Several groups have characterized the pharmacology of α4- or α6β3δ-containing GABAA receptors expressed in different cell systems. We have previously demonstrated that the pharmacological profiles of a series of GABAA receptor agonists are highly dependent on the α subunit and little on the β and γ subunits, so to further understand the contribution of the different subunits in the GABAA receptor complex, we characterized a series of full agonists, partial agonists, and antagonists at α4β3, α4β3δ, and α6β3δ receptors expressed in Xenopus oocytes. Little or no difference was seen when the compounds were compared at αβ- and αβδ-containing receptors, whereas a significant reduction in both potency and relative efficacy was observed compared with αβγ-containing receptors described in the literature. These data clearly confirm that the presence of the δ subunit in heterotrimeric receptors is a strong determinant of the increased pharmacological activity of compounds with agonist activity. The very similar agonist pharmacology of αβ- and αβδ-containing receptors, which is significantly different from that of αβγ-containing receptors, shows that whereas the presence of a γ subunit impairs the response to an agonist stimulation of the αβ receptor complex, the δ subunit does not affect this in any way. Taken together, these data are well in line with the idea that α4β3δ 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 GABAA agonists.
The major inhibitory neurotransmitter within the mammalian brain, GABA, mediates its fast transmission via the GABAA receptors. These ligand-gated ion channels assume a pentameric structure formed by coassembly of subunits from seven different classes (α1–6, β1–3, γ1–3, δ, ϵ, θ, and ρ1–3). Most native GABAA receptors are composed of two α, two β, and a γ, δ,or ϵ subunit (for a recent review, see Sieghart and Sperk, 2002). During the last decade, it has become increasingly evident that the subcellular localization of these receptors may be of crucial importance to the functional consequences of receptor activation. Thus, in addition to the synaptically located GABAA receptors, which are usually constituted by αβγ subunits, αβδ-containing receptors may be selectively targeted to perior extrasynaptic sites (Stell et al., 2003; Wei et al., 2003). It is generally agreed that the physiological role of these extrasynaptic receptors is to respond to synaptic spillover, thereby acting as sensors for extracellular GABA (Brickley et al., 2001). Whereas receptors implicated in the direct synaptic transmission mediate phasic inhibitory signals, extrasynaptic receptors mediate tonic currents, which are thought to play a major role in refinement of the neuronal firing pattern (Brickley et al., 2001; Mody, 2001).
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 GABAA receptors may be distinctly different from that of extrasynaptic receptors. One of the most clear-cut examples is the α6βδ subtype, which is exclusively present at extrasynaptic sites in cerebellar granule cells, whereas the synaptically located receptor subtypes in these cells are dominated by α1βγ2 and α1α6βγ2 subunits (Nusser et al., 1998). Studies in transgenic mice, in which the expression of the α6 subunit has been obliterated, have demonstrated a post-translational loss of δ 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 α4 and δ subunits (Sur et al., 1999; Peng et al., 2002). It has been speculated whether the cerebral homolog of α6, the α4 subunit, is also located preferentially at extrasynaptic locations. This seems reasonable because both α 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 α4 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 GABAA receptor agonist gaboxadol may be the extrasynaptically located receptors. Expression of α4β3δ-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 α1-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 α4β3δ 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 α1–6βxγ2 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 δ-containing GABAA receptors, namely the receptor subtype composed of α6β3δ subunits, to determine whether the altered pharmacology could be ascribed to the presence of the α4 or the δ subunit.
Materials and Methods
cRNA Preparation. Cloning and sequencing of cDNAs encoding human α4, α6, β3, and δ GABAA 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 (α4, β3, and δ) or a pCDM8 (α6) 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 α4/δ, β3, and α6 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 α4-, β3-, and δ-encoding cRNA (α4 and δ: 32.2 ng/oocyte; β3: 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 MΩ 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 Imax, elicited from that particular cell and fitted to eq. 1: where Emax 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 nH is the slope factor.
The competitive GABAA receptor antagonists were characterized with respect to their ability to shift a GABA concentration-response curve rightward when present in a fixed concentration. Ki 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: where [I] is the (fixed) antagonist concentration. Geometric mean values of the Ki 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: 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:
With an F test comparing the χ2 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.
α4β3- and α4β3δ-Expressing Xenopus Oocytes. Various combinations of GABAA receptor α4, β3, and δ subunits encoding cRNAs were injected in Xenopus oocytes. Whereas injection of α4β3 and α4β3δ subunit combinations gave rise to functional receptors with maximal GABA currents, GABA Imax, of 314 ± 48 (mean ± S.E.M.) and 103 ± 29 nA, respectively, no GABA-elicited currents could be measured in β3δ- and α4δ-injected oocytes.
Because the δ 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 α4β3δ encoding cRNA really do contain the δ subunit. In an attempt to increase the fraction of δ-containing receptor complexes as much as possible, a 10-fold excess of δ-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 GABAA receptor subunit assemblies, whereas other subunit combinations seem to be less sensitive to these cations. Zn2+ has been reported to inhibit recombinant α1βx receptors, whereas their γ2-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 α4β3γ2S- and α4β3δ-containing receptors stably expressed in L(tk) cells and a less significant difference between the same constructs toward Zn2+, with α4β3γ2S being the most sensitive.
Therefore, concentration-inhibition studies were performed by application of increasing concentrations of the cations in the presence of an EC50 GABA concentration (determined for each individual cell). Data were fitted to eq. 3.
In our system, the α4β3δ combination proved to be sensitive to La3+ with an IC50 value of 5.7 μM, whereas the δ-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 α4β3δ receptors and because the degree of inhibition of the α4β3 receptor complexes turned out to be variable, La3+ would to all appearances not be the most suitable tool for discrimination between δ-deficient and δ-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 α4β3-containing receptors (IC50 = 62 nM), whereas α4β3δ-containing receptors are 100-fold less sensitive (IC50 = 5.3 μM). This finding demonstrates that the δ 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 α4β3δ receptors yields a response of approximately 80% of the EC50 response or less, the receptor population presumably will contain a considerable fraction of α4β3 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 α1–6βxγ2S receptors (Ebert et al., 1994, 1997, 2001), were tested at α4β3δ-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 Emax 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 Emax 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).
To investigate whether the highly efficacious behavior of gaboxadol would be due to a δ-specific receptor interaction, this agonist was tested at α4β3 GABAA 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 α4β3δ-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 α4β3δ-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).
The potencies of the two standard competitive antagonists, bicuculline methochloride (BMC) and SR 95531 (gabazine), were established from α4β3δ-expressing oocytes, using the ability of a fixed concentration of these compounds to cause a parallel, rightward shift of a GABA concentration-response curve. A representative example of such a shift is seen in Fig. 4. Coanalysis of the concentration-response curves in the absence and presence of the antagonists (using eq. 2, see Materials and Methods) revealed Ki values of 1.16 μM and 95 nM for BMC and SR 95531, respectively (Table 3). Likewise, the antagonist properties of the two very low efficacy partial agonists, 4-PIOL and thio-4-PIOL, were assessed by the same procedure, yielding Ki values of 13.5 and 3.33 μM, respectively (Table 3).
α6β3δ-Expressing Xenopus Oocytes. To evaluate the agonist pharmacology at another major extrasynaptic receptor subtype, the cerebellar α6βδ-containing GABAA receptors (Nusser et al., 1998; Sassoe-Pognetto et al., 2000), Xenopus oocytes were injected with α6β3δ-encoding cRNAs in a subunit ratio of 10:1:10. As for the α4β3δ-containing receptors, the full incorporation of the δ subunit into the expressed receptors was a matter of some concern. Surprisingly, control experiments revealed that injection of δ-deficient α6β3 cRNAs did not give rise to formation of functional receptor complexes sensitive to GABA or other agonists, whereas robust expression was obtained with α6β3δ-encoding cRNAs (GABA Imax = 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 α6β3 expression might indicate that formation of this receptor by-product in α6β3δ-injected oocytes is not a crucial problem. However, to prove the presence of δ subunits in α6β3δ-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, nH, in Table 4, the course of the curve was not as steep as that seen at α4β3δ 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.
When fitting the data to the traditional one-site equation (eq. 3), the IC50 value obtained from the α6β3δ-expressing oocytes (8.7 μM) was in the same range as that derived from α4β3δ-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 α6β3- and α6β3δ-expressing oocytes. Conclusively, these data suggest that the δ subunit is indeed present along with α6 and β3 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 α4β3δ-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 α6β3δ than with α4β3δ. With respect to the Emax 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.
We have suggested previously that the GABAA 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 α1β2/3γ2-containing receptors whereas gaboxadol's main target seems to be extrasynaptic α4β3δ receptors (Ebert et al., 2002; Storustovu and Ebert, 2003; Voss et al., 2003). To shed further light on gaboxadol's apparent functional selectivity for GABAA receptors composed of α4β3δ subunits, we expressed this subunit combination in Xenopus oocytes.
Because the δ 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 δ-deficient versus δ-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 δ subunit is not successfully incorporated or that the δ 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 α4β3 (IC50 = 62 nM)- and α4β3δ (IC50 = 5.3 μM)-containing receptors. These findings are in perfect agreement with results from analogous studies in α6β3- and α6β3δ-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 γ2-containing receptor complexes (for which the presence of a γ subunit renders the receptor complex less sensitive to agonists (for a recent review, see Sieghart and Sperk, 2002), the incorporation of a δ subunit does not seem to lead to alteration of the agonist-gating properties of the receptor complex.
At α6β3δ-containing receptors, gaboxadol, muscimol, and isoguvacine showed even higher relative efficacy and potency levels than at α4β3δ-containing receptors. The expression of α6 is confined to granule cells of the cerebellum where it assembles with β2/3 and δ subunits (Jones et al., 1997; Jechlinger et al., 1998; Nusser et al., 1999; Sassoe-Pognetto et al., 2000) to form receptor complexes almost exclusively found at extrasynaptic locations (Nusser et al., 1999; Sassoe-Pognetto et al., 2000). Because cerebellum plays a key role in the refinement of motor action, an involvement of α6-containing receptors in motor coordination could be expected. However, studies in transgenic mice, in which the expression of α6 has been obliterated, have not revealed any major phenotypic deficits. Thus, these α6 knockout mice seem to breed and develop normally (Homanics et al., 1997; Jones et al., 1997) and exhibit no difference in motor performance from their wild-type littermates (Jones et al., 1997). Thus, at present, the functional importance of α6β2/3δ-containing GABAA receptors remains enigmatic.
In Fig. 7, the concentration-response relationship for gaboxadol at α4β3δ- and α6β3δ-containing receptors is compared with those of α1–6β3γ2S-containing GABAA receptors. As seen from the figure, gaboxadol interacts much more potently and efficaciously with the α4/6β3δ-containing receptors than with any of the γ2-containing receptors. Curiously, the highly efficacious behavior of gaboxadol at the former subunit combinations could seem to be a δ-specific property, because this behavior is not found at α4β3γ2S-expressing oocytes or at any other of the γ2-containing receptors. On the other hand, the high relative efficacy level is also found at the δ-deficient α4β3 receptors, indicating that the γ2 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 δ- and γ2-containing receptors possess dissimilar gating properties, which is in line with the observation that the former receptors are insensitive toward BZD modulation whereas the γ subunit confers BZD sensitivity to α1–3,5βδ receptor complexes.
In previous publications on α4β3δ-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 α4β3δ-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 α4β3-, α4β3δ-, and α6β3δ-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 GABAA 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 α4β3δ- and α6β3δ-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 GABAA 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 GABAA 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 GABAA receptors underlies the novel pharmacological profile of gaboxadol.
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.
- Received July 11, 2005.
- Accepted September 16, 2005.
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.
- The American Society for Pharmacology and Experimental Therapeutics