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NEUROPHARMACOLOGY

Pharmacological Characterization of Agonists at -Containing GABA_A Receptors: Functional Selectivity for Extrasynaptic Receptors Is Dependent on the Absence of ₂

Department of Electrophysiology, H. Lundbeck A/S, Valby, Denmark

Received July 11, 2005; accepted September 16, 2005.

	Abstract

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Several groups have characterized the pharmacology of ₄- or ₆₃-containing GABA_A receptors expressed in different cell systems. We have previously demonstrated that the pharmacological profiles of a series of GABA_A receptor agonists are highly dependent on the subunit and little on the and subunits, so to further understand the contribution of the different subunits in the GABA_A receptor complex, we characterized a series of full agonists, partial agonists, and antagonists at ₄₃, ₄₃, and ₆₃ receptors expressed in Xenopus oocytes. Little or no difference was seen when the compounds were compared at - and -containing receptors, whereas a significant reduction in both potency and relative efficacy was observed compared with -containing receptors described in the literature. These data clearly confirm that the presence of the subunit in heterotrimeric receptors is a strong determinant of the increased pharmacological activity of compounds with agonist activity. The very similar agonist pharmacology of - and -containing receptors, which is significantly different from that of -containing receptors, shows that whereas the presence of a subunit impairs the response to an agonist stimulation of the receptor complex, the subunit does not affect this in any way. Taken together, these data are well in line with the idea that ₄₃ may contribute to the pharmacological action of exogenously applied agonists and may explain why systemically active compounds such as gaboxadol and muscimol in vivo appear to act as selective extrasynaptic GABA_A agonists.

Much of the current knowledge about the existence of extrasynaptic receptors originates from immunocytochemical studies combined with high-resolution electron microscopy. These studies have revealed that the subunit composition of synaptically located GABA_A receptors may be distinctly different from that of extrasynaptic receptors. One of the most clear-cut examples is the ₆ subtype, which is exclusively present at extrasynaptic sites in cerebellar granule cells, whereas the synaptically located receptor subtypes in these cells are dominated by ₁₂ and ₁₆₂ subunits (Nusser et al., 1998). Studies in transgenic mice, in which the expression of the ₆ subunit has been obliterated, have demonstrated a post-translational loss of subunit protein in the cerebellum (Jones et al., 1997; Nusser et al., 1999; Brickley et al., 2001). A similar subunit partnership has been suggested between ₄ and subunits (Sur et al., 1999; Peng et al., 2002). It has been speculated whether the cerebral homolog of ₆, the ₄ subunit, is also located preferentially at extrasynaptic locations. This seems reasonable because both subunits render the receptor complexes insensitive toward benzodiazepines and because they exhibit complementary regional expression (cerebrum versus cerebellum) (Sieghart and Sperk, 2002). Indeed, Sun et al. (2004) have recently demonstrated that ₄ is located extrasynaptically in hippocampus.

Functional studies have shown that the extrasynaptic receptors act as low conductance channels (Bai et al., 2001). However, importantly, the potency of GABA is relatively high and the level and speed of desensitization are very small (Saxena and Macdonald, 1994). A series of functional studies have all suggested that the main site of action for the GABA_A receptor agonist gaboxadol may be the extrasynaptically located receptors. Expression of ₄₃-containing receptors and subsequent electrophysiological or biochemical characterization have shown that gaboxadol possesses both high potency and efficacy at these receptors (Adkins et al., 2001; Brown et al., 2002). In tissue sections of rats, no synergistic interactions with benzodiazepines (mediating their effects via the synaptically located ₁-containing receptors) are seen (Storustovu and Ebert, 2003). In addition, Liang et al. (2004) have shown that gaboxadol activates tonic currents elicited by extrasynaptic receptors in hippocampi of rats. It therefore seems very plausible that extrasynaptic receptors may be the main target for gaboxadol.

To further understand the mechanism of action of gaboxadol and to attempt to address what drives this previously unpredicted functional selectivity of gaboxadol, we have conducted the present study. We have expressed ₄₃ receptors in Xenopus oocytes and extended the findings by Adkins et al. (2001) and Brown et al. (2002) with thorough characterization of a broad spectrum of agonists, which in _1–6x2 receptors expressed in Xenopus oocytes have been found to behave as full and partial agonists (Ebert et al., 1994, 1997, 2001). These compounds include, in addition to GABA, muscimol, isoguvacine, gaboxadol, piperidine-4-sulfonic acid (P4S), imidazole-4-acetic acid (IAA) and 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL). Furthermore, we have investigated the interaction of the same agonists with the cerebellar equivalent of -containing GABA_A receptors, namely the receptor subtype composed of ₆₃ subunits, to determine whether the altered pharmacology could be ascribed to the presence of the ₄ or the subunit.

	Materials and Methods

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cRNA Preparation. Cloning and sequencing of cDNAs encoding human ₄, ₆, ₃, and GABA_A receptor subunit proteins have been described elsewhere (Hadingham et al., 1993, 1996; Wafford et al., 1996; Thompson et al., 1997). The cDNAs were engineered into a pcDNAI/Amp (₄, ₃, and ) or a pCDM8 (₆) vector (Invitrogen, San Diego, CA). DNA was a kind gift from Dr. Paul Whiting, Merck Sharp and Dohme, Terlings Park, Harlow, UK. Large-scale cDNA preparation and purification were undertaken using a QIAGEN Plasmid Maxi kit (QIAGEN GmbH, Hilden, Germany). Plasmids were linearized using HpaI, XbaI, and NotI restriction enzymes for ₄/, ₃, and ₆ cDNAs, respectively, and transcribed and capped in vitro (mMESSAGE mMACHINE T7 kit; Ambion, Austin, TX). The RNAs were precipitated with LiCl, redissolved in sterile RNase-free water, and stored at –80°C. cRNA was kindly supplied by Jan Egebjerg and Lene Heding, Department of Molecular Genetics, H. Lundbeck A/S, Valby, Denmark.

Oocyte Isolation and Injection. An adult female Xenopus laevis was anesthetized by immersion in a 0.4% (w/v) 3-aminobenzoic acid ethyl ester solution (Sigma Chemical, St. Louis, MO) for 15 to 20 min. Through an incision in the abdominal wall two to three ovarian lobes were removed, and stage V and VI oocytes were manually defolliculated with watchmaker's fine forceps. After mild collagenase treatment [type IA (Sigma), 0.5 mg/ml for 6 min] to remove remaining follicle cells, each oocyte was injected with various combinations of ₄-, ₃-, and -encoding cRNA (₄ and : 32.2 ng/oocyte; ₃: 3.2 ng/oocyte). Oocytes were incubated for at least 4 days in modified Barth's saline [88 mM NaCl, 1 mM KCl, 15 mM HEPES, 2.4 mM NaHCO₃, 0.41 mM CaCl₂, 0.82 mM MgSO₄, and 0.3 mM Ca(NO₃)₂] supplemented with 2 mM sodium pyruvate, 0.1 U/l penicillin, and 0.1 µg/l streptomycin and filtered through nitrocellulose.

Two-Electrode Voltage Clamping. Oocytes were placed in a 60-µl bath and perfused with Ringer's solution (115 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1.8 mM CaCl₂, and 0.1 mM MgCl₂, pH 7.5). Cells were impaled with agar-plugged 0.5 to 1 M electrodes containing 3 M KCl and voltage-clamped at –70 mV by a GeneClamp 500B amplifier (Molecular Devices Corporation, Sunnyvale, CA) with a gain setting of ÷1 or ÷10. The cells were continuously perfused with Ringer's buffer at 4 to 6 ml/min, and the drugs were applied in the perfusate. Agonist-containing solutions were applied until the peak of the response was observed, usually after 30 s or less. A 6-to 10-min washout period between agonist applications was allowed to minimize desensitization. To ensure complete binding, antagonists were preapplied alone for 1 min before their coapplication with GABA.

Data Analysis. Agonist concentration-response curves obtained from Xenopus oocytes were fitted by use of the nonlinear, least-squares fitting program, GraFit 5.0.10 (Erithacus Software, Horley, Surrey, UK). Data series from each individual oocyte were normalized with respect to the maximum GABA current, GABA I_max, elicited from that particular cell and fitted to eq. 1:

(1)

where E_max is the maximum response, [L] is the concentration of the ligand, EC₅₀ is the concentration of the ligand eliciting 50% of the maximal response, and n_H is the slope factor.

The competitive GABA_A receptor antagonists were characterized with respect to their ability to shift a GABA concentration-response curve rightward when present in a fixed concentration. K_i values for these antagonists were derived from coanalysis of data obtained from each individual oocyte in the absence and presence of antagonist by use of the Waud equation:

(2)

where [I] is the (fixed) antagonist concentration. Geometric mean values of the K_i values were calculated.

Concentration-inhibition curves for Zn²⁺ and La³⁺ were performed by application of increasing concentrations of the cations in the presence of a fixed concentration of GABA corresponding to the exact EC₅₀ value determined for each individual cell before the concentration-inhibition experiment. Data were normalized with respect to the GABA EC₅₀ response and fitted to eq. 3:

(3)

where IC₅₀ is the inhibitor concentration yielding 50% inhibition of the GABA EC₅₀ response and background is an arbitrary constant.

Experiments were performed on at least four different oocytes stemming from at least two different oocyte batches. When a putative two-site behavior was in question, the data were fitted to eq. 4:

(4)

With an F test comparing the ² values obtained from the two fits (using eq. 3 or 4, respectively) and the degrees of freedom in each, we statistically tested whether the two-site equation fitted the data significantly better than the one-site equation.

Drugs and Solutions. Stock solutions of GABA (1 M) were made in double-distilled water and stored in aliquots at –20°C. The remaining compounds were dissolved freshly in double-distilled water at a concentration of 10 to 100 mM. Final drug solutions were made by dilution in Ringer's solution. All chemicals were purchased from conventional commercial sources except for 5-(4-piperidyl)-3-isoxazolol (4-PIOL) and thio-4-PIOL, which were kind gifts from Bente Frølund, Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark.

	Results

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₄₃- and ₄₃-Expressing Xenopus Oocytes. Various combinations of GABA_A receptor ₄, ₃, and subunits encoding cRNAs were injected in Xenopus oocytes. Whereas injection of ₄₃ and ₄₃ subunit combinations gave rise to functional receptors with maximal GABA currents, GABA I_max, of 314 ± 48 (mean ± S.E.M.) and 103 ± 29 nA, respectively, no GABA-elicited currents could be measured in ₃- and ₄-injected oocytes.

Because the subunit is notoriously difficult to coexpress in recombinant systems, it can be questioned whether or not all the expressed receptor complexes in oocytes injected with ₄₃ encoding cRNA really do contain the subunit. In an attempt to increase the fraction of -containing receptor complexes as much as possible, a 10-fold excess of -encoding cRNA was injected as previously reported by SundströmPoromaa et al. (2002). Furthermore, we strove to find a pharmacological tool capable of discriminating between the two different assemblies of subunits, thus giving a means of assessing the homogeneity of the receptor population. The polyvalent cations Zn²⁺ and La³⁺ are well established negative allosteric modulators of certain GABA_A receptor subunit assemblies, whereas other subunit combinations seem to be less sensitive to these cations. Zn²⁺ has been reported to inhibit recombinant _1x receptors, whereas their ₂-containing counterparts are virtually insensitive to inhibition (e.g., Smart et al., 1991; Wooltorton et al., 1997; Nagaya and Macdonald, 2001). In addition, Brown et al. (2002) have recently demonstrated a pronounced difference in the sensitivity toward La³⁺ between _432S- and ₄₃-containing receptors stably expressed in L(tk) cells and a less significant difference between the same constructs toward Zn²⁺, with _432S being the most sensitive.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Lanthanum (left panel) and zinc (right panel) concentration-inhibition curves obtained from Xenopus oocytes expressing GABAA receptors composed from 43 (n = 4) and 43 (n = 4) subunits. Data were normalized with respect to the GABA EC50 response and fitted to eq. 3. Fitting results are presented in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Fitting results from Zn2+ and La3+ concentration-inhibition curves (inhibiting GABA EC50 responses) obtained from Xenopus oocytes expressing GABAA receptors composed from 43 and 43 subunits Data from each individual oocyte were normalized with respect to the GABA EC50 response obtained from that particular oocyte and fitted to eq. 3. IC50 values are presented as geometric mean values. The remaining values are arithmetic means ± S.E.M.

In our system, the ₄₃ combination proved to be sensitive to La³⁺ with an IC₅₀ value of 5.7 µM, whereas the -deficient receptors were practically insensitive (Fig. 1, left panel). However, because even high concentrations of La³⁺ could only bring about approximately 50% inhibition of the GABA EC₅₀ responses at ₄₃ receptors and because the degree of inhibition of the ₄₃ receptor complexes turned out to be variable, La³⁺ would to all appearances not be the most suitable tool for discrimination between -deficient and -containing receptor complexes. Therefore, similar experiments were carried out using Zn²⁺ instead. As illustrated in Fig. 1 (right panel) and Table 1, Zn²⁺ is a potent inhibitor of GABA responses at ₄₃-containing receptors (IC₅₀ = 62 nM), whereas ₄₃-containing receptors are 100-fold less sensitive (IC₅₀ = 5.3 µM). This finding demonstrates that the subunit is in fact incorporated into functional receptor complexes expressed at the cell surface.

Thus, as seen from Fig. 1, if coapplication of GABA and 0.1 µM Zn²⁺ to ₄₃ receptors yields a response of approximately 80% of the EC₅₀ response or less, the receptor population presumably will contain a considerable fraction of ₄₃ receptors. Now having an efficient means of assessing the receptor population purity, we proceeded with agonist concentration-responses curves.

A wide variety of agonists, which have proven to be full and partial agonists at _1–6x2S receptors (Ebert et al., 1994, 1997, 2001), were tested at ₄₃-expressing oocytes. As seen from Fig. 2a and Table 2, gaboxadol, isoguvacine, and muscimol appeared to elicit higher maximal responses than GABA, with gaboxadol being the most efficacious with an E_max value of 215 ± 10.6% compared with the 191 ± 6.7 and 144 ± 4.2% found for isoguvacine and muscimol, respectively. On the contrary, P4S and IAA behaved as partial agonists with maximal responses of about 65%. In addition, the low-efficacy partial agonists, thio-4-PIOL and 4-PIOL, were tested, with the former giving an E_max value of 4.4 ± 0.6%. For 4-PIOL, however, the agonist responses were so minute that they could not be adequately separated from the background noise. With EC₅₀ values in the low micromolar range, GABA and muscimol turned out to be 10-to 50-fold more potent than the remaining agonists (Table 2).

View larger version (25K): [in this window] [in a new window]   Fig. 2. a, concentration-response curves for a broad range of GABAA receptor agonists obtained in Xenopus oocytes expressing GABAA receptors composed of 43 subunits. The inset depicts an enlarged excerpt of the lower part of the graph containing the partial agonist concentration-response curves. b, comparison of agonist curves for THIP, GABA, and P4S at GABAA receptors composed from 43 and 43 subunits. All data were normalized with respect to GABA Imax and subsequently fitted to eq. 1. Derived parameters are seen in Table 2. c, representative example of a trace recorded from a Xenopus oocyte expressing 43 subunits. The horizontal bars indicate application of the gaboxadol or GABA concentration given above each bar.

View this table: [in this window] [in a new window]   TABLE 2 Fitting results from agonist concentration-response curves obtained from Xenopus oocytes expressing GABAA receptors consisting of 43 subunits and 43 subunits All data were normalized with respect to the GABA Imax from the same oocyte and fitted to eq. 1. Fitting results are presented as arithmetic means ± S.E.M. except for EC50, which is the geometric mean value.

To investigate whether the highly efficacious behavior of gaboxadol would be due to a -specific receptor interaction, this agonist was tested at ₄₃ GABA_A receptors. As reference compounds the full agonist GABA and the partial agonist P4S were chosen. Interestingly, these three compounds displayed the same potencies as at the ₄₃-containing receptors. In addition, no large discrepancies between relative efficacy levels of the agonists at the two different subunit combinations were found (Table 2).

In an attempt to further address the functional differences between gaboxadol and GABA, GABA was coapplied with a fixed concentration of gaboxadol at ₄₃-containing receptors. As illustrated in Fig. 3, GABA concentration dependently reduced the response to gaboxadol until the response corresponded to the maximum response to GABA. This reduction was not due to desensitization or down-regulation of receptors, because the response to gaboxadol after coapplication with GABA was similar to that before the coapplication (data not shown).

View larger version (11K): [in this window] [in a new window]   Fig. 3. a, concentration-response curves for GABA and gaboxadol in Xenopus oocytes expressing GABAA receptors composed of 43 subunits. b, at 43-containing receptors, GABA acts as a partial agonist. With increasing concentrations of GABA, the response to a fixed concentration of gaboxadol (300 µM) is suppressed approximately to the relative efficacy level of GABA alone (n = 4). All data were normalized with respect to GABA Imax and subsequently fitted to the eq. 3.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Example of a rightward parallel shift of a GABA concentration-response curve obtained with 10 µM bicuculline in Xenopus oocytes expressing GABAA receptors composed of 43 subunits. In the absence of the antagonist, an EC50 value of 2.7 µM was obtained (). In the presence of 10 µM bicuculline the EC50 was shifted approximately 10-fold to 32 µM (). Data were fitted to eq. 2.

View this table: [in this window] [in a new window]   TABLE 3 Fitting results from shift of GABA concentration-response curves obtained from Xenopus oocytes expressing GABAA receptors composed from 43 subunits Data were fitted to eq. 2. Ki values presented as geometric mean values. pKi values are arithmetic means ± S.E.M.

₆₃-Expressing Xenopus Oocytes. To evaluate the agonist pharmacology at another major extrasynaptic receptor subtype, the cerebellar ₆-containing GABA_A receptors (Nusser et al., 1998; Sassoe-Pognetto et al., 2000), Xenopus oocytes were injected with ₆₃-encoding cRNAs in a subunit ratio of 10:1:10. As for the ₄₃-containing receptors, the full incorporation of the subunit into the expressed receptors was a matter of some concern. Surprisingly, control experiments revealed that injection of -deficient ₆₃ cRNAs did not give rise to formation of functional receptor complexes sensitive to GABA or other agonists, whereas robust expression was obtained with ₆₃-encoding cRNAs (GABA I_max = 304 ± 48 nA, mean ± S.E.M.). Although in contrast to findings from other oocyte laboratories (e.g., Thompson et al., 1997; Wallner et al., 2003), the lack of ₆₃ expression might indicate that formation of this receptor by-product in ₆₃-injected oocytes is not a crucial problem. However, to prove the presence of subunits in ₆₃-expressing oocytes, a series of zinc experiments, performed as described previously, were conducted. As seen from the concentration-inhibition curve depicted in Fig. 5, left panel, and the slope factor, n_H, in Table 4, the course of the curve was not as steep as that seen at ₄₃ receptors. In fact, for all the oocytes, a more shallow curve was apparent, suggesting two distinct binding sites. However, when the goodness of the fits obtained from a one-site and a two-site equation were compared by an F test, the two-site equation was significantly better than the one-site equation only in two instances. A concentration-inhibition curve for one of these cells is seen in Fig. 5, right panel (p < 0.005 when a two-site fit was compared with a one-site fit). Interaction with the primary site yielded an IC₅₀ value of 2.6 µM (resolved by use of eq. 4) and a secondary low-affinity site (IC₅₀ = 240 µM) could also be identified. However, because of the high variability in the appearance of the two sites, we will refrain from speculating about its significance, which at the current time remains enigmatic.

View larger version (10K): [in this window] [in a new window]   Fig. 5. Zinc concentration-inhibition curves obtained from Xenopus oocytes expressing GABAA receptors composed from 63 subunits (n = 5). Data were normalized with respect to the GABA EC50 response and fitted to eq. 3. Fitting results are presented in Table 4.

View this table: [in this window] [in a new window]   TABLE 4 Fitting results from Zn2+ concentration-inhibition curves (inhibiting GABA EC50 responses) obtained from Xenopus oocytes expressing GABAA receptors composed from 43 subunits Data from each individual oocyte were normalized with respect to the GABA EC50 response obtained from that particular oocyte and fitted to eq. 3. The IC50 value is presented as a geometric mean value. The remaining values are arithmetic means ± S.E.M.

When fitting the data to the traditional one-site equation (eq. 3), the IC₅₀ value obtained from the ₆₃-expressing oocytes (8.7 µM) was in the same range as that derived from ₄₃-expressing oocytes (5.3 µM) and, furthermore, in agreement with findings of Thompson et al. (1997) who in similar experiments found IC₅₀ values of 0.42 and 8.6 µM in ₆₃- and ₆₃-expressing oocytes. Conclusively, these data suggest that the subunit is indeed present along with ₆ and ₃ in the expressed receptor complexes.

Next, a series of concentration-response studies were performed with GABA, gaboxadol, isoguvacine, muscimol, IAA, and P4S (Fig. 6; Table 5). As observed at ₄₃-containing receptors, gaboxadol, isoguvacine, and muscimol proved to elicit a higher maximal response than GABA, whereas IAA and P4S behaved as partial agonists. All compounds interacted more potently with ₆₃ than with ₄₃. With respect to the E_max values, a further polarization of the efficacy levels was observed in that gaboxadol, isoguvacine, and muscimol interacted more efficaciously with the former receptor subtype than with the latter whereas the reverse was the case for the partial agonists.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Concentration-response curves for a broad range of GABAA receptor agonists obtained in Xenopus oocytes expressing GABAA receptors composed of 63 subunits. All data were normalized with respect to GABA Imax and subsequently fitted to the eq. 1. Derived parameters are seen in Table 5.

View this table: [in this window] [in a new window]   TABLE 5 Fitting results from agonist concentration-response curves obtained from Xenopus oocytes expressing GABAA receptors consisting of 63 subunits All data were normalized with respect to GABAImax values from the same oocyte and fitted to eq. 1. Fitting results are presented as arithmetic means ± S.E.M. except for EC50, which is the geometric mean value.

	Discussion

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We have suggested previously that the GABA_A receptor agonist, gaboxadol, and the allosteric benzodiazepines site agonists, including the standard 1,4-benzodiazepines as well as the more recent compounds with nonbenzodiazepine structures such as zaleplon, zolpidem, zopiclone and indiplon, in addition to binding to different recognition sites also interact with two distinctly different receptor populations in the mammalian brain. According to our interpretation of data generated from the rat cortical wedge preparation and from rotarod studies in the rat, benzodiazepines primarily interact with synaptically located _12/32-containing receptors whereas gaboxadol's main target seems to be extrasynaptic ₄₃ receptors (Ebert et al., 2002; Storustovu and Ebert, 2003; Voss et al., 2003). To shed further light on gaboxadol's apparent functional selectivity for GABA_A receptors composed of ₄₃ subunits, we expressed this subunit combination in Xenopus oocytes.

Because the subunit has proven difficult to coexpress, our attention was focused on demonstrating that this subunit was actually incorporated into the expressed receptor complexes at the cell surface. Initially, in an attempt to elucidate potential differences in the agonist pharmacology of the -deficient versus -containing receptor complexes, potencies and relative efficacies for gaboxadol, GABA, and P4S were established from the two different receptor constructs. However, no differences in the potencies and only minor differences in the relative efficacies were found. This finding suggests either that the subunit is not successfully incorporated or that the subunit does not alter the agonist responsiveness. To clarify whether or not the former possibility was the case, we investigated the sensitivity of the two subunit combinations toward the negative modulators, Zn²⁺ and La³⁺, and found significant differences in the Zn²⁺ sensitivity between ₄₃ (IC₅₀ = 62 nM)- and ₄₃ (IC₅₀ = 5.3 µM)-containing receptors. These findings are in perfect agreement with results from analogous studies in ₆₃- and ₆₃-containing receptors expressed in Xenopus oocytes (Thompson et al., 1997). Here, Thompson et al. found no difference between GABA potencies at the two different subunit assemblies, whereas a 100-fold difference in Zn²⁺ sensitivities was observed. Thus, in contrast to ₂-containing receptor complexes (for which the presence of a subunit renders the receptor complex less sensitive to agonists (for a recent review, see Sieghart and Sperk, 2002), the incorporation of a subunit does not seem to lead to alteration of the agonist-gating properties of the receptor complex.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Concentration-response curves for gaboxadol obtained from Xenopus oocytes expressing 43- and 63-containing GABAA receptors compared with previously published oocyte results obtained at 1–632S-containing GABAA receptors (Ebert et al., 2001). The gray bar enclosed by dashed lines indicates the therapeutically relevant concentration area (Madsen et al., 1983).

In Fig. 7, the concentration-response relationship for gaboxadol at ₄₃- and ₆₃-containing receptors is compared with those of _1–632S-containing GABA_A receptors. As seen from the figure, gaboxadol interacts much more potently and efficaciously with the _4/63-containing receptors than with any of the ₂-containing receptors. Curiously, the highly efficacious behavior of gaboxadol at the former subunit combinations could seem to be a -specific property, because this behavior is not found at _432S-expressing oocytes or at any other of the ₂-containing receptors. On the other hand, the high relative efficacy level is also found at the -deficient ₄₃ receptors, indicating that the ₂ subunit would be the one that renders the receptors "resistant" to relative efficacy levels higher than that of GABA. This points to the fact the - and ₂-containing receptors possess dissimilar gating properties, which is in line with the observation that the former receptors are insensitive toward BZD modulation whereas the subunit confers BZD sensitivity to _1–3,5 receptor complexes.

In previous publications on ₄₃-containing receptors, the activity of gaboxadol was termed "superagonism" (Adkins et al., 2001; Brown et al., 2002). However, as demonstrated in Fig. 3, the relative increase in the maximal response of gaboxadol (relative to GABA) is primarily a consequence of a reduced maximal response to GABA, making this endogenous ligand a partial agonist at ₄₃-containing receptors. Thus, GABA, as predicted by the concept of partial agonism, is able to suppress the response to gaboxadol until a level of response corresponding to that of GABA is obtained. The mechanism underlying this partial agonist activity of GABA still remains to be addressed.

In conclusion, the present oocyte data suggest that gaboxadol interacts potently and efficaciously with ₄₃-, ₄₃-, and ₆₃-containing receptors, for all of which a predominantly extrasynaptic localization has been proposed (Nusser et al., 1998; Sun et al., 2004). Furthermore, since clinical observations (e.g., Faulhaber et al., 1997) have indicated that a single p.o. dose of 10 to 15 mg of gaboxadol modulates sleep, and plasma concentration at these doses lie within 0.5 to 2 µM (Madsen et al., 1983) (shown by a gray bar in Fig. 7), plasma concentrations relevant for the activation of extra synaptic GABA_A receptors are obtained in humans. It is not currently known whether gaboxadol is accumulated in the central nervous system after a single exposure. It may therefore be possible that a higher concentration of gaboxadol in the central nervous system than that in the plasma may be present. However, previous studies with gaboxadol in slices from rodents (e.g., Shen et al., 2005) have used concentrations in the low micromolar range, indicating that effects are obtained within this concentration range. In this low micromolar concentration range, gaboxadol activates only ₄₃- and ₆₃-containing receptors. These data are therefore in agreement with our previously published hypothesis that these extrasynaptically located and BZD-insensitive receptors are the primary sites of action of gaboxadol and underline the hypothesis that BZD site agonists and the GABA_A receptor agonist, gaboxadol, act at two different receptor populations. This may also explain why no cross-sensitization between gaboxadol and zolpidem was observed in a cross-tolerance study using the rotarod model in the rat (Voss et al., 2003). Similarly, lorazepam-trained rats and baboons have been shown not to occasion drug-lever responding in a food-maintained two-lever drug versus no-drug discrimination procedure when exposed to gaboxadol (Ator and Griffiths, 1986). In a similar experimental paradigm, using muscimol-trained rats, administration of gaboxadol produced full substitution for muscimol, whereas diazepam failed to produce full substitution (Jones and Balster, 1998). Analogously, in zolpidem-trained squirrel monkeys, full substitution was observed in at least half of the subjects studied after triazolam, lorazepam, midazolam, diazepam, and chlordiazepoxide (but not oxazepam) administration, in contrast to findings with i.a. muscimol, baclofen, and buspirone, which did not evoke substantial substitution (Rowlett et al., 1999). Hence, both rodents and primates do not appear to be able to distinguish the various benzodiazepine site agonists from each other, regardless of chemical structure, whereas drugs that bind to other (non-BZD) sites (i.e., gaboxadol and muscimol) are surprisingly discernible from the benzodiazepines, thus demonstrating the distinct molecular mechanisms of action of the two compound classes.

Taken together, several lines of evidence obtained from in vivo and in vitro studies have emphasized that gaboxadol seems to interact with a GABA_A receptor population, which is distinctly different from that targeted by the benzodiazepines. The present data add further evidence to our previous published hypothesis that a specific interaction with extrasynaptically located GABA_A receptors underlies the novel pharmacological profile of gaboxadol.

	Acknowledgements

 
We thank Jan Egebjerg, Head of Department of Molecular Genetics, H. Lundbeck A/S, for constructive discussions and for the generous supply of the cRNA used in the present study. We thank Lene Heding for excellent technical expertise in the RNA preparation and for outstanding collaboration. We thank Marianne Faber for grand help with the oocyte preparation. In addition, we thank Dr. Keith Wafford, Merck Sharp and Dohme Neuroscience Research Center, Harlow, Essex, UK, for fruitful discussions.

	Footnotes

 
doi:10.1124/jpet.105.092403.

ABBREVIATIONS: P4S, piperidine-4-sulfonic acid; IAA, imidazole-4-acetic acid; thio-4-PIOL, 5-(4-piperidyl)-3-isothiazolol; 4-PIOL, 5-(4-piperidyl)-3-isoxazolol; SR 95531, 2-(3-carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide (gabazine); BZD, benzodiazepine; MUSC, 5-(aminomethyl)-3-isoxazolol (muscimol); IGU, 1,2,3,6-tetrahydro-4-pyridine carboxylic acid (isoguvacine); THIP, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (gaboxadol); BMC, bicuculline methochloride.

Address correspondence to: Dr. Bjarke Ebert, Department of Electrophysiology, H. Lundbeck A/S, 9 Ottiliavej, DK-2500 Valby, Denmark. E-mail: bjeb{at}lundbeck.com

	References

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