. 861 at A PE T Jornals on M ay 0, 2017 m oharm .aspeurnals.org D ow nladed from with dimetrically opposite actions on 2and -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 Human GABAA 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 GABAA 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/ l of cDNA. Confirmation that all the subunits injected were being expressed was routinely checked using Zn , 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 g/ml gentamicin, 10 g/ml streptomycin, 10 units/ml penicillin, and 2 mM sodium pyruvate, for up to 6 days. For electrophysiological recordings, oocytes were placed in a 50l bath and continually perfused at 4 to 6 ml/min with MBS. Cells were impaled with two 1to 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 GABAevoked 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 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) Bmax/[1 (EC50/x) H], where x is the drug concentration, EC50 is the concentration of drug eliciting a half-maximal response, and nH is the Hill coefficient. For some receptor subtypes high concentrations of tracazolate or etazolate (e.g., 100 M etazolate on 1 3 2s, and 30 M tracazolate on 6 3 2s) 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 M. 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.